Use of atad3 inhibitors in the prevention and / or treatment of breast cancer

Abstract

The application discloses application of ATAD3 inhibitors in prevention and / or treatment of breast cancer. Specifically, application of nucleic acid molecules for silencing or knocking out ATAD3A and ATAD3B genes, or ATAD3A and ATAD3B antibodies in prevention and / or treatment of breast cancer is disclosed. The application knocks out ATAD3A and ATAD3B genes in breast cancer cells by using a CRISPR / Cas9 system, and results show that the ATAD3 inhibitor can significantly increase cGAS-STING signal pathway activation and STING protein transport, and can significantly inhibit proliferation of breast cancer cells and occurrence of tumors in vivo. Breast cancer cells with knocked out ATAD3A and ATAD3B genes completely lose the ability to form tumors in mice in vivo. The ATAD3 inhibitor developed by the application has wide clinical application value and prospect in prevention and treatment of breast cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of ATAD3 inhibitors in prevention and / or treatment of breast cancer. BACKGROUND

[0002] The cGAS-STING signaling pathway regulates various immune-related diseases, including exogenous pathogen infection, autoimmune diseases, anti-tumor immune response, and aging-related inflammatory response. The basis for the cGAS-STING signaling pathway to widely participate in biological processes is its ability to perceive the invasion of microbial exogenous DNA. When exogenous DNA invades, the cGAS protein is activated to catalyze the synthesis of the second messenger cGAMP. Subsequently, the resting STING protein on the endoplasmic reticulum captures cGAMP and undergoes oligomerization, and then translocates from the endoplasmic reticulum to the Golgi apparatus. The STING protein recruits the TBK1 protein in the cell, and the TBK1 is then autophosphorylated and phosphorylates the STING. The activated TBK1 recruits and activates IRF3, which in turn triggers the production of downstream type I interferons (IFNs). IFNs have an important influence on the anti-tumor effect of cells. On the one hand, IFNs induce cytotoxic T lymphocytes to migrate to the tumor, thereby recognizing and killing tumor cells; on the other hand, IFNs promote the presentation of tumor-specific antigens to CD8 + T cells, normalizing the tumor microenvironment and achieving an inhibitory effect on tumor cells.

[0003] Breast cancer is not only the most common malignant tumor in women worldwide, but also ranks second in female mortality. The treatment methods for breast cancer are diverse, mainly including surgical treatment, chemotherapy, radiotherapy, endocrine therapy, and targeted therapy dominated by trastuzumab in recent decades. Although the comprehensive treatment of breast cancer can reduce its recurrence and metastasis rate and mortality, and can 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 this complex disease, the medical community still faces great challenges. Therefore, continuous research and development of new breast cancer treatment drugs have far-reaching and important significance for improving the survival period and cure rate of breast cancer patients and changing the treatment pattern of breast cancer. SUMMARY

[0004] One of the purposes of the present application is to provide the application of cGAS-STING signaling pathway activators or ATAD3 inhibitors in prevention and / or treatment of breast cancer. The technical problems to be solved are not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0005] To achieve the above-mentioned object, the present application firstly provides an application of a cGAS-STING signaling pathway activator in any one of the following aspects:

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

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

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

[0009] A4) 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 capable of activating a protein molecule in the signaling pathway and thereby activating the signaling pathway, including but not limited to a cGAS activator, a STING activator, a TBK1 activator, and an IRF3 activator.

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

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

[0013] The present application also provides an application of an ATAD3 inhibitor in any one of the following aspects:

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

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

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

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

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

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

[0020] C2) a substance that inhibits or reduces the content, activity, and / or function of ATAD3A protein and ATAD3B protein.

[0021] The substance can be any substance that inhibits the activity and / or reduces the content of ATAD3A protein and ATAD3B protein through expression regulation at the genetic level or protein level.

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

[0023] The protein level regulation can include regulation of the activity and / or content of the protein through protein degradation, protein interaction, or other methods capable of regulating protein activity.

[0024] In the above applications, the substance includes a substance that causes the ATAD3A gene and the ATAD3B gene to be deleted or inactivated through 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 or inactivate the function of ATAD3A protein and ATAD3B protein.

[0025] It is well known to those skilled in the art to inhibit gene expression, silence, or knockout genes 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 or silence the gene at the post-transcriptional level or the translational level. The CRISPR-Cas system containing sgRNA and Cas protein can also be used to knockout the target gene. Alternatively, site-directed mutagenesis technology can be used to mutate the target gene to produce a frameshift mutation or premature translation termination, thereby inactivating or weakening the function of the target gene. In some embodiments of the present application, CRISPR gene editing technology is used to knockout the target gene (ATAD3A gene and ATAD3B gene).

[0026] Further, 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 lentiviral and adeno-associated viral vectors).

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

[0028] The antibodies can include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, Fab, Fab', F(ab')2, antibody variable region (Fv), disulfide-stabilized Fv (dsFv), single-chain antibody (ScFv), single-domain antibody (sdAb, i.e., nanobody), minibody, and minimal recognition unit (MRU).

[0029] As is well known to those skilled in the art, antibodies are commonly used protein inhibitors that can inhibit the activity of their ligands. And as is known to those skilled in the art, antibodies can be prepared using ATAD3A protein or ATAD3B protein, and the activity of the protein can be inhibited by the binding of the antibody to the protein.

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

[0031] D1) a nucleic acid molecule for silencing or knocking out ATAD3A gene and ATAD3B gene;

[0032] D2) an antibody that binds to ATAD3A protein and / or ATAD3B protein.

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

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

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

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

[0037] Further, the Cas9 proteins described herein include Streptococcus pyogenes Cas9 (spCas9, subtype II-A), spCas9-HF (high fidelity), nickase 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, and Cas9 orthologs from other organisms, but are not limited thereto. The Cas9 proteins can also include high-fidelity Cas9 mutants (e.g., SpCas9-HF1, eSpCas9-1.1, and TrueCut TM HiFi Cas9 proteins), etc.

[0038] It is known to those skilled in the art that Cas9 proteins, Cas9 protein mRNAs, Cas9 expression vectors (vectors containing and expressing DNA molecules encoding Cas9 proteins), sgRNAs, sgRNA expression vectors (vectors containing and expressing DNA molecules encoding sgRNAs) can be introduced into animal cells by various methods known in the art, for example, ex vivo animal cells can be introduced (transfected) by calcium phosphate coprecipitation, cationic polymer method (e.g., DEAE-dextran transfection), cationic liposome method, electroporation method (i.e., electroporation), microinjection, gene gun method, or viral-mediated method (e.g., retroviral infection, adenoviral infection, lentiviral infection), etc. In vivo animal cells can also be introduced by microinjection, viral vector method, etc.

[0039] When sgRNAs and Cas9 proteins are delivered using expression vectors, sgRNAs and Cas9 proteins can be expressed linked in different expression vectors, or can be expressed linked in the same expression vector.

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

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

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

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

[0044] E4) a recombinant microorganism containing or respectively containing the DNA molecule 1 and the DNA molecule 2 as described in E2).

[0045] In the above applications, the recombinant microorganism can be a recombinant virus (e.g., a recombinant lentivirus).

[0046] The present application also provides a biological material, which can be any one of the following:

[0047] F1) the sgRNA1 and the sgRNA2 as described herein;

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

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

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

[0051] F5) a recombinant host cell containing or respectively containing the DNA molecule 1 and the DNA molecule 2 as 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 vector includes, but is not limited to, an adeno-associated virus (AAV) vector, an adenovirus vector, a herpes simplex virus (HSV) vector, a lentivirus (LV) vector, a poxvirus vector, a retrovirus vector, a rhabdo virus (baculovirus) vector, a papillomavirus vector, a Sendai virus vector, and a Simian virus vector.

