Application of ATAD3 inhibitor in prevention and / or treatment of breast cancer

Abstract

The invention discloses application of an ATAD3 inhibitor in prevention and / or treatment of breast cancer. In particular discloses nucleic acid molecules for silencing or knocking out ATAD3A and ATAD3B genes, or application of ATAD3A and ATAD3B antibodies in prevention and / or treatment of breast cancer. According to the invention, ATAD3A and ATAD3B genes in breast cancer cells are knocked out by using a CRISPR / Cas9 system, and results show that the ATAD3 inhibitor can significantly increase cGAS-STING signal channel activation and STING protein transport, and can significantly inhibit proliferation of breast cancer cells and generation of in-vivo tumors, and the breast cancer cells with the ATAD3A gene and the ATAD3B gene knocked out completely lose the tumor formation ability in a mouse body. The ATAD3 inhibitor developed by the invention has wide clinical application value and prospect in prevention and treatment of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of ATAD3 inhibitors in preventing and / or treating breast cancer. Background Art

[0002] The cGAS-STING signaling pathway regulates a variety of immune-related diseases, including infection by exogenous pathogens, autoimmune diseases, anti-tumor immune responses, and aging-related inflammatory responses. The basis for the extensive involvement of the cGAS-STING signaling pathway in biological processes is its ability to sense cells' exogenous DNA from microorganisms. When exogenous DNA invades, the cGAS protein is activated and catalyzes the synthesis of the second messenger cGAMP. Subsequently, the resting STING protein on the endoplasmic reticulum captures cGAMP and undergoes oligomerization before leaving the endoplasmic reticulum and translocating to the Golgi apparatus. The STING protein recruits the TBK1 protein in the cell, and TBK1 then undergoes autophosphorylation and phosphorylates STING. The activated TBK1 recruits and activates IRF3, thereby initiating the production of downstream type I interferon I IFNs, and IFNs have an important effect on the anti-tumor effect of cells. On the one hand, IFNs induce cytotoxic T lymphocytes to migrate to tumors, thereby recognizing and killing tumor cells; on the other hand, they promote the expression of tumor-specific antigens to CD8 + The presentation of T cells normalizes the tumor microenvironment and achieves an inhibitory effect on tumor cells.

[0003] Breast cancer is not only the most common malignant tumor in women worldwide, but also the second highest tumor in terms of mortality rate among women. There are various treatments for breast cancer, including surgery, chemotherapy, radiotherapy, endocrine therapy, and targeted therapy based on trastuzumab that has emerged in recent decades. Although comprehensive treatment of breast cancer can reduce its recurrence and metastasis rate and mortality rate, and prolong the disease-free survival (DFS) and overall survival (OS) of patients, the treatment effect is still not ideal, and problems such as tumor recurrence and drug resistance are still prominent. In dealing with the complex disease of breast cancer, the medical community still faces huge challenges. Therefore, continuous research and development of new breast cancer treatment drugs has far-reaching and important significance for improving the survival and cure rate of breast cancer patients and changing the treatment pattern of breast cancer. Summary of the invention

[0004] One of the purposes of the present invention is to provide a use of a cGAS-STING signaling pathway activator or an ATAD3 inhibitor in the prevention and / or treatment of breast cancer. The technical problems to be solved are not limited to the described technical subjects, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0005] In order to achieve the above objectives, the present application first provides the use of a cGAS-STING signaling pathway activator in any of the following:

[0006] A1) Use in the preparation of products for preventing and / or treating breast cancer;

[0007] A2) Use in the preparation of a product for inhibiting the proliferation of breast cancer cells;

[0008] A3) Use in the preparation of a product for inhibiting the tumorigenicity of breast cancer cells;

[0009] A4) Use in the preparation of a product for inhibiting the occurrence or growth of breast cancer tumors.

[0010] The cGAS-STING signaling pathway activator includes any substance that can activate protein molecules in the signaling pathway and thus activate the signaling pathway, including but not limited to cGAS activators, STING activators, TBK1 activators and IRF3 activators.

[0011] Furthermore, the cGAS-STING signaling pathway activator may be an ATAD3 inhibitor.

[0012] The ATAD3 inhibitor can increase or promote the transport of STING protein from the endoplasmic reticulum to the Golgi apparatus in cells, activate STING protein, and then activate the cGAS-STING signaling pathway.

[0013] The present invention also provides the use of ATAD3 inhibitors in any of the following:

[0014] B1) Use in the preparation of products for preventing and / or treating breast cancer;

[0015] B2) Use in the preparation of a product for inhibiting breast cancer cell proliferation;

[0016] B3) Use in the preparation of a product for inhibiting the tumorigenicity of breast cancer cells;

[0017] B4) Use in the preparation of a product for inhibiting the occurrence or growth of breast cancer tumors;

[0018] The ATAD3 inhibitor includes any of the following:

[0019] C1) a substance that inhibits the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the ATAD3A gene and the ATAD3B gene;

[0020] C2) Substances that inhibit or reduce the content, activity and / or function of ATAD3A protein and ATAD3B protein.

[0021] The substance may be any substance that inhibits the activity of ATAD3A protein and ATAD3B protein and / or reduces the content of ATAD3A protein and ATAD3B protein by regulating expression at the gene level or regulating the protein level.

[0022] The expression regulation at the gene level may include expression regulation at the chromatin level (such as histone modification, chromatin remodeling), transcription level (such as regulation of promoters, transcription factors, co-regulators), post-transcriptional level (such as RNA splicing, microRNA regulation) and post-translational level (such as ubiquitination, SUMOylation, acetylation, glycosylation, phosphorylation, methylation, NEDD8 modification, etc.), etc.

[0023] The regulation of protein levels may include regulating the activity and / or content of the protein by protein degradation, protein interaction or other methods capable of regulating protein activity.

[0024] In the above application, the substance includes a substance that causes the ATAD3A gene and ATAD3B gene to be deleted or inactivated by site-directed mutagenesis technology, gene knockdown technology, gene editing technology and / or gene knockout technology, or a substance that specifically binds to ATAD3A protein and / or ATAD3B protein to reduce the content of ATAD3A protein and ATAD3B protein or inactivate its function.

[0025] It is well known to those skilled in the art to inhibit gene expression, silence or knock out genes by using site-directed mutagenesis technology (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis and cassette mutagenesis, etc.), gene knockdown technology (including RNA interference technology, Morpholino interference technology, antisense nucleic acid technology and ribozyme technology, etc.), gene editing technology (including zinc finger ribozyme gene editing technology, TALEN gene editing technology and CRISPR gene editing technology, etc.) or gene knockout technology (including complete gene knockout and conditional gene knockout). For example, shRNA, siRNA or miRNA targeting the target gene can be used to inactivate gene expression or silence the gene from the post-transcriptional level or translation level. The target gene can also be knocked out using a CRISPR-Cas system containing sgRNA and Cas protein. Alternatively, the target gene is mutated using site-directed mutagenesis technology to produce a frameshift mutation or premature termination of translation, thereby inactivating the target gene or weakening its function. In some embodiments of the present invention, CRISPR gene editing technology is used to knock out the target gene (ATAD3A gene and ATAD3B gene).

[0026] Furthermore, the substance includes nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, recombinant vectors (such as gene editing vectors), recombinant cells or viral vectors (such as lentivirus and adeno-associated virus vectors).

[0027] Furthermore, the nucleic acid molecules may include (1) double-stranded RNA (dsRNA), small interfering RNA (siRNA), micro RNA (miRNA) and short hairpin RNA (shRNA) used in RNA interference technology; (2) antisense RNA (asRNA) and antisense oligonucleotide (AON) used in antisense nucleic acid technology; (3) gRNA and sgRNA used in gene editing technology; (4) aptamer and ribozyme, etc.

[0028] The antibodies may include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, Fab, Fab′, F(ab′)2, antibody variable region (Fv), disulfide bond-stabilized Fv (dsFv), single-chain antibodies (ScFv), single-domain antibodies (sdAb, i.e., nanobodies), minibodies, and minimum recognition units (MRU).

[0029] It is well known to those skilled in the art that antibodies are commonly used protein inhibitors that can inhibit the activity of their ligands. It is also known to those skilled in the art that antibodies can be prepared using ATAD3A protein or ATAD3B protein to inhibit the activity of the protein through the binding of the antibody to the protein.

[0030] In the above application, the ATAD3 inhibitor includes any one of the following:

[0031] D1) Nucleic acid molecules for silencing or knocking out ATAD3A gene and ATAD3B gene;

[0032] D2) Antibodies that bind to ATAD3A protein and / or ATAD3B protein.

[0033] In the above application, the ATAD3 inhibitor includes a sgRNA composition targeting the ATAD3A gene and the ATAD3B gene or a CRISPR / Cas9 system containing the sgRNA composition.

[0034] The sgRNA composition includes an sgRNA targeting the ATAD3A gene and an sgRNA targeting the ATAD3B gene.

[0035] The CRISPR / Cas9 system also includes a Cas9 protein.

[0036] Furthermore, the Cas9 protein described herein is not limited to a specific protein, as long as it can be used in conjunction with the sgRNA of the present invention.

[0037] Further, the Cas9 proteins described in this article include Streptococcus pyogenes Cas9 (spCas9, subtype II-A), spCas9HF (high fidelity), notchase Cas9 (nCas9), Staphylococcus aureus Cas9 (saCas9, subtype II-A), Neisseria meningitidis Cas9 (NmCas9, subtype II-C), Francisella novicida Cas9 (FnCas9, subtype II-B), Streptococcus thermophilus Cas9 (St1Cas9, St3Cas9), Campylobacter jejuni Cas9 (CjCas9) and Treponema sp. Cas9, as well as Cas9 orthologs of other organisms but are not limited to these. The Cas9 protein may also include high-fidelity Cas9 mutants (such as SpCas9-HF1, eSpCas9-1.1 and TrueCut TM HiFi Cas9 protein) etc.

[0038] It is known to those skilled in the art that Cas9 protein, Cas9 protein mRNA, Cas9 expression vector (a vector containing and expressing a DNA molecule encoding Cas9 protein), sgRNA, and sgRNA expression vector (a vector containing and expressing a DNA molecule encoding sgRNA) can be transferred to animal cells by various methods known in the art, for example, calcium phosphate coprecipitation method, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e., electrotransfection method), microinjection, gene gun method, or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method), etc. can be introduced into ex vivo animal cells (transfection). It can also be introduced into in vivo animal cells by microinjection method, viral vector method, etc.

[0039] When using an expression vector to deliver sgRNA and Cas9 protein, sgRNA and Cas9 protein can be linked to different expression vectors for expression, or linked to the same expression vector for expression.

[0040] In the above application, the ATAD3 inhibitor includes any one of the following:

[0041] E1) sgRNA1 and sgRNA2, wherein the target sequence of sgRNA1 is shown in SEQ ID NO: 1; the target sequence of sgRNA2 is shown in SEQ ID NO: 2;

[0042] E2) DNA molecule 1 encoding the sgRNA1 described in E1) and DNA molecule 2 encoding the sgRNA2 described in E1);

[0043] E3) a recombinant vector containing or respectively containing the DNA molecule 1 and the DNA molecule 2 described in E2);

[0044] E4) A recombinant microorganism containing or separately containing the DNA molecule 1 and the DNA molecule 2 described in E2).