[0054] The recombinant vector includes a recombinant DNA molecule constructed by connecting the DNA molecule encoding the sgRNA1 and / or the sgRNA2 described herein with 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] Further, the recombinant vector can also be a recombinant DNA molecule constructed by connecting a DNA molecule encoding sgRNA1 and / or sgRNA2 described herein with a vector containing a gene encoding Cas9 protein 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 to perform the knockout function on the target gene.

[0056] The recombinant vector can be a gene editing vector expressing sgRNA and Cas9 protein described herein. The method for constructing a gene editing vector is well known to those skilled in the art, and the Cas9 protein matched (or combined) with the designed guide RNA (such as sgRNA of the present application) can be selected as long as the experimental purpose (such as gene knockout) can be achieved. For example, sgRNA and Cas9 protein can be connected into the same vector and started by double promoters, or sgRNA and Cas9 protein can be connected on 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 the 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] Further, the recombinant vector can be a recombinant viral vector.

[0058] Further, the recombinant viral vector can be a recombinant lentiviral vector.

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

[0060] The sgATAD3A#B recombinant vector 1 contains the editing target point of the ATAD3A gene (SEQ ID NO: 1, the target sequence of sgRNA1) and the gene encoding 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 PAM in the genome of the recipient cell, i.e. target the ATAD3A gene, and the Cas9 protein causes double-strand break of DNA at the target point of the ATAD3A gene. Through the organism's own DNA damage repair response mechanism, the broken DNA ends will be directly connected together during the repair process, resulting in random insertion or deletion of bases, thereby achieving knockout of the ATAD3A gene.

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

[0062] Although the embodiments provided by the present application utilize the skeleton vector as a lentivirus vector lentiCRISPRv2, the present application can not be limited to this specific vector. Those skilled in the art can 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] Further, the microorganism described herein includes but is 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 (baculovirus), papillomavirus, Sendai virus, and Simian virus.

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

[0066] Specifically, the recombinant lentivirus can 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 a packaging cell for packaging. The sgATAD3A#B recombinant lentivirus 2 is obtained by introducing the sgATAD3A#B recombinant vector 2 into a packaging cell for packaging.

[0068] The packaging cell is used to package a viral vector plasmid that lacks at least one gene encoding a protein required for viral packaging, which can provide comprehensive viral proteins for viral packaging into a recombinant virus, and which itself cannot produce any form of viral particles. Any cell that provides a protein or polypeptide lacking for viral packaging can be considered for use 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 application, the packaging cell is a HEK293T cell.

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

[0070] Further, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0071] The pharmaceutically acceptable carrier is selected from excipients, preservatives, protective agents, solubilizers, diluents (such as water, physiological saline, PBS (phosphate-buffered saline), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc.), wetting agents, disintegrants (such as dry starch, sodium carboxymethyl starch, croscarmellose, etc.), lubricants (such as sorbitan trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, etc.), fillers (such as starch, dextrin, etc.), binders (such as gelatin, pectin, gum arabic, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), and penetration enhancers (such as Brij-78). pH adjusters, stabilizers (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactants (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium lauryl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption enhancers (such as chitosan), thickeners (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidants (such as sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, etc.), plasticizers (such as glycerin, sorbitol, phthalates, etc.), propellants (such as hydrofluorocarbons, dimethyl ether, etc.), atomizing agents, suspending agents, dispersing agents, colorants (such as TiO2, pigments, etc.), and flavoring agents. Those skilled in the art will recognize that a carrier typically has multiple functions; for example, starch can act as both a disintegrant and a binder. Those skilled in the art can routinely select the above-mentioned carriers based on the properties of the drug and the route of administration.

[0072] Excipients are generally used in pharmaceutical products to make the pharmaceutical products into shape, change the physical state of the pharmaceutical products, and serve as a support. Excipients include, but are not limited to: (1) excipients for injection solutions: such as solvent water, alcohols, ethers, amides, sulfones, esters, etc.; (2) excipients for injection powders: such as sucrose, lactose, mannitol, etc.; (3) excipients for sprays: such as soybean lecithin, propylene glycol, menthol, 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, ethylenediaminetetraacetic acid disodium salt (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, acacia gum, etc.; (10) excipients for ointments: such as vaseline, paraffin, liquid paraffin, lanolin, lanolin alcohol, beeswax, lard, vegetable oil, silicone oil, silicone, soap, higher fatty alcohol, fatty alcohol sulfate, polyhydric alcohol, 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, acacia gum, polyvinyl alcohol compounds, acrylic acid copolymers, etc.

[0073] The dosage form of the pharmaceutical composition includes, but is not limited to, injections (including injection solutions and injection powders), gels, eye drops (including eye drop solutions and intraocular injection solutions), oral solutions, suppositories, effervescent tablets, capsules, ointments, creams, sprays, aerosols, external solutions, tablets, powders, pills, powders, scratch agents, granules, drops, paints, patches, and long-acting controlled-release preparations. Those skilled in the art know that various dosage forms can be prepared using active ingredients with suitable pharmaceutically acceptable carriers according to conventional preparation processes.

[0074] The dosage form of the pharmaceutical composition can be an injection preparation.

[0075] Injections generally refer to solutions, emulsions, and sterile powders for preparing solutions before use by injecting into the body after extraction and purification of medicinal materials, and include injection solutions and injection powders. The preparation method of injections is well known to those skilled in the art, for example, injection powders can be prepared by vacuum freeze-drying technology, spray drying technology, and spray freeze-drying technology. Injection solutions can be prepared by dilution or concentration of the drug with suitable diluents, cosolvents, and / or wetting agents, etc., followed by filtration (such as surface filtration and / or depth filtration), filling and sealing, sterilization, and other steps.

[0076] To prepare the pharmaceutical composition into an injection preparation such as a solution, an emulsion, a lyophilized powder, and a suspension, a diluent commonly used 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, to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerol, etc. can be added to the injection preparation, and in addition, a conventional co-solvent, a buffer, a pH adjuster, etc. can be added.

[0077] The administration method of the pharmaceutical composition includes, but is not limited to, injection administration (such as administration by injection form), mucosal administration (such as administration by spray, aerosol, tablet, eye drop, suppository, granule, capsule, etc.), and transdermal administration (such as administration by gel, ointment, patch, film, etc.).

[0078] The administration method of the pharmaceutical composition includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, intratumoral injection, intracranial injection, mucosal administration, oral administration, skin smearing, oral-nasal cavity spraying, aerosol inhalation, in vivo implantation, and in vitro carrying device administration.

[0079] The present application also provides the use of ATAD3A protein and ATAD3B protein as a target in the preparation of a product having any one of the following functions:

[0080] G1) binding to STING protein;

[0081] G2) regulating cGAS-STING signaling pathway;

[0082] G3) regulating the transport of STING protein from endoplasmic reticulum to 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 the mitochondria, and according to the topological structure prediction analysis, the N-terminal of ATAD3 extends outside the outer membrane of mitochondria, and the C-terminal 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 application takes breast cancer as the research object, uses the BioID proximity labeling technology to screen and identify the STING protein interacting with the N-terminal of ATAD3, and verifies the effect of ATAD3 deletion on the activation of the cGAS-STING signaling pathway in breast cancer cells, and further analyzes the role of ATAD3 in the translocation of STING protein, which provides a theoretical basis and data support for exploring the molecular mechanism of ATAD3 participating in the regulation of the cGAS-STING signaling pathway.

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

[0086] Term definition

[0087] In the present 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 the present application, the definitions and explanations of related terms are provided as follows.

[0088] The term "cGAS-STING signaling pathway" generally refers to a key pathway in cells responsible for recognizing endogenous and exogenous DNA and producing an immune response. The proteins in this signaling pathway mainly include cyclic GMP-AMP synthase (cGAS), interferon gene stimulator (STING), TANK binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3).

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

[0090] In this application, the term "ATAD3 inhibitor" can refer to a substance that inhibits the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of the ATAD3A and ATAD3B genes, or a substance that inhibits or reduces the content, activity, and / or function of the ATAD3A and ATAD3B proteins. The ATAD3 inhibitor includes substances that cause the deletion or inactivation of the ATAD3A and ATAD3B genes through techniques such as site-directed mutagenesis, gene knock-down, gene editing, and gene knock-out. The ATAD3 inhibitor also includes substances capable of targeting and binding to the ATAD3A and ATAD3B proteins, inhibiting their activity, or preventing them from performing their functions.

[0091] The term "site-directed mutagenesis" generally refers to altering one or more bases in a gene through site-directed mutation, 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," also known as gene knockdown reduction, generally refers to techniques that inactivate or silence gene expression at the post-transcriptional or translational level without altering the gene's DNA sequence. Gene knockdown includes techniques such as RNA interference, Morpholino interference, antisense nucleic acid techniques, and ribozyme techniques.

[0093] The term "gene editing" generally refers to the ability to alter specific gene sequences within any cell, including somatic cells, resulting in base deletions, duplications, insertions, frameshift mutations, and replacements or knockouts of target genes. This allows for the substitution, deletion, splicing, and single-base changes of the genome sequence—essentially, the technology to arbitrarily "edit" the genome or the sequence of a specific gene. Gene editing includes zinc finger ribozyme knockout technology, TALEN gene editing technology, and CRISPR gene editing technology.

[0094] The term "Cas9 protein" generally refers to a type II CRISPR system's Cas endonuclease that forms a complex with crRNA and tracrRNA or with a guide RNA for specific recognition and cleavage of a whole or partial DNA target sequence. The Cas9 protein has two distinct domains: an HNH domain and a RuvC domain. The HNH domain is responsible for cleaving the DNA strand (target strand) that is complementary paired with 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 a single RNA structure that is artificially engineered from a crRNA / tracrRNA complex (gRNA) with a double RNA structure, in which the crRNA and tracrRNA are directly (or through a linker) connected. The sgRNA is a component of the CRISPR-Cas system, responsible for guiding the Cas protein to recognize and cleave the target nucleic acid molecule. In actual gene editing applications, the sgRNA can be directly synthesized, or obtained through plasmid expression or in vitro transcription. The sgRNA includes a recognition region and a scaffold region. The scaffold region is known to those skilled in the art and is responsible for binding to the Cas protein, and the recognition region is responsible for binding to the target site of the target gene, guiding the Cas protein to the target site.

[0096] The term "target sequence" can refer to the target sequence of the sgRNA, which refers to a sequence located in the gene to be edited (usually the coding region), which is complementary to the sgRNA and is located near the position where cleavage is desired, and is the target 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 technology that uses an exogenous mutated gene to replace an endogenous normal homologous gene by homologous recombination, thereby inactivating the endogenous gene, including complete gene knock-out (such as complete mutation of the target gene based on a replacement-type targeting vector or an insertion-type targeting vector) and conditional gene knock-out (such as tissue-specific knock-out 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 mRNA degradation of a target gene that is homologously complementary to the dsRNA, silencing and closing the expression of the gene, thereby triggering post-transcriptional gene silencing (PTGS), achieving the purpose of preventing gene expression.

[0099] The term "Morpholino interference technology" generally refers to the substitution of a five-carbon sugar ring on a traditional nucleotide with a morpholino, and the original phosphate group is also changed, so that the whole molecule does not have any charge and cannot be recognized and degraded by RNase and DNase, and is extremely stable. Its principle is the same as that of antisense nucleic acid technology, that is, by binding to the homologous sequence of the target gene mRNA through complementation, the binding of other molecules and proteins to the specific mRNA nucleic acid sequence is hindered, and finally the target gene mRNA cannot be translated into protein.

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

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

[0102] The term "comprising" is not intended to be limiting, is intended to be inclusive and means that there can be additional elements other than the listed elements that are also contemplated. The term "comprising" encompasses the terms "consisting of" and "consisting essentially of." The terms "comprising" and "including" are used interchangeably in this document. BRIEF DESCRIPTION OF DRAWINGS

[0103] Figure 1 To pull the N-terminal interacting proteins of ATAD3 protein using BioID proximity labeling technology. Among them, (A) SnapGene software analyzes the similarity of ATAD3A and ATAD3B sequences. (B) Construction of a labeling vector expressing BioID-ATAD3A. (C) Immunoblotting detects the expression level of BioID-ATAD3A in 293T cells transfected with BioID-ATAD3A labeling vector. (D) Mass spectrometry screening of ATAD3A N-terminal interacting STING protein.

[0104] Figure 2 ATAD3 protein family members interact with STING protein. Among them, (A) Overexpression of ATAD3A-His and ATAD3B-His in MCF-7 cells, immunoprecipitation to capture ATAD3A-His and ATAD3B-His interacting proteins, and immunoblotting to detect STING protein in immunoprecipitation. (B) Immunoprecipitation to capture endogenous ATAD3 protein family members in MCF-7 wild-type cells, and immunoblotting to detect STING protein and endoplasmic reticulum transmembrane protein Sec61B in immunoprecipitation.

[0105] Figure 3 ATAD3 protein family members reduce the binding of STING protein under the stimulation of STING activator cGAMP. Among them, (A) In MCF-7 wild-type cells, after treatment with different concentrations of cGAMP (0 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL) for 2.5 hours, immunoprecipitation was used to capture ATAD3 protein family members, and immunoblotting was used to detect the change of STING protein level in immunoprecipitation. (B) ImageJ analysis of experimental results, the numerical value is expressed as mean ± SEM (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0106] Figure 4 The deletion of ATAD3 significantly increases the sensitivity of cells to DNA mimic poly(dA:dT). Among them, (A) After 1 μg / mL double-stranded DNA mimic poly(dA:dT) was treated for 1 hour and 2 hours in MDA-MB-231 control group (Ctrl) and DKO cells double-knocked out ATAD3A and ATAD3B, respectively, immunoblotting was used to detect the change of STING protein and downstream TBK1 protein expression level, and the change of STING protein and TBK1 protein phosphorylation level. (B), (C) ImageJ analysis of experimental results, the numerical value is expressed as mean ± SEM (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0107] Figure 5Deletion of ATAD3 significantly increased the sensitivity of cells to STING protein agonist cGAMP. Among them, (A) After MCF-7 control group (Ctrl) and ATAD3 DKO cells were treated with different concentrations of cGAMP (0 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL) for 2.5 hours, the changes of STING protein and downstream TBK1 protein expression levels, and the changes of STING protein and TBK1 protein phosphorylation levels were detected by immunoblotting. (B), (C) ImageJ analyzed the experimental results, the numerical values were expressed as mean ± SEM (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0108] Figure 6 Brefeldin A blocked the concentration-dependent increase of STING protein phosphorylation level in MCF-7 cells. MCF-7 control group (Ctrl) and ATAD3 DKO cells were treated with different concentrations of cGAMP (0 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL) for 2.5 hours in NC group, and MCF-7 Ctrl and DKO cells were treated with different concentrations of cGAMP (0 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL) plus 0.5 μg / mL Brefeldin A for 2.5 hours in BFA group, and the changes of STING protein expression level and phosphorylation level were detected by immunoblotting.