[0045] In the above applications, the recombinant microorganism may be a recombinant virus (such as a recombinant lentivirus).

[0046] The present invention also provides a biomaterial, which may be any of the following:

[0047] F1) sgRNA1 and sgRNA2 described herein;

[0048] F2) DNA molecule 1 and DNA molecule 2 described herein;

[0049] F3) a recombinant vector containing or respectively containing the DNA molecule 1 and the DNA molecule 2 described in F2);

[0050] F4) a recombinant microorganism containing or respectively containing the DNA molecule 1 and the DNA molecule 2 described in F2);

[0051] F5) A recombinant host cell containing or separately containing the DNA molecule 1 and the DNA molecule 2 described in F2).

[0052] The vector described herein can be a cloning vector or an expression vector. The vector can be a plasmid or a viral vector.

[0053] The viral vectors include, but are not limited to, adeno-associated virus (AAV) vectors, adenovirus vectors, herpes simplex virus (HSV) vectors, lentivirus (LV) vectors, poxvirus vectors, retrovirus vectors, rhabdo virus (baculovirus) vectors, papillomavirus vectors, Sendai virus vectors, and simian virus vectors.

[0054] The recombinant vector includes a recombinant DNA molecule constructed by connecting a DNA molecule encoding sgRNA1 and / or sgRNA2 described herein to a vector in vitro. The recombinant vector can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the sgRNA into a recipient cell or microorganism and can express the sgRNA.

[0055] Furthermore, the recombinant vector also includes a recombinant DNA molecule constructed by connecting the DNA molecule encoding the sgRNA1 and / or sgRNA2 described herein to a vector containing a Cas9 protein encoding gene in vitro. The recombinant vector can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the sgRNA into the recipient cell or microorganism and can express the sgRNA so that it can perform the knockout function of the target gene.

[0056] The recombinant vector can be a gene editing vector expressing sgRNA and Cas9 protein described herein. The method of constructing a gene editing vector is well known to those skilled in the art, and the Cas9 protein that matches (or combines with) it can be selected according to the designed guide RNA (such as sgRNA of the present invention), as long as the experimental purpose (such as gene knockout) can be achieved. For example, sgRNA and Cas9 protein can be connected to the same vector and started with a dual promoter, or sgRNA and Cas9 protein can be connected to different vectors, or a backbone vector (such as PX459 vector, PX458 vector, PX461 vector, PX462 vector, PX551 vector, PX552 vector, pGK1.1 vector, PX330 vector, PX335 vector, PX165 vector, eSpCas9 (1.1) vector, etc.) containing Cas9 protein gene can be selected as an expression vector of sgRNA, and the DNA molecule encoding sgRNA is cloned into the backbone vector to construct a gene editing vector targeting the target gene.

[0057] Furthermore, the recombinant vector may be a recombinant viral vector.

[0058] Furthermore, the recombinant viral vector may be a recombinant lentiviral vector.

[0059] Specifically, the recombinant lentiviral vector may be sgATAD3A#B recombinant vector 1 or sgATAD3A#B recombinant vector 2.

[0060] The sgATAD3A#B recombinant vector 1 contains the editing target site of the ATAD3A gene (SEQ ID NO: 1, the target sequence of sgRNA1) and the coding gene of the Cas9 protein on the vector backbone. After being packaged into a lentivirus and introduced into a recipient cell, the transcribed guide RNA (sgRNA1) can target the target sequence near the PAM of the recipient cell genome through base complementary pairing, that is, target the ATAD3A gene. The Cas9 protein causes a double-strand break in the DNA at the target site of the ATAD3A gene. Through the organism's own DNA damage repair response mechanism, during the repair process, the broken DNA ends will be directly connected together, resulting in random insertion or deletion of bases, thereby achieving knockout of the ATAD3A gene.

[0061] The sgATAD3A#B recombinant vector 2 contains the editing target site of the ATAD3B gene (SEQ ID NO: 2, the target sequence of sgRNA2) and the coding gene of the Cas9 protein on the vector backbone. After being packaged into a lentivirus and introduced into a recipient cell, the transcribed guide RNA (sgRNA2) can target the target sequence near the PAM of the recipient cell genome through base complementary pairing, that is, it targets the ATAD3B gene. The Cas9 protein causes a double-strand break in the DNA at the target site of the ATAD3B gene. Through the organism's own DNA damage repair response mechanism, during the repair process, the broken DNA ends will be directly connected together, resulting in random insertion or deletion of bases, thereby achieving knockout of the ATAD3B gene.

[0062] Although the backbone vector used in the embodiments provided by the present invention is a lentiviral vector lentiCRISPRv2, the present invention is not limited to this specific vector. Those skilled in the art may use other suitable vectors, such as adeno-associated virus vectors, adenovirus vectors, etc. As long as the vector can clone or express the DNA molecule 1 and / or the DNA molecule 2 described herein.

[0063] Furthermore, the microorganisms described herein include but are not limited to bacteria, viruses, fungi and actinomycetes.

[0064] Further, the microorganism described herein can be a virus. The virus includes but is not limited to adeno-associated virus (AAV), adenovirus, herpes simplex virus (HSV), lentivirus (LV), poxvirus, retrovirus, rhabdo virus (rhabdo virus), papillomavirus, Sendai virus and simian virus (Simian virus).

[0065] Furthermore, the recombinant microorganism may be a recombinant virus. The recombinant virus may be obtained by packaging a recombinant viral vector. Further, the recombinant virus may be a recombinant lentivirus.

[0066] Specifically, the recombinant lentivirus may be sgATAD3A#B recombinant lentivirus 1 or sgATAD3A#B recombinant lentivirus 2.

[0067] The sgATAD3A#B recombinant lentivirus 1 is obtained by introducing the sgATAD3A#B recombinant vector 1 into packaging cells for packaging. The sgATAD3A#B recombinant lentivirus 2 is obtained by introducing the sgATAD3A#B recombinant vector 2 into packaging cells for packaging.

[0068] The packaging cell is used to package a viral vector plasmid lacking at least one gene encoding a protein required for viral packaging, which can provide comprehensive viral proteins for viral packaging into recombinant viruses, and itself cannot produce any form of viral particles. Any cell that provides a protein or polypeptide lacking in viral packaging can be considered as a packaging cell. Suitable packaging cells are well known to those skilled in the art, such as Palt E cells, ECO cells, PG13 cells, HEK293 cells, HEK293T cells, CHO cells, 293T cells, 293 cells, MDCK cells, NIH3T3 cells, PA317 cells, COS cells, HeLa cells, Vero cells, PsiCRIP cells, etc. In one or more embodiments of the present invention, the packaging cell is a HEK293T cell.

[0069] The present invention also provides a pharmaceutical composition for preventing and / or treating breast cancer, which comprises any ATAD3 inhibitor described herein (e.g., sgRNA1 and sgRNA2 described in the present invention, or a gene editing vector containing a DNA molecule encoding the sgRNA1 and sgRNA2, or ATAD3A antibody and ATAD3B antibody).

[0070] Furthermore, the pharmaceutical composition also includes one or more pharmaceutically acceptable carriers.

[0071] The pharmaceutically acceptable carrier is selected from excipients, preservatives, protective agents, cosolvents, diluents (such as water, saline, PBS (phosphate buffer), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc.), wetting agents, disintegrants (such as dry starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, etc.), lubricants (such as sorbitan trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, etc.), fillers (such as starch, dextrin, etc.), adhesives (such as gelatin, pectin, gum arabic, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), penetration enhancers (such as Brij-78), pH regulator, stabilizer (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactant (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium lauryl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption promoter (such as chitosan), thickener (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidant (such as sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium thiosulfate, vitamin C, etc.), plasticizer (such as glycerol, sorbitol, phthalate, etc.), propellant (such as hydrofluoroalkane, dimethyl ether, etc.), aerosolizer, suspending agent, dispersant, colorant (such as TiO2, pigment, etc.) and flavoring agent. Those skilled in the art know that a carrier usually has multiple functions, for example, starch can be used as both a disintegrant and a binder. Those skilled in the art can make conventional selections of the above carriers according to the properties of the drug and the route of administration.

[0072] Excipients are usually used in medicines to shape the medicines, change the physical state of the medicines, and play a supporting role. Excipients include, but are not limited to: (1) Excipients for injections: such as solvent water, alcohols, ethers, amides, sulfoxides, esters, etc.; (2) Excipients for injection powders: such as sucrose, lactose, mannitol, etc.; (3) Excipients for sprays: such as soy lecithin, propylene glycol, borneol, ethanol, phenol, etc.; (4) Excipients for tablets: such as starch, sucrose, dextrin, methyl cellulose, gelatin, polyethylene glycol, tartaric acid, boric acid, etc.; (5) Excipients for eye drops: such as sodium hyaluronate, disodium ethylenediaminetetraacetic acid (EDTA-Na2), etc.; (6) Excipients for suppositories: such as cocoa butter, semi-synthetic or fully synthetic fatty acid glycerides, glycerol gelatin, polyethylene glycol, etc.; (7) Excipients for granules: such as corn flour, bentonite, zeolite powder, etc.; (8) Excipients for capsules: such as gelatin, etc.; (9) Excipients for gels: such as gelatin, pectin, gum arabic, etc.; (10) Excipients for ointments: such as vaseline, paraffin, liquid paraffin, lanolin, lanolin alcohol, beeswax, lard, vegetable oil, silicone oil, silicone, soaps, higher fatty alcohols, fatty alcohol sulfates, polyols, polyethylene glycol, FAPG, etc.; (11) Excipients for patches: such as ethylene-vinyl acetate copolymer (EVA), pressure-sensitive adhesive (PSA), etc.; (12) Excipients for films: such as gelatin, shellac, gum arabic, polyvinyl alcohol compounds, acrylic acid copolymers, etc.

[0073] The dosage form of the pharmaceutical composition includes, but is not limited to, injection (including injection solution and powder for injection), gel, eye drops (including eye drops and intraocular injection solution), oral solution, suppository, effervescent tablet, capsule, ointment, cream, spray, aerosol, external solution, tablet, powder, pill, powder, scarification agent, granule, drop, coating, patch and long-acting sustained-release preparation. Those skilled in the art know that the above-mentioned various dosage forms can be prepared by using active components, with suitable pharmaceutically acceptable carriers, according to conventional preparation processes of preparations.

[0074] The pharmaceutical composition may be in the form of an injection preparation.

[0075] Injection generally refers to a solution or emulsion for injection into the body made from extracted and purified medicinal materials, as well as a sterile powder prepared into a solution before use, which includes injection and powder injection for injection. The preparation method of injection is well known to those skilled in the art, for example, powder injection for injection can be prepared by vacuum freeze drying technology, spray drying technology, spray freeze drying technology. The drug can be prepared into a solution by a concentrated preparation method or a diluted preparation method with a suitable diluent, a cosolvent and / or a wetting agent, and then the injection can be prepared by filtering (such as surface filtration and / or deep filtration), encapsulation, sterilization and other steps.

[0076] In order to prepare the pharmaceutical composition into an injection preparation, such as a solution, emulsion, lyophilized powder injection and suspension, a commonly used diluent in the art, such as water, physiological saline, PBS (phosphate buffered saline), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc., can be used as a solvent to prepare a solution. In addition, in order to prepare an isotonic injection, an appropriate amount of a carrier such as sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, a conventional cosolvent, buffer, pH adjuster and other carriers can also be added.