[0109] Figure 7 Deletion of ATAD3 significantly increased the colocalization of STING protein with Golgi. Among them, (A) After MCF-7 control group (Ctrl) and ATAD3 DKO cells were treated with cGAMP (2 μg / mL) for 5 hours, 4% paraformaldehyde was used for fixation at 37°C for 15 min, and then the cells were immunofluorescence 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, and then imaged by FV3000 confocal microscope, scale bar 50 μm. (B) ImageJ analyzed the results of three independent repeated experiments, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0110] Figure 8The absence of ATAD3 led to an increase in STEEP protein, which binds to STING protein in cells. Specifically, (A) MCF-7 control (Ctrl) and ATAD3DKO cells were treated with cGAMP (2 μg / mL) for 2.5 hours, followed by immunoprecipitation to capture endogenous STING protein. The changes in STEEP protein levels in the immunoprecipitate were detected by Western blotting. (B) MDA-MB-231 control (Ctrl) and ATAD3DKO cells were treated with cGAMP (2 μg / mL) for 2.5 hours, followed by immunoprecipitation to capture endogenous STEEP protein. The changes in STING protein levels in the immunoprecipitate were detected by Western blotting. (C) and (D) ImageJ analysis of the experimental results, with values ​​expressed as mean ± SEM (n=3); *, P<0.05; **, P<0.01; ***, P<0.001.

[0111] Figure 9 The in vitro proliferation capacity of MCF-7 and MDA-MB-231 knockout cell lines was evaluated. (A) Results of Western blotting analysis of ATAD3A and ATAD3B reinjection in the MCF-7 cell line. (B) Results of Western blotting analysis of ATAD3A and ATAD3B reinjection in the MDA-MB-231 cell line. (C) Proliferation capacity of the control, DKO, and Rescue cell lines in the MCF-7 cell line (6 replicates per group). (D) Proliferation capacity of the control, DKO, and Rescue cell lines in the MDA-MB-231 cell line (6 replicates per group). *, P<0.05; **, P<0.01; ***, P<0.001.

[0112] Figure 10 The results are from a subcutaneous tumorigenesis experiment in mice. In (A) and (B), control and DKO cells, respectively, were injected into nude mice with MCF-7 and MDA-MB-211. Each injection contained 10... 6 Cells / 100μL PBS. n(MCF-7)=10, n(MDA-MB-231)=9. Detailed Implementation

[0113] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0114] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0115] The following examples use the GraghPad Prism 9 statistical software to process the data, and the experimental results are expressed as mean ± standard deviation, and P<0.05 is considered to be a significant difference between groups. The quantitative experiments in the following examples, unless otherwise specified, are set up for three biological replicates.

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

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

[0118] In the following examples, the ATAD3 protein refers to the ATAD3A protein and the ATAD3B protein.

[0119] In the following examples, the human breast cancer MCF7 cell line is described in the following literature: Wong C Y A, Jiang H, Abel P W, 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 document: 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 promote 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 (hereinafter referred to as pLVX-puro plasmid) in the following examples was purchased from UBIOBIOSCIENCES, item number VT1465.

[0122] The myc-BioID2-MCS plasmid in the following examples is described in the following document: Kim DI, Jensen SC, Noble KA, Kc B, Roux KH, Motamedchaboki K, Roux KJ. An improved smaller biotin ligase 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] This example uses a lentivirus vector based on the CRISPR / Cas9 gene editing system to construct a recombinant lentivirus for knocking out the ATAD3A gene and the ATAD3B gene, and the specific steps are as follows:

[0125] 1. Recombinant vector preparation

[0126] (1) sgRNA design: Based on the nucleotide sequences of the ATAD3A gene (NM_001170535.3) and the ATAD3B gene (NM_031921.6), sgRNA targets were designed at their first start codons. Two target sites (named target 1 and target 2, respectively) and one control target site 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 the ATAD3A gene, and the target gene corresponding to sgRNA2 is the ATAD3B gene. The nucleotide sequence of the control target site 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) Annealing sgATAD3A#B4-F and sgATAD3A#B4-R yields oligo dimer 1. Annealing sgATAD3A#B32-F and sgATAD3A#B32--R yields oligo dimer 2. Annealing sgNTC-F and sgNTC-R yields oligo dimer 3.

[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 are connected using T4 DNA ligase (NEB) to obtain sgATAD3A#B recombinant vector 1. The vector backbone and oligo dimer 2 are connected using T4 DNA ligase (NEB) to obtain sgATAD3A#B recombinant vector 2. The vector backbone and oligo dimer 3 are connected 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 is co-transfected with the lentivirus packaging vector psPAX2 (Addgene, #12260) and pMD2G (Addgene, #12259) into HEK293T cells to obtain sgATAD3A#B recombinant lentivirus 1. The specific steps are as follows:

[0134] (1) The sgATAD3A#B recombinant vector 1, the lentivirus packaging vector psPAX2 and pMD2G are co-transfected into HEK293T cells using Lipo3000 transfection reagent kit (Thermo Scientific) (the transfection ratio is: 293T cells plated in 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 is added and incubated in a 37°C cell incubator for 8 hours.

[0135] (2) After step (1) is completed, the old culture medium is replaced with fresh HEK293T cell culture medium, and the 37°C cell incubator is continued for 72 hours. At 24, 48 and 72 hours of culture, the culture solution is collected and filtered with a 0.45 μm filter membrane to remove dead cells and cell debris, etc.

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

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

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

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

[0140] 1. Using human breast cancer MCF7 cell lines as test cells, infecting control lentivirus sgNTC, sgATAD3A#B recombinant lentivirus 1 and sgATAD3A#B recombinant lentivirus 2, respectively. The specific method is: adding 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 (polybrene) into a 10 cm diameter culture dish of MCF7 breast cancer cell line. 5 Personal breast cancer cell line MCF7 culture dish (10 cm in diameter) is added with 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 (polybrene).

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

[0142] 3. The positive cells screened above are trypsinized, counted with a cell counter, and the cell suspension is diluted and plated into a 96-well plate at a density of 1 cell per well. The growth of cells in each well is observed at regular intervals, and the cells are periodically expanded. At the same time, the knockout of the target protein is detected by immunological methods (note that the internal reference protein is used as a control group), and the DKO cells of the human breast cancer MCF7 cell line lacking ATAD3 protein (ATAD3A protein and ATAD3B protein) are screened, named MCF-7 DKO cells, which are used for subsequent experiments.

[0143] According to the above steps, the human breast cancer MCF7 cell line is replaced with the human breast cancer MDA-MB-231 cell line, and the other steps are unchanged, to obtain DKO cells of the human breast cancer MDA-MB-231 cell line lacking ATAD3 protein, named MDA-MB-231 DKO 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) design primer ATAD3A-His-F: 5'-at ctcgagctcaagcttcgaattcGCCACCatgtcgtggctcttcggcat-3' and ATAD3A-His-R: 5'-ATGGTGATGGTGATGATGGCCTCCggatggggagggctcgtccc-3', with MCF7 cell line cDNA as template, ATAD3A-His-F and ATAD3A-His-R as primer pair for PCR amplification, obtain ATAD3A-His gene sequence PCR product; according to the nucleotide sequence of ATAD3B gene (NM_031921.6) design primer ATAD3B-His-F: 5'-atctcgagctcaagcttcgaattcGCCACCatgtcgtggctcttcggcgt-3' and ATAD3B-His-R: 5'-GGTGATGGTGATGATGGCCTCCcaacagggggtgccctgggg-3', with MCF7 cell line cDNA as template, ATAD3B-His-F and ATAD3B-His-R as primer pair for PCR amplification, obtain ATAD3B-His gene sequence PCR product; with EcoRI endonuclease (Neb) enzyme cut pLVX-puro plasmid, use homologous recombination enzyme (Neb) to connect the PCR product to the MCS region of the enzyme cut pLVX-puro plasmid, obtain pLVX-ATAD3A-His plasmid and pLVX-ATAD3B-His plasmid.

[0146] 2、Use packaging plasmid (psPAX2 and pMD2) and backbone plasmid (pLVX-ATAD3A-His plasmid or pLVX-ATAD3B-His plasmid) to transfect HEK293T cell line, after transfection, continuously culture for 36h, without trypsin digestion of cells, collect supernatant, mark as A liquid, replace fresh culture medium, after 72h of culture, collect supernatant again, mark as B liquid, after mixing A liquid and B liquid, filter with 0.45μM filter screen, it is virus infection stock solution.

[0147] 3、Mix virus infection stock solution and cell culture medium according to 1:4 ratio, add to the adherent human breast cancer cell line MCF7 to be infected, after 24h of culture, discard virus infection liquid, add fresh culture medium, after 24h of continuous culture, add a certain concentration of puromycin to screen positive cells, it is MCF7 cell overexpressing ATAD3A-His protein and MCF7 cell overexpressing ATAD3B-His protein.

[0148] Example 4, Western Blot

[0149] 1. First, prepare the cell lysate. Add 10 μL protease inhibitor (Yeasen) to 1 mL RIPA cell lysate (Solebio) and store on ice. Then, discard the culture medium of the adherent cells, add 1 mL PBS to the cell surface, wash the cells and discard the PBS. Add an appropriate amount of cell lysate according to the cell density, place on ice for 3 min, then scrape the cells from the cell culture dish and store on ice for 20 min. Then collect the cell lysate and centrifuge at 12000 rpm, 4°C for 20 min. Collect the supernatant.

[0150] 2. Prepare the reaction solution according to the BCA protein quantification guide of Thermo. Then add 10 μL of protein standard and 10 μL of the protein to be tested to 90 μL of the reaction solution. React at 37°C for 30 min, then use the enzyme label instrument to detect OD 562 . According to the protein standard, draw a standard curve and calculate the concentration of the protein to be tested.

[0151] 3. Separate 20 μg of protein by SDS-PAGE gel electrophoresis, then transfer it to a PVDF membrane (Millipore). Block with 5% skim milk (Yeasen) at room temperature for 1 hour. Incubate with the primary antibody at 4°C overnight. Then incubate with horseradish peroxidase (HRP) labeled secondary antibody at room temperature for 1 hour.

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

[0153] 5、The primary antibodies for Western blotting experiments are 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), His antibody (Proteintech, 66005-1-Ig). The secondary antibodies are 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 E. coli and biotinylates only a single cellular protein. In 2004, scientists reported a mutant BirA that attaches 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. The mutant BirA also self-biotinylates in vivo and in vitro. The wild-type BirA protein is much less active in these reactions. The biotinylation reaction is proximity dependent, as greater degrees of biotinylation are observed when the mutant ligase is coupled to the acceptor protein than when the acceptor is free in solution. This approach allows the interacting proteins to be easily detected and recovered by existing avidin / streptavidin technology.

[0156] 1. According to the nucleotide sequence of ATAD3A gene (NM_001170535.3), the primers ATAD3-BioID-F: 5'-agcctgagaagaagcGGTGGCGGAGGGAGCatgtcgtggctcttcggca-3' and ATAD3-BioID-R: 5'-tggcggccgctcgagtcaggatggggaggg-3' are designed, and the full-length PCR product of ATAD3A is obtained by PCR amplification with the cDNA of MCF7 cell line as a template and the primers ATAD3-BioID-F and ATAD3-BioID-R; the myc-BioID2-MCS plasmid is single-enzymatically cut by NotI endonuclease (Neb), and the PCR product is ligated to the MCS region of the single-enzymatically cut myc-BioID2-MCS plasmid by using a homologous recombinase to obtain myc-BioID-ATAD3A.

[0157] 2. Transfected cell line

[0158] ①According to the transfection reagent manual of polyplus company, the transfection system is prepared, and for example, for a 6cm dish, the transfection system is as follows:

[0159] Table 1. Transfection system

[0160] DNA 4 ng Buffer 200 μL Transfection reagen 8 μL

[0161] ②The transfection system is placed at room temperature for 5min;

[0162] ③The transfection system is added to the culture medium of HEK-293T cells, and the culture medium is replaced after 4h.

[0163] 3. Extraction of cell mitochondria

[0164] ①Prepare solutions:

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

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

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

[0168] ②Collect cells

[0169] Wash the cells with 1mL PBS, completely digest the cells with trypsin, centrifuge at 800g for 3min;

[0170] 3. Wash the cells

[0171] Resuspend the cells with pre-cooled PBS, after the completion of the wash, 800g, 4℃, centrifuge for 3min, collect the cells;

[0172] 4. Crude mitochondria

[0173] Resuspend the cells with 2mL solution A (with protease inhibitors added), grind the cells with a Dounce grinder for about 30 strokes on ice, 1000g, 4℃, centrifuge for 10min, transfer the supernatant to a new centrifuge tube, 3500g, 4℃, centrifuge for 10min, carefully aspirate the supernatant, the precipitate is the isolated mitochondria, aspirate the supernatant, 12000g, 4℃, centrifuge for 10min, the obtained supernatant is the cytoplasmic protein;

[0174] 5. Purify the mitochondria

[0175] Wash the crude mitochondria with pre-cooled solution B for 3 times, finally resuspend with 400μL solution B, spread the crude mitochondria on a percoll gradient, ultracentrifuge at 18000rpm, 4℃, centrifuge for 45min; carefully aspirate the middle layer to a new centrifuge tube, resuspend with 1mL solution B, 15000rpm, 4℃, centrifuge for 10min, carefully wash the supernatant, lyse the purified mitochondria with 200μL RIPA cell lysis buffer.

[0176] 4. Streptavidin affinity purification of the protein

[0177] 1. Equilibrate the magnetic beads: take 20μL hydrophilic streptavidin magnetic beads (Neb) in a centrifuge tube, place the centrifuge tube on a magnetic stand, after 1-2s, aspirate the supernatant; resuspend the magnetic beads with 200μL RIPA cell lysis buffer (Solabio), equilibrate on a mixer for 10min at 4℃, repeat this step three times;

[0178] 2. Incubate the protein: aspirate the resuspended magnetic beads, incubate with the lysed mitochondria at 4℃ for 4h on a mixer;

[0179] 3. Wash the magnetic beads: place the centrifuge tube containing the magnetic beads and mitochondria on a magnetic stand, after 1-2s, aspirate the supernatant, resuspend the magnetic beads with 500μL TBS, wash for 10min at 4℃ on a mixer, repeat this step three times;

[0180] 4. Elute the protein: place the centrifuge tube on a magnetic stand, after 1-2s, aspirate the TBS, resuspend the magnetic beads with 20μL 1x loading buffer, place in a 95℃ metal bath for 10min, after a brief centrifugation, place the centrifuge tube on a magnetic stand, after 1-2s, carefully aspirate the supernatant to a new centrifuge tube.

[0181] 5. Mass spectrometry analysis

[0182] ①: In-gel digestion: The Coomassie-stained bands were destained, reduced with DTT and alkylated with iodoacetamide, and then digested with trypsin overnight. The peptides were extracted from the gel with 60% CAN.