[0077] The administration methods of the pharmaceutical composition include, but are not limited to, injection (such as administration in the form of injection), mucosal administration (such as administration in the form of sprays, aerosols, tablets, eye drops, suppositories, granules, capsules, etc.) and transdermal administration (such as administration in the form of gels, ointments, patches, films, etc.).

[0078] The administration methods of the pharmaceutical composition include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, intratumor injection, intracranial injection, mucosal administration, oral administration, skin application, oral and nasal spraying, aerosol inhalation, in vivo implantation, and in vitro device administration.

[0079] The present invention also provides the use of ATAD3A protein and ATAD3B protein as targets in the preparation of products having any of the following functions:

[0080] G1) binds to STING protein;

[0081] G2) regulates the cGAS-STING signaling pathway;

[0082] G3) regulates the transport of STING protein from the endoplasmic reticulum to the Golgi apparatus.

[0083] The ATAD3 described herein includes ATAD3A protein and ATAD3B protein. In mitochondria, ATAD3A and ATAD3B can form an ATAD3B-ATAD3A-mtDNA complex.

[0084] The ATAD3 protein family is a transmembrane protein on mitochondria. According to the topological structure prediction analysis, the N-terminus of ATAD3 extends outside the outer membrane of mitochondria, and the C-terminus extends into the mitochondrial matrix. The ATAD3 protein family has three family members, namely ATAD3A, ATAD3B and ATAD3C, but so far, no research has reported the expression and localization of ATAD3C in humans. The present invention takes breast cancer as the research object, screens and identifies the STING protein that interacts with the ATAD3N terminus using BioID proximity labeling technology, and verifies the effect of the loss of ATAD3 on the activation of the cGAS-STING signaling pathway in breast cancer cells, and further analyzes the role of ATAD3 in the translocation of the STING protein, providing a theoretical basis and data support for exploring the molecular mechanism of ATAD3's involvement in regulating the cGAS-STING signaling pathway.

[0085] The present invention uses ATAD3 inhibitors for the treatment of breast cancer for the first time. The present invention designs sgRNA for knocking out ATAD3A gene and ATAD3B gene, and knocks out ATAD3A gene and ATAD3B gene in breast cancer cells based on CRISPR / Cas9 system. Experimental results show that the use of ATAD3 inhibitors can significantly increase the activation of cGAS-STING signaling pathway in cells, significantly increase the transport of STING protein from the endoplasmic reticulum to the Golgi apparatus in cells, and ATAD3 inhibitors can significantly inhibit the proliferation of breast cancer cells and the occurrence of tumors in vivo. Breast cancer cells with ATAD3A gene and ATAD3B gene knocked out completely lose the ability to form tumors in mice. The ATAD3 inhibitor developed by the present invention has a wide range of clinical application value and prospects in the prevention and treatment of breast cancer.

[0086] Definition of terms

[0087] In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand this application, the definitions and explanations of relevant terms are provided below.

[0088] The term "cGAS-STING signaling pathway" generally refers to the key pathway in cells that is responsible for recognizing exogenous and exogenous DNA and generating immune responses. The proteins in this signaling pathway mainly include cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3).

[0089] The term "inhibitor (also called antagonist)" has a well-known meaning in the art and may refer to any substance that reduces (downregulates) the level and / or activity of a target protein or gene. The inhibitor may also refer to any substance (including phosphorylation inhibitors) that inhibits post-translational modification of a target gene.

[0090] The term "ATAD3 inhibitor" in the present application may be a substance that inhibits the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the ATAD3A gene and the ATAD3B gene, or a substance that inhibits or reduces the content, activity and / or function of the ATAD3A protein and the ATAD3B protein. The ATAD3 inhibitor includes a substance that deletes or inactivates the ATAD3A gene and the ATAD3B gene by techniques such as site-directed mutagenesis, gene knock-down, gene editing, and gene knock-out. The ATAD3 inhibitor also includes a substance that can target and bind to the ATAD3A protein and the ATAD3B protein, inhibit the activity of the ATAD3A protein and the ATAD3B protein, or prevent the ATAD3A protein and the ATAD3B protein from performing their functions.

[0091] The term "site-directed mutagenesis" generally refers to changing one or several bases in a gene through site-directed mutagenesis, resulting in a change in the amino acid composition of the corresponding protein. Site-directed mutagenesis methods include oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis.

[0092] The term "gene knock-down" is also called gene knockdown, which generally refers to the technology of inactivating gene expression or silencing gene at the post-transcriptional level or translation level without changing the DNA sequence of the gene. Gene knock-down includes RNA interference technology, Morpholino interference technology, antisense nucleic acid technology and ribozyme technology.

[0093] The term "gene editing" generally refers to the technology that can change a specific gene sequence in any cell, including somatic cells, and can cause base deletion, duplication, insertion, frameshift mutation, replacement and knockout of target genes, and achieve replacement, deletion, shearing and single base change of genomic sequences, that is, the technology of "editing" the genome or the sequence of a specific gene at will. Gene editing includes zinc finger nuclease gene knockout technology, TALEN gene editing technology and CRISPR gene editing technology.

[0094] The term "Cas9 protein" generally refers to the Cas endonuclease of the type II CRISPR system that forms a complex with crRNA and tracrRNA or with guide RNA for specific recognition and cleavage of all or part of the DNA target sequence. The Cas9 protein has two different domains: the HNH domain and the RuvC domain. The HNH domain is responsible for cleaving the DNA strand (target strand) that is complementary to the crRNA (or gRNA), while the RuvC domain is responsible for cleaving the non-complementary strand (non-target strand).

[0095] The term "sgRNA (single-guide RNA)" generally refers to the structure of a single RNA formed by artificially modifying the crRNA / tracrRNA complex (gRNA) with a dual RNA structure, and directly connecting crRNA and tracrRNA (or through a linker). sgRNA is a component of the CRISPR-Cas system, responsible for guiding the Cas protein to recognize and cut the target nucleic acid molecule. In actual gene editing applications, sgRNA can be directly synthesized or obtained by plasmid expression or in vitro transcription. sgRNA includes a recognition region and a framework region (scaffold). The framework region is known to those skilled in the art and is responsible for binding to the Cas protein. The recognition region is responsible for binding to the target site of the target gene and guiding the Cas protein to the target site.

[0096] The term "target sequence" herein refers to the target sequence of sgRNA, which refers to a sequence located in the gene to be edited (usually the coding region), which is complementary to the sgRNA, located near the desired location for cutting, and is the target site recognized by the sgRNA. The target sequence can determine the location and specificity of gene editing.

[0097] The term "gene knock-out" generally refers to a technique in which an exogenous mutated gene is used to replace an endogenous normal homologous gene through homologous recombination, thereby inactivating the endogenous gene, including complete gene knockout (e.g., complete mutation of the target gene based on a replacement targeting vector or an insertion targeting vector) and conditional gene knockout (e.g., tissue-specific knockout based on the Cre-LoxP recombinase system or the FLP-FRT recombinase system).

[0098] The term "RNA interference (RNAi)" generally refers to a technology that uses double-stranded RNA (dsRNA) to induce the degradation of mRNA of a target gene that is homologous and complementary to it, thereby silencing and shutting down gene expression, thereby inducing post-transcription gene silencing (PTGS) and achieving the purpose of preventing gene expression.

[0099] The term "Morpholino interference technology" usually refers to the use of morpholino to replace the pentose ring on traditional nucleotides, and the original phosphate group is also changed, so that the molecule as a whole does not carry any charge, cannot be recognized and degraded by RNase and DNase, and has extremely strong stability. Its principle is the same as that of antisense nucleic acid technology, which binds to the mRNA molecule by complementing the homologous sequence of the target gene mRNA, thereby hindering the binding of other molecules and proteins to the specific mRNA nucleic acid sequence, and ultimately preventing the target gene mRNA from being translated into protein.

[0100] The term "antisense nucleic acid technology" generally refers to the technology of inhibiting the expression of specific genes by artificially synthesizing antisense RNA or introducing its gene into cells, based on the principle that antisense RNA can complementarily bind to specific mRNA molecules with homologous sequences, thereby inhibiting the processing and translation of the mRNA. Antisense nucleic acid technology mainly includes antisense RNA (antisense RNA, asRNA) and antisense oligonucleotide (antisense oligonucleotide, AON).

[0101] The term "ribozyme technology" generally refers to the technology of using ribozymes to cut and degrade target RNA molecules. Ribozymes are a type of RNA molecules with biocatalytic activity that can specifically bind to and cut target RNA molecules, and can achieve degradation of specific RNA molecules, thereby inhibiting the expression of target genes. Ribozymes include hammerhead ribozymes, hairpin ribozymes, hepatitis D virus ribozymes, VS (Varkud satellite) ribozymes, and class I intron ribozymes.

[0102] The term "comprising" is not intended to be limiting, but is intended to be inclusive and means that there may be other elements besides the listed elements, which can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 To extract the N-terminal interacting proteins of ATAD3 protein using BioID proximity tagging technology. (A) SnapGene software analyzes the similarity of ATAD3A and ATAD3B sequences. (B) Construction of a tagging vector expressing BioID-ATAD3A. (C) Immunoblotting to detect the expression level of BioID-ATAD3A in 293T cells transfected with the BioID-ATAD3A tagging vector. (D) STING proteins that interact with the N-terminus of ATAD3A screened by mass spectrometry.

[0104] Figure 2 The ATAD3 protein family members interact with the STING protein. (A) ATAD3A-His and ATAD3B-His were overexpressed in MCF-7 cells, and the ATAD3A-His and ATAD3B-His interacting proteins were captured by immunoprecipitation, and the STING protein in the immunoprecipitation was detected by immunoblotting. (B) Endogenous ATAD3 protein family members were captured by immunoprecipitation in MCF-7 wild-type cells, and the STING protein and the endoplasmic reticulum transmembrane protein Sec61B in the immunoprecipitation were detected by immunoblotting.

[0105] Figure 3 The STING protein bound by ATAD3 protein family members under the stimulation of STING activator cGAMP is reduced. Among them, (A) After 2.5 hours of treatment with different concentrations of cGAMP (0μg / mL, 1μg / mL, 2μg / mL, 5μg / mL) in MCF-7 wild-type cells, ATAD3 protein family members were captured by immunoprecipitation, and the changes in STING protein levels in the immunoprecipitate were detected by immunoblotting. (B) ImageJ analysis of experimental results, values ​​are expressed as mean ± SEM (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0106] Figure 4 The loss of ATAD3 significantly increased the sensitivity of cells to the DNA mimic poly(dA:dT). (A) After the MDA-MB-231 control group (Ctrl) and DKO cells with double knockout of ATAD3A and ATAD3B were treated with 1 μg / mL double-stranded DNA mimic poly(dA:dT) for 1 hour and 2 hours, the changes in the expression levels of STING protein and downstream TBK1 protein, and the changes in the phosphorylation levels of STING protein and TBK1 protein were detected by immunoblotting. (B) and (C) ImageJ analysis of experimental results, values ​​are expressed as mean ± SEM (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0107] Figure 5The loss of ATAD3 significantly increases the sensitivity of cells to the STING protein agonist cGAMP. Among them, (A) After treatment with different concentrations of cGAMP (0μg / mL, 1μg / mL, 2μg / mL, 5μg / mL) for 2.5 hours in MCF-7 control group (Ctrl) and ATAD3DKO cells, immunoblotting was used to detect changes in the expression levels of STING protein and downstream TBK1 protein, and changes in the phosphorylation levels of STING protein and TBK1 protein. (B), (C) ImageJ analysis of experimental results, values ​​are expressed as mean ± SEM (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0108] Figure 6 Brefeldin A blocked the cGAMP concentration-dependent increase in STING protein phosphorylation in MCF-7 cells. The NC group treated MCF-7 control group (Ctrl) and ATAD3DKO cells with different concentrations of cGAMP (0μg / mL, 1μg / mL, 2μg / mL, 5μg / mL) for 2.5 hours, and the BFA group treated MCF-7Ctrl and DKO cells with different concentrations of cGAMP (0μg / mL, 1μg / mL, 2μg / mL, 5μg / mL) superimposed with 0.5μg / mL Brefeldin A for 2.5 hours, and the changes in STING protein expression and phosphorylation levels were detected by immunoblotting.