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

[0184] Example 6, Immunoprecipitation and co-immunoprecipitation

[0185] 1. Immunoprecipitation

[0186] (1) Protein extraction: Prepare cell lysis solution, add 10 μL protease inhibitor (Yeasen) to 1 mL non-denaturing cell lysis solution (Solabio) and store on ice. Then, discard the culture medium of adherent cells, add 1 mL PBS to the cell surface, wash the cells and discard the PBS. Add an appropriate amount of non-denaturing cell lysis solution according to the cell density, place the cells on ice for 3 min, then scrape the cells from the cell culture dish and place them on ice for 20 min. Then collect the cell lysate and centrifuge tube, centrifuge at 12000 rpm, 4°C for 20 min, and collect the supernatant;

[0187] (2) Protein quantification: Prepare the reaction solution according to the BCA protein quantification guide of Thermo company, then take 10 μL of protein standard and 10 μL of protein to be tested and add them to 90 μL of reaction solution, 37°C reaction for 30 min, then use the enzyme label instrument to detect OD 562 , draw the standard curve according to the protein standard and calculate the concentration of the protein to be tested;

[0188] (3) Preparation of Input Sample: Take 100 μL of the prepared protein sample into a new centrifuge tube, add 25 μL of 5× loading buffer, mix well, place on a 95℃ metal bath for 5 min, centrifuge at 12000 rpm for 3 min, and freeze in a -80℃ freezer for subsequent Western blot detection.

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

[0190] (5) Balancing magnetic beads: Take 45 μL of protein A / G magnetic beads (MCE) into a centrifuge tube, place the centrifuge tube on a magnetic rack, let stand for 1-2 seconds, then aspirate the supernatant, resuspend the magnetic beads with 600 μL of TBS, equilibrate on a mixer at 4℃ for 10 min, place the centrifuge tube on a magnetic rack, let stand for 1-2 seconds, then aspirate the supernatant, resuspend the magnetic beads with 600 μL of non-denatured cell lysis buffer, equilibrate on a mixer at 4℃ for 10 min, place the centrifuge tube on a magnetic rack, let stand for 1-2 seconds, then aspirate the supernatant;

[0191] (6) Binding of magnetic beads to antibody-antigen: Mix the overnight incubated antibody-antigen mixture with magnetic beads. After the magnetic beads are fully resuspended, incubate at 4°C on a mixer for 2 hours.

[0192] (7) Cleaning the magnetic beads: Place the centrifuge tube containing 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-denatured cell lysis buffer to resuspend the magnetic beads, and wash at 4°C on a mixer for 10 min. Repeat this step 6 times. Add 600 μL of TBS to resuspend the magnetic beads, and wash at 4°C on a mixer for 10 min. 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. Resuspend the magnetic beads in 20 μL of 1×loading Buffer, place it on a 95℃ metal bath for 10 min, and after a short centrifugation, place the centrifuge tube on a magnetic rack, let it stand for 1-2 seconds, and carefully aspirate the supernatant into a new centrifuge tube. This part of the protein is used for subsequent Western blot detection.

[0194] 2. Immunoprecipitation

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

[0196] (2) Preparation of Input Sample: Refer to the experimental steps in immunoprecipitation (3);

[0197] (3) Equilibrium magnetic beads: Take 45 μL anti-His magnetic beads (Bi Yun Tian) in a centrifuge tube, place the centrifuge tube on the magnetic stand, and let stand for 1-2 s before aspirating the supernatant. Resuspend the magnetic beads with 600 μL TBS, equilibrate for 10 min on the mixer at 4°C, then place the centrifuge tube on the magnetic stand, let stand for 1-2 s, and aspirate the supernatant. Resuspend the magnetic beads with 600 μL non-denaturing cell lysate, equilibrate for 10 min on the mixer at 4°C, then place the centrifuge tube on the magnetic stand, let stand for 1-2 s, and aspirate the supernatant.

[0198] (4) Incubate the protein: Take 500 μL of the protein resuspended and equilibrated magnetic beads, incubate for 3 h on the mixer at 4°C.

[0199] (5) Wash the magnetic beads: Place the centrifuge tube containing the incubated magnetic beads and protein on the magnetic stand, let stand for 1-2 s, and aspirate the supernatant. Resuspend the magnetic beads with 600 μL of non-denaturing cell lysate, wash for 10 min on the mixer at 4°C, and repeat this step 3 times. Resuspend the magnetic beads with 600 μL TBS, wash for 10 min on the mixer at 4°C, and repeat this step 3 times.

[0200] (6) Protein elution: Place the centrifuge tube on the magnetic stand, let stand for 1-2 s, and aspirate the TBS. Resuspend the magnetic beads with 20 μL 1x loading buffer, place in a 95°C metal bath for 10 min, and then place the centrifuge tube on the magnetic stand, let stand for 1-2 s, and carefully aspirate the supernatant into a new centrifuge tube. This portion of the protein is used for subsequent Western blot detection.

[0201] Example 7, Immunofluorescence

[0202] (1) Inoculate cells: Carefully place the cell slides in a 6-well plate, and inoculate the normally growing cells in the 6-well plate at a density of 100,000 cells / well. Use the "cross method" to spread the cells, let stand for 1 min, and then incubate in a 37°C, 5% CO2 cell incubator for 24 h.

[0203] (2) Fix the cells: Aspirate the culture medium, and add 2 mL of 37°C preheated 4% paraformaldehyde to each well. Incubate at 37°C for 15 min, then add 2 mL of PBS to each well after fixation is complete, let stand for 10 min, and repeat three times.

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

[0205] (4) Incubate the primary antibody: Dilute the antibody according to the antibody manual, and add 100 μL of the primary antibody to the cell slides. Incubate at 4°C, let stand, and incubate overnight.

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

[0207] (6) Incubate the secondary antibody: dilute the antibody according to the antibody manual, add 100 μL of the secondary antibody to the cell climbing sheet, stand at room temperature, incubate for 2 h.

[0208] (6) Wash the secondary antibody: aspirate the secondary antibody, add 2 mL PBS to each well, stand for 10 min, repeat three times

[0209] (8) DAPI staining and mounting: take 5 μL of DAPI-containing anti-fluorescence quenching mounting medium in the center of the glass slide, invert the cell climbing sheet in the center of the glass slide, and carefully avoid air bubbles. Use transparent nail polish to fix the cell climbing sheet at the four corners.

[0210] (9) Imaging: take pictures of the treated cell climbing sheet with a laser confocal microscope FV 3000. The excitation light is 405 nm, 488 nm and 561 nm respectively.

[0211] (10) The primary antibody used in the immunofluorescence experiment is: GM130 (BD) and STING (RD).

[0212] Example 8, subcutaneous tumor experiment in nude mice

[0213] The 6-8 week old female Balb / c nude mice in the experiment were from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental operation strictly followed the experimental animal ethics regulations of the Institute of Biophysics, Chinese Academy of Sciences, and the regulations of the Animal Care and Use Committee (IACUC-IBP).

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

[0215] (2) After 3 weeks of injection, the mice were sacrificed, and the xenograft tumor was removed, washed with PBS once, and the tumor size and weight were observed and recorded, and photographed.

[0216] Example 9, ATAD3 protein and STING protein interaction

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

[0218] Based on the topological structures of ATAD3A and ATAD3B, the N-termini of both ATAD3A and ATAD3B are located in the cytoplasm, and the sequences extending into the cytoplasm are essentially identical. The N-terminus of the ATAD3 protein family was labeled using the BioID (Bio-Proximity Identifier) ​​technique, but due to the extreme similarity between the N-termini of ATAD3A and ATAD3B... Figure 1 A) We selected the ATAD3A protein as the BioID marker target, added the BioID tag to the N-terminus of the full-length ATAD3A protein, and constructed a BioID-ATAD3A neighboring marker vector. Figure 1 B), and the expression level of BioID-ATAD3A was detected by immunoblotting to ensure the effectiveness of the labeling. Figure 1 C). Subsequently, we enriched the N-terminal interacting proteins of ATAD3A using streptomycin affinity chromatography and screened for STING protein ( Figure 1 D).

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

[0220] To ensure the accuracy of the enrichment results, we used the MCF-7 cell line overexpressing ATAD3A-His and ATAD3B-His proteins prepared in Example 3 for immunoprecipitation experiments.