[0109] Figure 7 The loss of ATAD3 significantly increased the colocalization of STING protein with the Golgi apparatus. Among them, (A) MCF-7 control group (Ctrl) and ATAD3DKO cells were treated with cGAMP (2μg / mL) for 5 hours, and then fixed with 4% paraformaldehyde at 37°C for 15 minutes. The cells were immunofluorescently stained with STING, GM130 and DAPI. STING was labeled with green light, GM130 was labeled with red light, and DAPI was labeled with blue light. The images were taken by FV3000 confocal microscope, and the scale was 50μm. (B) ImageJ statistics of three independent repeated experiments, *, P<0.05; **, P<0.01; ***, P<0.001.

[0110] Figure 8The loss of ATAD3 leads to an increase in the STEEP protein bound to the STING protein in cells. Among them, (A) After the MCF-7 control group (Ctrl) and ATAD3DKO cells were treated with cGAMP (2μg / mL) for 2.5 hours, the endogenous STING protein was captured by immunoprecipitation, and the changes in the STEEP protein level in the immunoprecipitate were detected by immunoblotting. (B) After the MDA-MB-231 control group (Ctrl) and ATAD3DKO cells were treated with cGAMP (2μg / mL) for 2.5 hours, the endogenous STEEP protein was captured by immunoprecipitation, and the changes in the STING protein level in the immunoprecipitate were detected by immunoblotting. (C), (D) ImageJ analysis of experimental results, values ​​are expressed as mean ± SEM (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0111] Fig. 9 The in vitro proliferation capacity of MCF-7 and MDA-MB-231 knockout cell lines. Among them, (A) Western blot detection of ATAD3A and ATAD3B complementation in MCF-7 cell line. (B) Western blot detection of ATAD3A and ATAD3B complementation in MDA-MB-231 cell line. (C) The proliferation capacity of the control group, DKO, and Rescue cell lines in MCF-7 cell line (six replicates in each group). (D) The proliferation capacity of the control group, DKO, and Rescue cell lines in MDA-MB-231 cell line (six replicates in each group). *, P<0.05; **, P<0.01; ***, P<0.001.

[0112] Fig.10 The results of the mouse subcutaneous tumor formation experiment. In (A) and (B), nude mice were injected with MCF-7 and MDA-MB-211 control group and DKO cells, with 10 6 cells / 100 μL PBS. n(MCF-7)=10, n(MDA-MB-231)=9. DETAILED DESCRIPTION

[0113] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0114] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0115] The following examples used GraphPad Prism 9 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation, and P < 0.05 was considered to be significantly different between the groups. The quantitative experiments in the following examples were set up with three biological replicates unless otherwise specified.

[0116] In the following examples, the Gene ID of ATAD3A protein is: 55210; the GenBank accession number of the gene encoding ATAD3A protein (ie, ATAD3A gene) is: NM_001170535.3; the amino acid sequence of ATAD3A protein: Genbank accession number: NP_001164006.1.

[0117] The Gene ID of ATAD3B protein is: 83858, and the GenBank accession number of the gene encoding ATAD3B protein (ie, ATAD3B gene) is: NM_031921.6. The amino acid sequence of ATAD3B protein: Genbank accession number: NP_114127.3.

[0118] The ATAD3 proteins in the following examples refer to the ATAD3A protein and the ATAD3B protein.

[0119] The human breast cancer MCF7 cell line in the following examples is described in the following document: Wong CYA, Jiang H, Abel PW, et al. Phorbol myristate acetate suppresses breast cancer cell growth via down-regulation of P-Rex1 expression [J]. Protein & Cell, 2016 (6). DOI: 10.1007 / S13238-016-0261-X.

[0120] The human breast cancer MDA-MB-231 cell line in the following examples is described in the following literature: Qianqian Chen, Min Xiao, Fei Dai, Ye Zhang, Jiayun Li, Yanwu Huo, Zhen Huang, Yi Fang, Taotao Wei, Mitochondrial elongation factor 4 modulates energy metabolism and promotes sbreast cancer metastasis by orchestration of mitochondrial translation. Archives of Biochemistry and Biophysics [J]. Volume 737, 2023. http: / / dx.doi.org / 10.1016 / j.abb.2023.109556.

[0121] The pLVX-MCS-puro plasmid in the following examples (hereinafter referred to as pLVX-puro plasmid) was purchased from Ubest Biotech, with the catalog number VT1465.

[0122] The myc-BioID2-MCS plasmid in the following examples is described in the following literature: Kim DI, Jensen SC, Noble KA, Kc B, Roux KH, Motamedchaboki K, Roux KJ. An improved smaller biotinligase for BioID proximity labeling [J] Mol Biol Cell. 2016 Feb 24. pii: mbc.E15-12-0844.10.1091 / mbc.E15-12-0844 PubMed 26912792.

[0123] Example 1: Preparation of recombinant lentivirus for knocking out human ATAD3A and ATAD3B proteins

[0124] In this example, a lentiviral vector based on the CRISPR / Cas9 gene editing system was used to construct a recombinant lentivirus for knocking out the ATAD3A gene and the ATAD3B gene. The specific steps are as follows:

[0125] 1. Preparation of recombinant vector

[0126] (1) Design of sgRNA: According to the nucleotide sequences of ATAD3A gene (NM_001170535.3) and ATAD3B gene (NM_031921.6), the target of sgRNA was designed at the first start codon. Two targets (named as target 1 and target 2) and a control target were selected for the experiment. The nucleotide sequence of target 1 is: 5'-CGAGCACCGTCAGACCGTCT-3' (SEQ ID NO: 1, i.e., the target sequence of sgRNA1), and the nucleotide sequence of target 2 is: 5'-GCTCTTGAGCTGCTCCACGG-3 (SEQ ID NO: 2, i.e., the target sequence of sgRNA2). The target gene corresponding to sgRNA1 is ATAD3A gene, and the target gene corresponding to sgRNA2 is ATAD3B gene. The nucleotide sequence of the control target is: 5'-GTAGGCGCGCCGCTCTCTAC-3' (i.e., the target sequence of the control sgRNA).

[0127] (2) Based on target 1, sgATAD3A#B4-F: 5'-CACCGCGAGCACCGTCAGACCGTCT-3' and sgATAD3A#B4-R: 5'-AAACAGACGGTCTGACGGTGCTCGC-3' were designed and synthesized. Based on target 2, sgATAD3A#B32-F: 5'-CACCGGCTCTTGAGCTGCTCCACGG-3' and sgATAD3A#B32-R: 5'-AAACCCGTGGAGCAGCTCAAGAGCC-3' were designed and synthesized. Based on the control target, sgNTC-F: 5'-CACCGGTAGGCGCGCCGCTCTCTAC-3' and sgNTC-R: 5'-CGTAGAGAGCGGCGCGCCTACCAAA-3' were designed and synthesized.

[0128] (3) After annealing sgATAD3A#B4-F and sgATAD3A#B4-R, oligo dimer 1 was obtained. After annealing sgATAD3A#B32-F and sgATAD3A#B32--R, oligo dimer 2 was obtained. After annealing sgNTC-F and sgNTC-R, oligo dimer 3 was obtained.

[0129] (4) The lentiCRISPRv2 vector (Addgene, #52961) was digested with Esp3I (Thermo Scientific) to obtain the vector backbone.

[0130] (5) The vector backbone and oligo dimer 1 were ligated using T4 DNA ligase (NEB) to obtain sgATAD3A#B recombinant vector 1. The vector backbone and oligo dimer 2 were ligated using T4 DNA ligase (NEB) to obtain sgATAD3A#B recombinant vector 2. The vector backbone and oligo dimer 3 were ligated using T4 DNA ligase (NEB) to obtain a control recombinant vector.

[0131] The sgATAD3A#B recombinant vector 1 and the sgATAD3A#B recombinant vector 2 can be used to reduce the content of human ATAD3A protein and ATAD3B protein.

[0132] 2. Preparation of recombinant lentivirus

[0133] The sgATAD3A#B recombinant vector 1 was co-transfected with the lentiviral packaging vectors psPAX2 (Addgene, #12260) and pMD2G (Addgene, #12259) into HEK293T cells to obtain the sgATAD3A#B recombinant lentivirus 1. The specific steps are as follows:

[0134] (1) HEK293T cells were co-transfected with sgATAD3A#B recombinant vector 1, lentiviral packaging vector psPAX2 and pMD2G using Lipo3000 transfection kit (Thermo Scientific) (the transfection ratio was: 293T cells covering a 15 cm diameter culture dish: 21 μg sgATAD3A#B recombinant vector 1, 14 μg psPAX2 and 7 μg pMD2G), and HEK293T cell culture medium was added and cultured in a cell culture incubator at 37°C for 8 h.

[0135] (2) After completing step (1), the old culture medium was replaced with fresh HEK293T cell culture medium and cultured in a 37°C cell culture incubator for 72 hours. The culture fluid was collected at 24, 48, and 72 hours of culture and filtered with a 0.45 μm filter membrane to remove dead cells and cell debris.

[0136] (3) After completing step (2), the culture medium was placed in an ultracentrifuge and centrifuged at 4°C and 19400 rpm for 2 hours and 15 minutes. The precipitate was collected and resuspended with PBS to obtain the sgATAD3A#B recombinant lentivirus 1 concentrate.

[0137] According to the above steps, the sgATAD3A#B recombinant vector 1 was replaced with the sgATAD3A#B recombinant vector 2, and the other steps remained unchanged to obtain the sgATAD3A#B recombinant lentivirus 2 concentrate.

[0138] According to the above steps, the sgATAD3A#B recombinant vector 1 was replaced with the control recombinant vector, and the other steps remained unchanged to obtain the control lentivirus sgNTC concentrate.

[0139] Example 2. Preparation of human breast cancer cell lines lacking ATAD3A and ATAD3B proteins

[0140] 1. Human breast cancer MCF7 cell line was used as test cells, and control lentivirus sgNTC, sgATAD3A#B recombinant lentivirus 1 and sgATAD3A#B recombinant lentivirus 2 were infected respectively. The specific method was: inoculate 5×10 5 6 μl of lentivirus concentrate (sgATAD3A#B recombinant lentivirus 1 concentrate, sgATAD3A#B recombinant lentivirus 2 concentrate or control lentivirus sgNTC concentrate) and 10 μl of Polybrene were added to a culture dish (10 cm in diameter) of the personal breast cancer cell line MCF7.

[0141] 2. After culturing the cells infected with lentivirus obtained in step 1 for 24 hours, add MCF7 culture medium containing 1 μg / mL puromycin (Invivogen) to screen positive cells.

[0142] 3. Digest the above-screened positive cells with trypsin, count them with a cell counter, dilute the cell suspension, and spread the cell suspension into a 96-well plate at a density of 1 cell per well. Observe the cell growth in each well regularly, expand the cultured cells regularly, and use immunoassays to detect the knockout of the target protein (note that the internal reference protein is used as the control group). DKO cells of the human breast cancer MCF7 cell line lacking ATAD3 protein (ATAD3A protein and ATAD3B protein) were screened and named MCF-7DKO cells for subsequent experiments.

[0143] According to the above steps, the human breast cancer MCF7 cell line was replaced with the human breast cancer MDA-MB-231 cell line, and the other steps remained unchanged to obtain DKO cells of the human breast cancer MDA-MB-231 cell line lacking ATAD3 protein, which were named MDA-MB-231DKO cells.

[0144] Example 3: Construction of human breast cancer cell lines expressing ATAD3A-His and ATAD3B-His proteins

[0145] 1. According to the nucleotide sequence of ATAD3A gene (NM_001170535.3), primers ATAD3A-His-F: 5'-at ctcgagctcaagcttcgaattcGCCACCatgtcgtggctcttcggcat-3' and ATAD3A-His-R: 5'-ATGGTGAT GGTGATGATGGCCTCCggatggggagggctcgtccc-3' were designed. The cDNA of MCF7 cell line was used as template and ATAD3A-HisF and ATAD3A-His-R were used as primer pair for PCR amplification to obtain the PCR product of ATAD3A-His gene sequence; according to the nucleotide sequence of ATAD3B gene (NM_031921.6), primers ATAD3B-His-F: 5'-atctcgagctcaagct tcgaattcGCCACCatgtcgtggctcttcggcgt-3' and ATAD3B-His-R: 5'-GGTGATGGTGATGATGGCCTCC caacagggggtgccctgggg-3', using the cDNA of the MCF7 cell line as a template, PCR amplification was performed with ATAD3B-His-F and ATAD3B-His-R as primer pairs to obtain the PCR product of the ATAD3B-His gene sequence; the pLVX-puro plasmid was digested with EcoRI endonuclease (Neb), and the PCR product was ligated to the MCS region of the pLVX-puro plasmid after digestion using homologous recombinase (Neb) to obtain pLVX-ATAD3A-His plasmid and pLVX-ATAD3B-His plasmid.

[0146] 2. Transfect the HEK293T cell line with packaging plasmids (psPAX2 and pMD2) and backbone plasmids (pLVX-ATAD3A-His plasmid or pLVX-ATAD3B-His plasmid), and culture continuously for 36 hours after transfection. Collect the supernatant without digesting the cells with trypsin, mark it as liquid A, replace it with fresh culture medium, and collect the supernatant again after culturing for 72 hours, mark it as liquid B. Mix liquid A and liquid B, and filter it through a 0.45μM filter to obtain the virus infection stock solution.

[0147] 3. After mixing the virus infection stock solution with the cell culture medium in a ratio of 1:4, add it to the attached human breast cancer cell line MCF7 to be infected. After culturing for 24 hours, discard the virus infection solution and add fresh culture medium. After continuous culturing for 24 hours, add a certain concentration of puromycin to screen positive cells, namely MCF7 cells overexpressing ATAD3A-His protein and MCF7 cells overexpressing ATAD3B-His protein.

[0148] Example 4: Western Blot

[0149] 1. First, prepare the cell lysis buffer. Add 10 μL of protease inhibitor (Yeasen) to every 1 mL of RIPA cell lysis buffer (Solebo Bio) and store it on ice. Then, discard the culture medium of the adherent cells, add 1 mL of PBS to the cell surface, wash the cells and discard the PBS, add an appropriate amount of cell lysis buffer according to the cell density, place on ice for 3 minutes, scrape the cells from the cell culture dish, and place on ice for 20 minutes. Then collect the cell lysis buffer after the cells are lysed and put it into a centrifuge tube. Centrifuge at 12000 rpm, 4°C for 20 minutes, and collect the supernatant.

[0150] 2. Prepare the reaction solution in proportion according to Thermo's BCA protein quantification guide. Then add 10 μL of protein standard and protein to be tested to 90 μL of reaction solution. React at 37°C for 30 minutes and use a microplate reader to detect OD. 562 , draw a standard curve based on the protein standard and calculate the concentration of the protein to be tested.

[0151] 3. 20 μg of protein was separated by SDS-PAGE gel electrophoresis, then electrotransferred to a PVDF membrane (Millipore), blocked with 5% skim milk (Yeasen) at room temperature for 1 hour, incubated with the primary antibody overnight at 4°C, and then incubated with the secondary antibody labeled with horseradish peroxidase (HRP) at room temperature for 1 hour.

[0152] 4. The ChemiDoc XRS system (Bio-Rad) was used for data acquisition and data processing, and ImageJ was used for data analysis.

[0153] 5. The primary antibodies used for western blotting experiments were ATAD3 antibody (abclonal, A8230), Actin antibody (Proteintech, 66009-1-Ig), GAPDH antibody (Proteintech, 60004-1-Ig), STING antibody (Cell Signaling Technology, 13647), TBK1 antibody (Cell Signaling Technology, 3504), Phospho-STING antibody (Cell Signaling Technology, 50907), Phospho-TBK1 antibody (Cell Signaling Technology, 5483), SEC61B antibody (abclonal, A15788), STEEP antibody (Cell Signaling Technology, 35136), and His antibody (Proteintech, 66005-1-Ig). The HRP-labeled secondary antibodies were goat anti-mouse antibody (ZSGB-BIO, ZB2305) and goat anti-rabbit antibody (ZSGB-BIO, ZB2301).

[0154] Example 5: Biotin proximity labeling and combined mass spectrometry analysis (BioID-MS)

[0155] Biotin protein ligases (BPLs) are enzymes with extraordinary specificity. BirA is the BPL of Escherichia coli that biotinylates only a single cellular protein. In 2004, scientists reported a mutant Bir A that can attach biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobulin heavy and light chains, and RNAse A in vitro. Mutant BirA also self-biotinylates in vivo and in vitro. Wild-type BirA protein is much less active in these reactions. The biotinylation reaction is proximity-dependent, as a greater degree of biotinylation is observed when the mutant ligase is coupled to a receptor protein than when the receptor is free in solution. This approach allows for easy detection and recovery of interacting proteins using existing avidin / streptavidin technology.

[0156] 1. According to the nucleotide sequence of ATAD3A gene (NM_001170535.3), primers ATAD3-BioID-F: 5'-a gcctgagaagaagcGGTGGCGGAGGGAGCatgtcgtggctcttcggca-3' and ATAD3-BioID-R: 5'-tggcggc cgctcgagtcaggatggggaggg-3' were designed. The cDNA of MCF7 cell line was used as template and PCR amplification was performed with primers ATAD3-BioID-F and ATAD3-BioID-R to obtain the full-length PCR product of ATAD3A gene; the myc-BioID2-MCS plasmid was digested with NotI endonuclease (Neb), and the PCR product was connected to the MCS region of the myc-BioID2-MCS plasmid after single digestion using homologous recombinase to obtain myc-BioID-ATAD3A.

[0157] 2. Transfected cell lines

[0158] ① Prepare the transfection system according to the transfection reagent manual of Polyplus. Taking a 6 cm dish as an example, the transfection system is as follows:

[0159] Table 1. Transfection system

[0160] DNA 4ng Buffer 200μL Transfectionreagen 8μL

[0161] ②The transfection system was placed at room temperature for 5 minutes;

[0162] ③ Add the transfection system to the culture medium of HEK-293T cells and replace the culture medium after 4 hours.

[0163] 3. Extract cell mitochondria

[0164] ①Prepare solution:

[0165] Solution A: 225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA, 30 mM Tris-HCl pH 7.4;

[0166] Solution B: 225 mM mannitol, 75 mM sucrose, 30 mM Tris-HCL pH 7.4;

[0167] Percoll gradient: 3 mL 50% percoll + 8 mL 25% percoll (percoll was diluted with solution B);

[0168] ② Collect cells

[0169] The cells were washed with 1 mL of PBS, completely digested with trypsin, and centrifuged at 800 g for 3 min;

[0170] ③ Wash cells

[0171] Resuspend the cells with pre-cooled PBS. After rinsing, centrifuge at 800g, 4°C for 3 min to collect the cells.

[0172] ④ Crude extraction of mitochondria

[0173] Resuspend the cells with 2 mL of solution A (with protease inhibitors added), grind the cells on ice for about 30 times using a Dounce grinder, centrifuge at 1000 g, 4°C for 10 min, transfer the supernatant to a new centrifuge tube, centrifuge at 3500 g, 4°C for 10 min, carefully aspirate the supernatant, which is the separated mitochondria, and centrifuge at 12000 g, 4°C for 10 min. The supernatant obtained is the cytoplasmic protein;

[0174] ⑤ Purification of mitochondria

[0175] The crude mitochondria were washed three times with pre-cooled solution B and finally resuspended with 400 μL solution B. The crude mitochondria were spread on the percoll gradient and ultracentrifuged at 18,000 rpm, 4°C for 45 min. The middle layer was carefully pipetted into a new centrifuge tube, resuspended with 1 mL solution B, and centrifuged at 15,000 rpm, 4°C for 10 min. The supernatant was carefully washed and the purified mitochondria were lysed with 200 μL RIPA cell lysis buffer.

[0176] 4. Protein purification by streptavidin affinity

[0177] ① Balance the magnetic beads: take 20 μL of hydrophilic streptavidin affinity magnetic beads (Neb) in a centrifuge tube, place the centrifuge tube on a magnetic rack, let it stand for 1-2 seconds, then remove the supernatant; take 200 μL of RIPA cell lysis buffer (Solebio Biotechnology) to resuspend the magnetic beads, balance on a mixer at 4°C for 10 minutes, and repeat this step three times;

[0178] ② Protein incubation: remove all lysed mitochondria and resuspend the balanced magnetic beads, incubate on a mixer at 4°C for 4 hours;

[0179] ③ Wash the magnetic beads: Place the centrifuge tube containing the magnetic beads and mitochondria on a magnetic rack, let it stand for 1-2 seconds, then remove the supernatant, add 500 μL TBS to resuspend the magnetic beads, wash on a mixer at 4°C for 10 minutes, and repeat this step three times;

[0180] ④ Elution of protein: Place the centrifuge tube on the magnetic rack, let it stand for 1-2 seconds, then remove TBS, use 20μL 1× loading buffer to resuspend the magnetic beads, place it in a 95℃ metal bath to cook the sample for 10 minutes, after a brief centrifugation, place the centrifuge tube on the magnetic rack, let it stand for 1-2 seconds, and carefully aspirate the supernatant into a new centrifuge tube.

[0181] 5. Mass spectrometry

[0182] ①: In-gel digestion: After the stained bands were decolorized, reduced with DTT and alkylated with iodoacetamide, trypsin was added for overnight digestion, and then the in-gel peptides were extracted using 60% CAN.