[0221] Experimental results show that, under exogenous overexpression of ATAD3A and ATAD3B, members of the ATAD3 protein family interact with the STING protein. Figure 2 To further validate our experimental results, we used immunoprecipitation to examine the interaction between ATAD3 and STING proteins in the MCF-7 wild-type cell line. We also selected the transmembrane protein Sec61B from the endoplasmic reticulum as a quality control protein to verify whether ATAD3 interacts with STING through its interaction with the endoplasmic reticulum. The results showed that ATAD3 and STING interact at the endogenous cellular level, and that ATAD3 did not exhibit a clear interaction with the endoplasmic reticulum transmembrane protein Sec61B. This indicates that the binding of ATAD3 to STING is not due to the interaction between ATAD3 and the endoplasmic reticulum.

[0222] 3. Stimulation by STING activators reduces the amount of STING protein that binds to ATAD3.

[0223] To further elucidate the relationship between ATAD3 and STING proteins, we attempted to examine whether their interaction changed upon STING protein activation. In this part of the experiment, we used cGAMP as the STING protein activator and employed immunoprecipitation to detect whether the interaction between endogenous ATAD3 protein family members and STING protein was altered.

[0224] We treated the MCF-7 wild-type cell line with different cGAMP concentrations, enriched proteins that interact with ATAD3 protein family members using immunoprecipitation, and detected changes in STING proteins that interact with ATAD3 protein family members using Western blotting. Figure 3 The experimental results showed that treatment with the STING protein agonist cGAMP significantly reduced the amount of STING protein interacting with ATAD3 protein family members compared to the untreated group (cGAMP concentration of 0 μg / mL). At cGAMP concentrations of 1 μg / mL and 2 μg / mL, the amount of STING protein interacting with ATAD3 protein family members decreased with increasing concentration, reaching 80% and 70% of the untreated group, respectively. However, when the cGAMP concentration reached 5 μg / mL, the amount of STING protein bound by ATAD3 protein family members actually increased slightly compared to the lower cGAMP concentration groups. At a cGAMP concentration of 2 μg / mL, the amount of STING protein bound by ATAD3 protein family members was the lowest compared to 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 their interaction with STING protein.

[0225] Example 10: ATAD3 deficiency significantly increased the activation of the cGAS-STING signaling pathway in cells.

[0226] In Example 9, we demonstrated the interaction between ATAD3 protein and STING protein in the cytoplasm, and that activation of STING protein led to a significant reduction in STING protein binding to members of the ATAD3 protein family in the cell. Therefore, we hypothesize that ATAD3 protein may act as a negative regulator of STING protein, participating in the activation of the cGAS-STING signaling pathway and downstream response factors.

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

[0228] The experimental results showed that, without treatment with the DNA mimic poly(dA:dT), the phosphorylation level of STING protein in MDA-MB-231DKO cells was higher than that in the MDA-MB-231 control group cells, but there was no significant difference in the phosphorylation level of TBK1 protein between the MDA-MB-231 control group and the MDA-MB-231DKO group cells. Under exogenous poly(dA:dT) treatment, the total STING protein level and TBK protein level in both the MDA-MB-231 control group and the MDA-MB-231DKO group cells did not change significantly. However, with increasing poly(dA:dT) treatment time, the phosphorylation levels of STING protein and TBK protein in both the MDA-MB-231 control group and the MDA-MB-231DKO group cells increased significantly. Most importantly, the phosphorylation level of STING protein in MDA-MB-231DKO cells treated with poly(dA:dT) was significantly higher than that in the MDA-MB-231 control group cells. Furthermore, after 1 and 2 hours of poly(dA:dT) treatment, the phosphorylation level of TBK1 protein, a downstream response factor of STING protein, was also significantly higher in the MDA-MB-231DKO group cells than in the MDA-MB-231 control group cells. These experimental results lead to the conclusion that ATAD3 protein deficiency makes cells more sensitive to activation of the cGAS-STING signaling pathway. Therefore, we believe that members of the ATAD3 protein family play an important role in the activation of the cGAS-STING signaling pathway in cells.

[0229] Subsequently, to further validate our hypothesis, we treated MCF-7 control group (Ctrl) and ATAD3DKO knockout group cells with the STING protein activator cGAMP. We then used Western blotting to detect the activation of STING protein and its downstream factor TBK1 protein in the cGAS-STING signaling pathway. Figure 5 ).

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

[0231] Example 11, Deletion of ATAD3 significantly increases the transport of STING protein in cells

[0232] STING protein is a protein located in the endoplasmic reticulum in the resting state and has 4 transmembrane domains. When the natural ligand of STING protein exists (such as cGAMP), the conformation of STING protein changes, leading to the dimerization of STING. The activation of STING protein is a step composed of multiple biological events, including the dimerization, oligomerization, departure from the endoplasmic reticulum, translocation to the Golgi body, and finally phosphorylation on the Golgi body. We have inferred from the above experimental results that ATAD3 protein may negatively regulate the activation of cGAS-STING signaling pathway and the activation of downstream TBK1 protein by interacting with resting STING protein on the endoplasmic reticulum. And it has been reported that the translocation of STING protein from the endoplasmic reticulum to the Golgi body is an important rate-limiting step for the activation of STING protein. Then, will the deletion of ATAD3 protein affect the transport of STING protein from the endoplasmic reticulum to the Golgi body?

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

[0234] The experimental results show that after cGAMP treatment at different concentrations, the phosphorylation level of STING protein in both control and DKO group cells increases in a cGAMP concentration-dependent manner, without changes in the total protein level of STING protein. And when the concentration of cGAMP is 1 μg / mL and 2 μg / mL, the phosphorylation level of STING protein in DKO group cells is significantly higher than that in control cells. However, when different concentrations of cGAMP and Brefeldin A coexist, although the phosphorylation of STING protein in both control and DKO group cells can still be detected, and the phosphorylation level of STING protein in DKO cells is higher than that in control cells, the phosphorylation level of STING protein does not increase with the increase of cGAMP concentration. This is because the presence of Brefeldin A blocks the transport of STING protein from endoplasmic reticulum to Golgi, thereby inhibiting the increase of STING protein phosphorylation level in control and DKO group cells stimulated by different concentrations of cGAMP. After blocking the translocation of STING, the phosphorylation level of STING protein in DKO cells is higher than that in control cells, which may be due to the fact that before the addition of blocking agent Brefeldin A, more STING protein in DKO group cells is translocated to Golgi and phosphorylated than in control cells.

[0235] To further detect the effect of ATAD3 deletion on the translocation of STING protein from endoplasmic reticulum to Golgi, we used immunofluorescence technology to label STING protein and Golgi membrane protein GM130 in cells, and detected the colocalization of STING protein and GM130 protein by cGAMP treatment of MCF-7 control and DKO group cells, to verify whether the absence of ATAD3 protein family members leads to more STING protein being translocated to Golgi Figure 7

[0236] ​​The results of immunofluorescence showed that there was no significant difference between the colocalization of STING protein with Golgi apparatus in DKO cells and control cells without the presence of STING protein agonist. After cGAMP treatment, the colocalization of STING protein with Golgi apparatus in both control cells and DKO cells was significantly increased compared to the untreated cells. Most importantly, the colocalization of STING protein with Golgi apparatus in DKO cells was significantly higher than that in control cells in the presence of cGAMP, which means that more STING protein was transported from endoplasmic reticulum to Golgi apparatus in the cells lacking ATAD3. This indicates that the deletion of ATAD3 protein family members affects the transport of STING protein from endoplasmic reticulum to Golgi apparatus, thereby affecting the activation of STING protein on Golgi apparatus.

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

[0238] We explored the effect of the deletion of ATAD3 protein on the binding of STING protein and STEEP protein when STING protein was activated. After cGAMP treatment of MCF-7 control cells and DKO cells, we used immunoprecipitation technique to detect the interaction between STING protein and STEEP protein in the cells Figure 8 ). The experimental results showed that the level of STEEP protein pulled by STING protein in MCF-7 ATAD3 DKO cells was slightly higher than that in control cells without cGAMP treatment; after cGAMP activated STING protein, the level of STEEP protein bound by STING protein in MCF-7 control cells and DKO cells was higher than that in untreated cells, and the level of STEEP protein bound by STING protein in MCF-7 DKO cells was higher than that in control cells Figure 8A). To further confirm the reliability of our experimental results, we also performed the immunoprecipitation experiment of the reverse pull of STEEP protein on STING protein in MDA-MB-231 control group and DKO group cells under cGAMP treatment. The experimental results are the same as those in the MCF-7 cell line. When there is no cGAMP treatment, the amount of STING protein combined with STEEP protein in MDA-MB-231 DKO group cells is higher than that in control group cells; when cGAMP exists, the amount of STEEP protein combined with STING protein in MDA-MB-231 control group and DKO group cells is increased, and the amount of STEEP protein combined with STING protein in MDA-MB-231 DKO group cells is higher than that in control group cells Figure 8 B). Therefore, we conclude that the deletion of ATAD3 protein leads to the recruitment of more STEEP protein to STING protein, so that STING protein is more likely to "escape" from the endoplasmic reticulum and be transported to the Golgi body.

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

[0240] Example 12, Application of ATAD3 inhibitor in prevention and / or treatment of breast cancer

[0241] The ATAD3 inhibitor can be a substance that inhibits the expression of ATAD3A gene and ATAD3B gene, or silences or knocks out the ATAD3A gene and ATAD3B gene, or a substance that inhibits or reduces the content and / or activity of ATAD3A protein and ATAD3B protein. This embodiment verifies the application of ATAD3 inhibitor (such as sgRNA for knocking out ATAD3A gene and ATAD3B gene, or CRISPR / Cas9 system containing the sgRNA) in prevention and / or treatment of breast cancer by knocking out ATAD3A gene and ATAD3B gene based on CRISPR / Cas9 gene editing system.

[0242] 1. Inhibition of breast cancer cell proliferation in vitro by ATAD3 deletion

[0243] We complemented ATAD3A and ATAD3B in DKO cells of MCF-7 cell line and MDA-MB-231 cell line (MCF-7 DKO cells and MDA-MB-231 DKO cells prepared in Example 2), we infected MCF7 and 231 DKO cell lines with lentivirus carrying ATAD3A and ATAD3B synonymous mutant genes, first we constructed lentivirus expression vector of ATAD3A and ATAD3B synonymous mutant genes. According to the nucleotide sequence of ATAD3A gene (NM_001170535.3), the primers of ATAD3A synonymous mutant gene were designed and synthesized: 3Afrg1-F: 5'-gcgctaccggactcagatctcgagGCCACCatgtcgtggctcttcggcatta-3'; 3Afrg1-R: 5'-ActTttCagTtgTtcGacAgcCgcctcatactctttgagcttg-3' and 3Afrg2-F: 5'-cGgcTgtCgaAcaActGaaAagTgagcagatccgggcgcaggc-3'; 3Afrg2-R: 5'-cTaaCacCgtTtgGcgAtgTtcTgccgccttcaggcggatctg-3', with cDNA of MCF7 cell line as template, 3Afrg1-F and 3Afrg1-R as primers, PCR amplification obtained PCR fragment 3Afrg1, cDNA of MCF7 cell line as 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 fragments were ligated 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), the primers of ATAD3B synonymous mutation gene 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', and the PCR fragment 3Bfrg1 was amplified by PCR with the cDNA of MCF7 cell line as the template and 3Bfrg1-F and 3Bfrg1-R as the primers, and the PCR fragment 3Bfrg1 was amplified by PCR with the cDNA of MCF7 cell line as the template and 3Bfrg2-F and 3Bfrg2-R as the primers; 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. The packaging plasmid (psPAX2 and pMD2) and the backbone plasmid (pLVX-ASM plasmid or pLVX-BSM plasmid) were used to transfect the HEK293T cell line, and after transfection, the cells were cultured for 36 h, without trypsin digestion, the supernatant was collected, the supernatant of pLVX-ASM packaging was labeled as A, the supernatant of pLVX-BSM packaging was labeled as B, and after mixing A and B in a ratio of 1:1 and filtering with a 0.45 μM filter, the virus stock solution was obtained. The virus stock solution was mixed with the cell culture medium in a ratio of 1:4, and then added to the adherent human breast cancer cell line MCF7 or MDA-MB-231 to be infected, and after 24 h of culture, the virus infection solution was discarded, fresh culture medium was added, and after 24 h of continuous culture, a certain concentration of puromycin was added to screen positive cells, which were the rescue cell lines of ATAD3A and ATAD3B.

[0244] The rescue effect was detected by immunoblotting Figure 9 A, B). Then we detected the in vitro proliferation ability of the cell lines, and found that the proliferation ability of the DKO cell line lacking ATAD3 was significantly inhibited compared with the control group, and after the rescue of ATAD3A and ATAD3B, the phenomenon of cell proliferation inhibition was alleviated.

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

[0246] In addition to in vitro experiments, to further verify that ATAD3A and ATAD3B are key factors in tumor development, we also used MCF7-WT, MCF-7DKO, MB231-WT (i.e. MDA-MB-231 wild-type cells), and MB231-DKO (i.e. MDA-MB-231 DKO cells) four cell lines to perform subcutaneous tumor formation experiments in nude mice (Example 8). After three weeks of injection, we detected xenograft tumors in mice. The results showed that MCF-7 cells and MDA-MB-231 cells double-knocked out ATAD3A and ATAD3B completely lost the ability to form tumors in mice Figure 10 ).

[0247] It can be seen that the ATAD3 inhibitor 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 lack of ATAD3 increases the sensitivity of breast cancer cells to exogenous DNA and STING agonists, allowing the STING protein to bind more STEEP proteins and more easily leave the endoplasmic reticulum, 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 inflammation, resulting in the inhibition of the in vitro proliferation of breast cancer cells and the complete loss of the ability to form tumors in vivo.

[0249] The above describes the present application in detail. For those skilled in the art, without departing from the spirit and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in this application.

Claims

1. Use of an ATAD3 inhibitor in any of the following: B1) in the manufacture of a product for preventing and / or treating breast cancer; B2) in the manufacture of a product for inhibiting proliferation of breast cancer cells; B3) in the manufacture of a product for inhibiting tumorigenic capacity of breast cancer cells; B4) in the manufacture of a product for inhibiting tumorigenesis or growth of breast cancer; The ATAD3 inhibitor is a targeted... ATAD3A Genes and ATAD3B The sgRNA composition of a gene or a CRISPR / Cas9 system containing the sgRNA composition, wherein the sgRNA composition comprises sgRNA1 and sgRNA2, wherein the target sequence of sgRNA1 is shown in SEQ ID NO:1; and the target sequence of sgRNA2 is shown in SEQ ID NO:

2.

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

3. The biomaterial of claim 2, wherein, The recombinant microorganism is a recombinant virus.

4. Use of the biological material as described in any of F2) - F5) of claim 2 in any of the following: B1) in the manufacture of a product for preventing and / or treating breast cancer; B2) in the manufacture of a product for inhibiting proliferation of breast cancer cells; B3) in the manufacture of a product for inhibiting tumorigenic capacity of breast cancer cells; B4) in the manufacture of a product for inhibiting tumorigenesis or growth of breast cancer.

5. A pharmaceutical composition for preventing and / or treating breast cancer, characterized by, The pharmaceutical composition comprises the ATAD3 inhibitor as described in claim 1.

Images