[0183] ②: LC-MS / MS analysis and database search: The peptide mixture obtained by enzymatic hydrolysis was first subjected to liquid chromatography-tandem mass spectrometry analysis, and then the database search and protein identification were performed in the Uniprot_proteome_human_2018 database using the SEQUEST HT search engine of Thermo Proteome Discoverer (1.4.0.288). Search parameters: Trypsin enzymatic hydrolysis, 2 missed cleavage sites, precursor ion mass error less than 10ppm, and fragment ion mass error less than 20mDa. Set cysteine ​​alkylation as a fixed modification and methionine oxidation as a variable modification. Search result filtering parameters: Use Percolator to filter the spectrum, Delta Cn less than 0.1, FDR set to 1%, and peptide confidence as High for peptide filtering parameters.

[0184] Example 6. Immunoprecipitation and co-immunoprecipitation

[0185] 1. Immunoprecipitation

[0186] (1) Protein extraction: Prepare cell lysis buffer, add 10 μL of protease inhibitor (Yeasen) to every 1 mL of non-denaturing cell lysis buffer (Solebo Biotechnology), and store on ice. Then, discard the culture medium of the adherent cells, add 1 mL of PBS to the cell surface, wash the cells and discard the PBS, add an appropriate amount of non-denaturing cell lysis buffer according to the cell density, place on ice for 3 minutes, scrape the cells from the cell culture dish, and place on ice for 20 minutes. Then collect the cell lysate after the cells are lysed and put it into a centrifuge tube. Centrifuge at 12000 rpm, 4°C for 20 minutes, and collect the supernatant;

[0187] (2) Protein quantification: Prepare the reaction solution in proportion according to the BCA protein quantification guide of Thermo Fisher Scientific. Then, add 10 μL of protein standard and protein to be tested to 90 μL of the reaction solution. After reacting at 37°C for 30 min, use a microplate reader to measure the OD. 562 , draw a standard curve based on the protein standard and calculate the concentration of the protein to be tested;

[0188] (3) Preparation of input sample: Take 100 μL of the determined protein sample into a new centrifuge tube, add 25 μL of 5× loading buffer, mix well, place in a 95°C metal bath to cook for 5 min, centrifuge at 12,000 rpm for 3 min, and freeze in a -80°C refrigerator for subsequent Western blot detection.

[0189] (4) Antibody-antigen binding: Add antibodies to cell lysates according to the ratio recommended in the antibody use guide, and incubate the antibody-antigen mixture at 4°C on a mixer overnight;

[0190] (5) Equilibration of magnetic beads: put 45 μL protein A / G magnetic beads (MCE) into a centrifuge tube, place the centrifuge tube on a magnetic rack, let it stand for 1-2 seconds, then remove the supernatant, take 600 μL TBS to resuspend the magnetic beads, equilibrate on a mixer at 4°C for 10 minutes, place the centrifuge tube on a magnetic rack, let it stand for 1-2 seconds, then remove the supernatant, take 600 μL non-denaturing cell lysis buffer to resuspend the magnetic beads, equilibrate on a mixer at 4°C for 10 minutes, place the centrifuge tube on a magnetic rack, let it stand for 1-2 seconds, then remove the supernatant;

[0191] (6) Binding of magnetic beads to antibody-antigen: The antibody-antigen mixture incubated overnight was mixed with the magnetic beads. After the magnetic beads were fully resuspended, they were incubated on a mixer at 4°C for 2 h.

[0192] (7) Washing the magnetic beads: Place the centrifuge tube of the incubated magnetic bead-antigen-antibody mixture on a magnetic rack, let it stand for 1-2 seconds, then remove the supernatant, add 600 μL of non-denaturing cell lysis buffer to resuspend the magnetic beads, wash on a mixer at 4°C for 10 minutes, and repeat this step 6 times; add 600 μL of TBS to resuspend the magnetic beads, wash on a mixer at 4°C for 10 minutes, and repeat this step 6 times;

[0193] (8) Protein elution: Place the centrifuge tube on a magnetic rack, let it stand for 1-2 seconds, then remove the TBS, use 20 μL of 1× loading buffer to resuspend the magnetic beads, place it in a 95°C metal bath for 10 minutes, and after a brief centrifugation, place the centrifuge tube on a magnetic rack, let it stand for 1-2 seconds, and carefully pipette the supernatant into a new centrifuge tube. This portion of protein will be used for subsequent protein immunoblotting detection.

[0194] 2. Co-immunoprecipitation

[0195] (1) Protein extraction and quantification: refer to the experimental steps in immunoprecipitation (1)(2);

[0196] (2) Prepare input sample: refer to the experimental steps in immunoprecipitation (3);

[0197] (3) Equilibration of magnetic beads: 45 μL of anti-His magnetic beads (Biyuntian) were placed in a centrifuge tube, and the centrifuge tube was placed on a magnetic rack. After standing for 1-2 seconds, the supernatant was removed. 600 μL of TBS was taken to resuspend the magnetic beads. After equilibration on a mixer at 4°C for 10 minutes, the centrifuge tube was placed on a magnetic rack. After standing for 1-2 seconds, the supernatant was removed. 600 μL of non-denaturing cell lysis buffer was taken to resuspend the magnetic beads. After equilibration on a mixer at 4°C for 10 minutes, the centrifuge tube was placed on a magnetic rack. After standing for 1-2 seconds, the supernatant was removed.

[0198] (4) Protein incubation: Take 500 μL of protein and resuspend the balanced magnetic beads, incubate on a mixer at 4°C for 3 h;

[0199] (5) Washing the magnetic beads: Place the centrifuge tube containing the magnetic beads and proteins on a magnetic rack, let it stand for 1-2 seconds, then remove the supernatant, add 600 μL of non-denaturing cell lysis buffer to resuspend the magnetic beads, wash on a mixer at 4°C for 10 minutes, and repeat this step 3 times; add 600 μL of TBS to resuspend the magnetic beads, wash on a mixer at 4°C for 10 minutes, and repeat this step 3 times;

[0200] (6) Protein elution: Place the centrifuge tube on a magnetic rack, let it stand for 1-2 seconds, then remove the TBS, use 20 μL 1× loading buffer to resuspend the magnetic beads, place it in a 95°C metal bath for 10 minutes, and after a brief centrifugation, place the centrifuge tube on a magnetic rack, let it stand for 1-2 seconds, and carefully pipette the supernatant into a new centrifuge tube. This portion of protein will be used for subsequent Western blot detection.

[0201] Example 7: Immunofluorescence

[0202] (1) Cell seeding: Carefully place the cell slide in a 6-well plate, seed the normally growing cells at a density of 100,000 cells / well in the 6-well plate, spread the cells using the "cross method", let them stand for 1 min, and then culture them in a cell culture incubator at 37°C and 5% CO2 for 24 h;

[0203] (2) Fixing cells: aspirate the culture medium, add 2 mL of 4% paraformaldehyde preheated at 37°C to each well, incubate at 37°C for 15 min. After fixation, add 2 mL of PBS to each well and let stand for 10 min. Repeat three times.

[0204] (3) Blocking: Add 2 mL of blocking solution (blocking solution: PBS + 0.3% Trion-X100 + 5% BSA) to each well and let stand at room temperature for 1 h;

[0205] (4) Incubation with primary antibody: dilute the antibody according to the antibody manual, add 100 μL of primary antibody to the cell slide, and incubate overnight at 4°C.

[0206] (5) Wash the primary antibody: remove the primary antibody, add 2 mL PBS to each well, let stand for 10 min, and repeat three times.

[0207] (6) Incubation with secondary antibody: dilute the antibody according to the antibody manual, add 100 μL of secondary antibody to the cell slide, and incubate at room temperature for 2 h.

[0208] (6) Washing the secondary antibody: remove the secondary antibody, add 2 mL PBS to each well, let stand for 10 min, and repeat three times.

[0209] (8) DAPI staining and sealing: Pipette 5 μL of anti-fluorescence quenching sealing medium containing DAPI in the center of the slide, place the cell slide upside down in the center of the slide, be careful to avoid bubbles, and use transparent nail polish to fix the four corners of the cell slide.

[0210] (9) Imaging: The treated cell slides were photographed and observed using a laser confocal microscope FV 3000. The excitation light was 405 nm, 488 nm, and 561 nm.

[0211] (10) The primary antibodies used in immunofluorescence experiments were GM130 (BD) and STING (RD).

[0212] Example 8: Nude mouse subcutaneous tumor formation experiment

[0213] The 6- to 8-week-old female Balb / c nude mice in the experiment were obtained from Beijing Weitonglihua Laboratory Animal Co., Ltd. The experimental procedures strictly complied with the experimental animal ethics regulations of the Institute of Biophysics, Chinese Academy of Sciences and the regulations of the International Animal Care and Use Committee (IACUC-IBP).

[0214] (1) Resuspend 1×10 7 cells (MCF7-WT, MCF-7DKO, MB231-WT (i.e., MDA-MB-231 wild-type cells), MB231-DKO (i.e., MDA-MB-231DKO cells)), 100 μL of the cell suspension was injected subcutaneously into nude mice.

[0215] (2) Three weeks after injection, the mice were killed, and the xenograft tumors were removed and washed once with PBS. The size and weight of the tumors were observed and recorded, and photographs were taken for preservation.

[0216] Example 9: Interaction between ATAD3 protein and STING protein

[0217] 1. Pulling and enrichment of ATAD3 interacting proteins in the cytoplasmic matrix

[0218] According to the topological structure of ATAD3A and ATAD3B, the N-termini of ATAD3A and ATAD3B are both in the cytoplasm, and the sequences of ATAD3A and ATAD3B extending to the cytoplasm are basically the same. The N-termini of the ATAD3 protein family were labeled using the proximity labeling BioID technology, but because the N-termini of ATAD3A and ATAD3B are extremely similar ( Figure 1 A), we selected ATAD3A protein as the BioID labeling target, added the BioID tag to the N-terminus of the full-length ATAD3A, and constructed the BioID-ATAD3A proximity labeling vector ( Figure 1 B), and the expression level of BioID-ATAD3A was detected by immunoblotting to ensure the effectiveness of labeling ( Figure 1 C). We then enriched the interacting proteins at the N-terminus of ATAD3A using streptavidin affinity chromatography and screened for the STING protein ( Figure 1 D).

[0219] 2. Identification of ATAD3-interacting proteins in the cytoplasm

[0220] In order to ensure the accuracy of the pull-up enrichment results, we used the MCF-7 cell line overexpressing ATAD3A-His and ATAD3B-His proteins prepared in Example 3 to perform co-immunoprecipitation experiments.

[0221] The experimental results showed that when ATAD3A and ATAD3B were overexpressed, ATAD3 protein family members interacted with STING protein ( Figure 2 ). To further verify our experimental results, we used immunoprecipitation to verify the interaction between ATAD3 and STING in the MCF-7 wild-type cell line. We also selected the transmembrane protein Sec61B on the endoplasmic reticulum as a quality control protein to verify whether ATAD3 interacts with STING through interaction with the endoplasmic reticulum. The experimental results showed that at the endogenous level of the cell, ATAD3 interacted with STING, and ATAD3 did not interact clearly with the endoplasmic reticulum transmembrane protein Sec61B, which indicated that the binding of ATAD3 to STING was not caused by the interaction between ATAD3 and the endoplasmic reticulum.

[0222] 3. The amount of STING protein bound by ATAD3 is reduced under the stimulation of STING activator

[0223] In order to further reveal the relationship between ATAD3 protein and STING protein, we tried to detect whether there were some changes in the interaction between ATAD3 protein and STING protein when STING protein was activated. In this part of the experiment, we selected cGAMP as the activator of STING protein and used immunoprecipitation to detect whether the interaction between endogenous ATAD3 protein family members and STING protein was changed.

[0224] We treated the MCF-7 wild-type cell line with different cGAMP concentrations, enriched the proteins interacting with ATAD3 protein family members by immunoprecipitation, and used immunoblotting to detect the changes of STING protein interacting with ATAD3 protein family members ( Figure 3 ). The experimental results showed that under the treatment of STING protein agonist cGAMP, the STING protein interacting with ATAD3 protein family members was significantly reduced compared with the untreated group (cGAMP concentration of 0μg / mL). When the cGAMP treatment concentration was 1μg / mL and 2μg / mL, the STING protein interacting with ATAD3 protein family members decreased with the increase of treatment concentration, which was 80% and 70% of the untreated group, respectively. However, when the cGAMP treatment concentration reached 5μg / mL, the amount of STING protein bound by ATAD3 protein family members actually increased compared with the experimental group with lower cGAMP treatment concentration. When the cGAMP treatment concentration was 2μg / mL, the amount of STING protein bound by ATAD3 protein family members was the lowest compared with the other two treatment groups. Therefore, we infer that ATAD3 protein family members may participate in the regulation of the cGAS-STING signaling pathway through interaction with STING protein.

[0225] Example 10. Loss of ATAD3 significantly increases activation of the cGAS-STING signaling pathway in cells

[0226] In Example 9, we have confirmed the relationship between ATAD3 protein and STING protein in the cytoplasmic matrix, and activation of STING protein will lead to a significant reduction in STING protein binding to ATAD3 protein family members in cells. Therefore, we speculate that ATAD3 protein may participate in the cGAS-STING signaling pathway and the activation of downstream response factors as a negative regulator of STING protein.

[0227] To verify our hypothesis, we tested the sensitivity of ATAD3 knockout DKO cells to cGAS-STING signaling pathway activators. First, we used exogenous dsDNA poly(dA:dT) to treat MDA-MB-231 control group (Ctrl) and ATAD3 knockout DKO group cells, and used immunoblotting to detect the activation of STING protein in the cGAS-STING signaling pathway and the activation of downstream factor TBK1 protein ( Figure 4 ).

[0228] The experimental results showed that when the DNA mimic poly(dA:dT) was not added, the phosphorylation level of STING protein in MDA-MB-231DKO cells was higher than that in MDA-MB-231 control cells, but there was no significant difference in the phosphorylation level of TBK1 protein between MDA-MB-231 control and MDA-MB-231DKO cells. Under exogenous poly(dA:dT) treatment, the total protein level of STING and the protein level of TBK in MDA-MB-231 control and MDA-MB-231DKO cells did not change significantly, but with the increase of poly(dA:dT) treatment time, the phosphorylation levels of STING and TBK proteins in MDA-MB-231 control and MDA-MB-231DKO cells increased significantly. Most importantly, the phosphorylation level of STING protein in MDA-MB-231DKO group cells under poly(dA:dT) treatment was significantly higher than that in MDA-MB-231 control group cells, and the phosphorylation level of TBK1 protein, a response factor downstream of STING protein, in MDA-MB-231DKO group cells was also significantly higher than that in MDA-MB-231 control group cells after 1 and 2 hours of poly(dA:dT) treatment. The above experimental results show that the loss of ATAD3 protein makes cells more sensitive to the activation of the cGAS-STING signaling pathway. Therefore, we believe that members of the ATAD3 protein family are of great significance for the activation of the cGAS-STING signaling pathway in cells.

[0229] Subsequently, to further verify our hypothesis, we used the STING protein activator cGAMP to treat the MCF-7 control group (Ctrl) and the ATAD3DKO knockout group cells, and also used immunoblotting to detect the activation of STING protein in the cGAS-STING signaling pathway and the activation of the downstream factor TBK1 protein ( Figure 5 ).

[0230] The experimental results showed that the expression level of STING protein in cells did not change significantly after cGAMP treatment, but the phosphorylation level of STING protein was in an increasing state in both the MCF-7 control group and the DKO group. When the treatment concentration of cGAMP was 2μg / mL, the phosphorylation level of STING protein in the control group and the DKO group was significantly increased compared with the untreated group cells, but when the treatment concentration of cGAMP was 5μg / mL, the increase in the phosphorylation level of STING protein in the cells was inhibited. Comparing the phosphorylation level of STING protein in the MCF-7 control group and the DKO group cells after cGAMP treatment, we found that when the treatment concentration of cGAMP was 1μg / mL and 2μg / mL, the phosphorylation level of STING protein in the DKO group cells was significantly higher than that in the control group cells. In addition to the STING protein and the phosphorylation level of STING protein, we also detected the expression level of TBK1 protein, a response factor downstream of the STING protein, and the phosphorylation level of TBK1 protein. We found that after cGAMP treatment, the expression level of TBK1 protein in the control group and DKO group cells did not change significantly, but the phosphorylation level of TBK1 protein was increased. Moreover, when the treatment concentration of cGAMP was 1μg / mL and 2μg / mL, the phosphorylation level of TBK1 in the DKO group cells was significantly higher than that in the control group cells. Similar to the trend of changes in the phosphorylation level of STING protein in cells, when the treatment concentration of cGAMP was 5μg / mL, the increase in the phosphorylation level of TBK1 protein in cells was inhibited.

[0231] Example 11. Loss of ATAD3 significantly increases the transport of STING protein in cells

[0232] In the resting state, the STING protein is a protein localized in the endoplasmic reticulum of the cell and has four transmembrane domains. When the natural ligand of the STING protein is present (such as cGAMP), the conformation of the STING protein changes, causing STING to dimerize. The activation of the STING protein is a step consisting of multiple biological events, including the dimerization, oligomerization, departure from the endoplasmic reticulum, translocation to the Golgi apparatus, and finally phosphorylation on the Golgi apparatus. We have inferred from the above experimental results that the ATAD3 protein may negatively regulate the activation of the cGAS-STING signaling pathway and the activation of the downstream TBK1 protein by interacting with the resting STING protein on the endoplasmic reticulum. In addition, there are literature reports that the translocation of the STING protein from the endoplasmic reticulum to the Golgi apparatus is an important rate-limiting step in the activation of the STING protein. So, does the loss of ATAD3 protein affect the transport of the STING protein from the endoplasmic reticulum to the Golgi apparatus?

[0233] In order to detect whether the loss of ATAD3 protein affects the transport of STING protein from the endoplasmic reticulum to the Golgi apparatus, we used Brefeldin A (BFA) to block the transport of secretory proteins and membrane proteins from the endoplasmic reticulum to the Golgi apparatus, and used immunoblotting to detect whether STING protein in the MCF-7 control group (Ctrl) and the DKO group with double knockout of ATAD3A and ATAD3B proteins could still be stimulated by its natural ligand cGAMP and phosphorylated ( Figure 6 ).

[0234] The experimental results showed that after treatment with different concentrations of cGAMP, the total protein level of STING protein did not change. The phosphorylation level of STING protein increased in a cGAMP concentration-dependent manner in both the control group cells and the DKO group cells. And when the cGAMP concentration was 1μg / mL and 2μg / mL, the phosphorylation level of STING protein in the DKO group cells was significantly higher than that in the control group cells. However, when different concentrations of cGAMP and Brefeldin A were present at the same time, the phosphorylation of STING protein in the cells could still be detected in both the control group cells and the DKO group cells, and the phosphorylation level of STING protein in the DKO cells was higher than that in the control group cells, but the phosphorylation level of STING protein did not increase due to the increase in cGAMP treatment concentration. This is because the presence of Brefeldin A blocks the transport of STING protein from the endoplasmic reticulum to the Golgi apparatus, thereby inhibiting the increase in the phosphorylation level of STING protein in the control group cells and DKO group cells under the stimulation of different concentrations of cGAMP. After blocking the translocation of STING, the phosphorylation level of STING protein in DKO cells was higher than that in the control group cells. This may be because before the addition of the blocker Brefeldin A, more STING protein was translocated to the Golgi apparatus and phosphorylated in the DKO group cells than in the control group cells.

[0235] To further examine the effect of ATAD3 deficiency on the translocation of STING protein from the endoplasmic reticulum to the Golgi apparatus, we used immunofluorescence to label the STING protein and the Golgi membrane protein GM130 in the cells, treated the MCF-7 control group and the DKO group with cGAMP, and detected the co-localization of STING protein and GM130 protein to verify whether the deficiency of ATAD3 protein family members leads to more STING protein being translocated to the Golgi apparatus ( Figure 7 ).

[0236] The results of immunofluorescence showed that in the absence of STING protein agonists, although the amount of STING protein co-localized with the Golgi apparatus in the DKO group cells lacking ATAD3 protein was higher than that in the control group cells, there was no significant difference between the two. After cGAMP treatment, the co-localization of STING protein with the Golgi apparatus in both the control group cells and the DKO group cells was significantly increased compared with the untreated group cells. Most importantly, in the presence of cGAMP, the co-localization of STING protein with the Golgi apparatus in the DKO group cells was significantly higher than that in the control group cells, that is, more STING protein may be transported from the endoplasmic reticulum to the Golgi apparatus in cells lacking ATAD3. This suggests that the loss of ATAD3 protein family members affects the transport of STING protein from the endoplasmic reticulum to the Golgi apparatus, thereby affecting the activation of STING protein on the Golgi apparatus.

[0237] Our experimental results above have shown that ATAD3A and ATAD3B play an important role in the transport of STING protein from the endoplasmic reticulum to the Golgi apparatus. So, does the interaction between ATAD3 protein family members and STING protein affect the process of STING protein leaving the endoplasmic reticulum? We have discovered a newly reported STEEP protein that is closely related to the departure of STING protein from the endoplasmic reticulum. In this part of the experiment, we used the changes in the interaction between STEEP protein and STING protein to explore whether the loss of ATAD3 protein affects the biological process of STING protein leaving the endoplasmic reticulum.

[0238] We investigated the effect of ATAD3 deficiency on the binding of STING and STEEP proteins when STING is activated. After treating MCF-7 control and DKO cells with cGAMP, we used immunoprecipitation to detect the interaction between STING and STEEP proteins in cells ( Figure 8 ). The experimental results showed that when cGAMP was not treated, the level of STEEP protein pulled by STING protein in MCF-7ATAD3DKO group cells was slightly higher than that in control group cells; after cGAMP activated STING protein, the level of STEEP protein bound by STING protein in MCF-7 control group and DKO group cells was higher than that in the untreated group, and the level of STEEP protein bound by STING protein in MCF-7DKO cells was higher than that in control group cells ( Figure 8A). To further confirm the reliability of our experimental results, we also performed immunoprecipitation experiments of STEEP protein pulling back STING protein in MDA-MB-231 control group and DKO group cells under cGAMP treatment. The experimental results were the same as those in the MCF-7 cell line. When cGAMP was not treated, the amount of STING protein bound to STEEP protein in MDA-MB-231DKO group cells increased compared with that in control group cells; while in the presence of cGAMP, the amount of STING protein bound to STEEP protein in both MDA-MB-231 control group and DKO group cells increased, and the level of STING protein interacting with STEEP protein in MDA-MB-231DKO group cells was higher than that in control group cells ( Figure 8 B). Therefore, we concluded that the loss of ATAD3 protein allows STING protein to recruit more STEEP protein, which makes it easier for STING protein to "escape" from the endoplasmic reticulum and be transported to the Golgi apparatus.

[0239] The above series of experimental results show that ATAD3 protein interacts with the resting STING protein on the endoplasmic reticulum through its N-terminus extending into the cytoplasmic matrix, thereby affecting the binding of STING protein with STING-endoplasmic reticulum exit protein - STEEP protein, further affecting the translocation of STING protein from the endoplasmic reticulum to the Golgi apparatus, and ultimately leading to the inhibition of STING protein activation.

[0240] Example 12: Use of ATAD3 inhibitors in the prevention and / or treatment of breast cancer

[0241] The ATAD3 inhibitor can be a substance that inhibits the expression of the ATAD3A gene and the ATAD3B gene, or a substance that silences or knocks out the ATAD3A gene and the ATAD3B gene, or a substance that inhibits or reduces the content and / or activity of the ATAD3A protein and the ATAD3B protein. This example uses a cell line in which the ATAD3A gene and the ATAD3B gene are knocked out based on the CRISPR / Cas9 gene editing system to verify the use of an ATAD3 inhibitor (e.g., an sgRNA for knocking out the ATAD3A gene and the ATAD3B gene, or a CRISPR / Cas9 system containing the sgRNA) in the prevention and / or treatment of breast cancer.

[0242] 1. Loss of ATAD3 inhibits the proliferation of breast cancer cells in vitro

[0243] We complemented ATAD3A and ATAD3B in DKO cells of MCF-7 cell line and MDA-MB-231 cell line (MCF-7DKO cells and MDA-MB-231DKO cells prepared in Example 2). We infected MCF7 and 231DKO cell lines with lentivirus carrying synonymous mutant genes of ATAD3A and ATAD3B, and first constructed lentiviral expression vectors of synonymous mutant genes of ATAD3A and ATAD3B. According to the nucleotide sequence of ATAD3A gene (NM_001170535.3), primers for synonymous mutation of ATAD3A gene were designed and synthesized: 3Afrg1-F: 5′-gcgctaccggactcagatctcgagGCCACCatgtcgtggctcttcgg catta-3′; 3Afrg1-R: 5′-ActTttCagTtgTtcGacAgcCgcctcatactctttgagcttg-3′ and 3Afrg2-F: 5′-cGgcTgtCgaAcaActGaaAagTgagcagatccgggcgcaggc-3′; 3Afrg2-R: 5′-cTaaCacCg tTtgGcgAtgTtcTgccgccttcaggcggatctg-3', using the cDNA of the MCF7 cell line as a template, 3Afrg1-F and 3Afrg1-R as primers, PCR amplification obtained PCR fragment 3Afrg1, and using the cDNA of the MCF7 cell line as a template, 3Afrg2-F and 3Afrg2-R as primers, PCR amplification obtained PCR fragment 3Afrg1; then, the pLVX-puro plasmid was digested with EcoRI endonuclease (Neb), and the PCR fragment was connected to the pLVX-puro plasmid using homologous recombinase (Neb) to obtain the ATAD3A synonymous mutant plasmid pLVX-ASM.According to the nucleotide sequence of ATAD3B gene (NM_031921.6), primers for synonymous mutation gene of ATAD3B were designed and synthesized: 3Bfrg1-F: 5'-gcgctaccggactcagatctcgagGCCACCatgtcgtggctcttcggc gtta-3'; 3Bfrg1-R: 5'-ActTttCagTtgTtcGacAgcCgcctcatactctttgagcttg-3' and 3Bfrg2-F: 5'-cGgcTgtCgaAcaActGaaAagTgagcagatccgggcgcaggc-3'; 3Bfrg2-R: 5'-cTaaCacCgt TtgGcgAtgTtcAgacgccttcaggcggatctg-3', using the cDNA of the MCF7 cell line as a template, 3Bfrg1-F and 3Bfrg1-R as primers, PCR amplification obtained PCR fragment 3Bfrg1, and using the cDNA of the MCF7 cell line as a template, 3Bfrg2-F and 3Bfrg2-R as primers, PCR amplification obtained PCR fragment 3Bfrg1; then, the pLVX-puro plasmid was digested with EcoRI endonuclease (Neb), and the PCR fragment was connected to the pLVX-puro plasmid using homologous recombinase (Neb) to obtain the ATAD3B synonymous mutation plasmid pLVX-BSM. HEK293T cell line was transfected with packaging plasmid (psPAX2 and pMD2) and backbone plasmid (pLVX-ASM plasmid or pLVX-B SM plasmid), and cultured continuously for 36 hours after transfection. The supernatant was collected without trypsin digestion of cells, and the supernatant of packaging pLVX-ASM was marked as liquid A, and the supernatant of packaging pLVX-BSM was marked as liquid B. Liquid A and liquid B were mixed in a ratio of 1:1, and filtered with a 0.45μM filter to obtain the virus infection stock solution. The virus infection stock solution was mixed with cell culture medium in a ratio of 1:4, and added to the human breast cancer cell line MCF7 or MDA-MB-231 to be infected that had already adhered to the wall. After culturing for 24 hours, the virus infection solution was discarded, and fresh culture medium was added. After continuous culture for 24 hours, a certain concentration of puromycin was added to select positive cells, which were the complement cell lines Rescue of ATAD3A and ATAD3B.

[0244] Immunoblotting was used to detect the complementation effect ( Fig. 9 A, B). We then tested the in vitro proliferation ability of the cell lines and found that, compared with the control group, the proliferation ability of the DKO cell line lacking ATAD3 was significantly inhibited, while after replenishing ATAD3A and ATAD3B, the inhibition of cell proliferation was alleviated.

[0245] 2. Breast cancer cells lacking ATAD3 lose their ability to form tumors in vivo

[0246] In addition to in vitro experiments, in order to further verify that ATAD3A and ATAD3B are key factors in the occurrence and development of tumors, we also used four cell lines, MCF7-WT, MCF-7DKO, MB231-WT (i.e., MDA-MB-231 wild-type cells), and MB231-DKO (i.e., MDA-MB-231DKO cells), to conduct a subcutaneous injection tumor-forming experiment in nude mice (Example 8). Three weeks after the injection, we detected xenograft tumors in mice. The results showed that MCF-7 cells and MDA-MB-231 cells with double knockout of ATAD3A and ATAD3B completely lost the ability to form tumors in mice ( Fig.10 ).

[0247] It can be seen that ATAD3 inhibitors can significantly inhibit the proliferation of breast cancer cells and the occurrence of tumors in vivo, and can be used for the prevention and / or treatment of breast cancer.

[0248] The above results show that ATAD3 negatively regulates the activation of the cGAS-STING signaling pathway by interacting with the transmembrane protein STING on the endoplasmic reticulum. The loss of ATAD3 increases the sensitivity of breast cancer cells to exogenous DNA and STING agonists, allowing STING protein to bind to more STEEP protein and leave the endoplasmic reticulum more easily, and then translocate to the Golgi apparatus, ultimately leading to the activation of the cGAS-STING signaling pathway and the activation of the downstream factor TBK1, leading to an increase in downstream inflammatory responses, thereby inhibiting the proliferation of breast cancer cells in vitro and completely losing the ability to form tumors in vivo.

[0249] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of cGAS-STING signaling pathway activator in any of the following: A1) Use in the preparation of products for preventing and / or treating breast cancer; A2) Use in the preparation of a product for inhibiting the proliferation of breast cancer cells; A3) Use in the preparation of a product for inhibiting the tumorigenicity of breast cancer cells; A4) Use in the preparation of a product for inhibiting the occurrence or growth of breast cancer tumors.

2. Use of ATAD3 inhibitors in any of the following: B1) Use in the preparation of products for preventing and / or treating breast cancer; B2) Use in the preparation of a product for inhibiting breast cancer cell proliferation; B3) Use in the preparation of a product for inhibiting the tumorigenicity of breast cancer cells; B4) Use in the preparation of a product for inhibiting the occurrence or growth of breast cancer tumors; The ATAD3 inhibitor includes any of the following: C1) a substance that inhibits the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the ATAD3A gene and the ATAD3B gene; C2) Substances that inhibit or reduce the content, activity and / or function of ATAD3A protein and ATAD3B protein.

3. The use according to claim 2, characterized in that: The substances include substances that delete or inactivate the ATAD3A gene and the ATAD3B gene through site-directed mutagenesis technology, gene knockdown technology, gene editing technology and / or gene knockout technology, or substances that specifically bind to the ATAD3A protein and / or the ATAD3B protein to reduce the content of the ATAD3A protein and the ATAD3B protein or inactivate their functions.

4. The use according to claim 2 or 3, characterized in that: The ATAD3 inhibitor includes any of the following: D1) Nucleic acid molecules for silencing or knocking out ATAD3A gene and ATAD3B gene; D2) Antibodies that bind to ATAD3A protein and / or ATAD3B protein.

5. The use according to any one of claims 2 to 4, characterized in that: The ATAD3 inhibitor includes an sgRNA composition targeting the ATAD3A gene and the ATAD3B gene or a CRISPR / Cas9 system containing the sgRNA composition.

6. The use according to any one of claims 2 to 5, characterized in that: The ATAD3 inhibitor includes any of the following: E1) sgRNA1 and sgRNA2, wherein the target sequence of sgRNA1 is shown in SEQ ID NO: 1; the target sequence of sgRNA2 is shown in SEQ ID NO: 2; E2) DNA molecule 1 encoding the sgRNA1 described in E1) and DNA molecule 2 encoding the sgRNA2 described in E1); E3) a recombinant vector containing or respectively containing the DNA molecule 1 and the DNA molecule 2 described in E2); E4) A recombinant microorganism containing or separately containing the DNA molecule 1 and the DNA molecule 2 described in E2).

7. The use according to claim 6, characterized in that: The recombinant microorganism is a recombinant virus.

8. Biomaterial, characterized in that The biological material is any of the following: F1) sgRNA1 and sgRNA2 described in claim 6; F2) DNA molecule 1 and DNA molecule 2 as described in claim 6; F3) a recombinant vector containing or respectively containing the DNA molecule 1 and the DNA molecule 2 described in F2); F4) a recombinant microorganism containing or respectively containing the DNA molecule 1 and the DNA molecule 2 described in F2); F5) A recombinant host cell containing or separately containing the DNA molecule 1 and the DNA molecule 2 described in F2).

9. A pharmaceutical composition for preventing and / or treating breast cancer, characterized in that: The pharmaceutical composition comprises the ATAD3 inhibitor according to any one of claims 2-6.

10. Use of ATAD3A protein and ATAD3B protein as targets in the preparation of products having any of the following functions: G1) binds to STING protein; G2) regulates the cGAS-STING signaling pathway; G3) regulates the transport of STING protein from the endoplasmic reticulum to the Golgi apparatus.

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