Application of MYO1F or Myo1f as target spot in preparation of products for diagnosing or treating tumors

Abstract

The invention discloses an application of MYO1F or Myo1f as a target spot in preparation of a product for diagnosing or treating tumors. By up-regulating the expression quantity of mRNA of Myo1f or MYO1F, amplification and migration infiltration of MDSCs can be inhibited, and immune checkpoint blocking is carried out on tumors. MYO1F and Myo1f are used as new targets for tumor immunotherapy, so that the tumor diagnosis and treatment thought is expanded, and a solid technical foundation is laid for solving the tumor treatment problem.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of MYO1F or Myo1f as a target in the preparation of products for diagnosing or treating tumors. Background Art

[0002] Immune checkpoints are immunosuppressive molecules that are expressed on immune cells and regulate the degree of immune activation. They are typically represented by programmed death receptor (PD-1) and its ligand PD-L1, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), etc. In the development of cancer, immune checkpoints inhibit T cell function and achieve immune escape. Therefore, blocking immune checkpoints and restarting the tumor immune cycle are important methods of immunotherapy. The development of immune checkpoint blockade (ICB) therapy is a revolutionary milestone in cancer treatment. However, for most cancer patients, ICB still does not achieve the expected therapeutic effect, and the overall pan-cancer remission rate of PD-1 inhibitors alone is only 20%. Therefore, further exploration and analysis of the mechanism of action of ICB is of great significance to promote the development of tumor immunotherapy.

[0003] The expansion of myeloid-derived suppressor cells (MDSCs) and their infiltration into the tumor microenvironment are the main obstacles to T cell anti-tumor immunity, and therefore become one of the key factors in ICB response. Currently, many studies have pointed out that MDSCs secrete a variety of substances that inhibit T lymphocyte immunity in the tumor microenvironment, and use a variety of molecular channels to enable tumor tissues to escape the body's immune surveillance, directly or indirectly promoting tumor growth, invasion and metastasis. (Chen Hongyan, Guo Hong. MDSCs and Tumor Microenvironment [J]. Journal of Immunology, 2015, 31(11): 996-1000).

[0004] The applicant's study also found that myosin MYO1F (MyosinIF) is closely related to ICB response in clinical and animal models, and that MYO1F deficiency directly promotes MDSCs expansion, while the expression of the MYO1F encoding gene Myo1f itself is also tightly regulated by upstream tumor microenvironment factors.

[0005] However, there are no reports on how MYO1F regulates the proliferation and function of MDSCs, thereby affecting the ICB response rate. Therefore, in-depth research on the molecular mechanism of MYO1F in the tumor immunosuppression circuit is of great significance for discovering new tumor immunotherapy targets for MDSCs, developing new tumor diagnosis and treatment programs, and improving the efficacy of clinical immunotherapy. Summary of the invention

[0006] In order to fully elucidate the molecular mechanism by which MYO1F regulates the expansion and function of MDSCs and develop new methods for tumor immunotherapy, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides the use of MYO1F or its encoding gene Myo1f as a biomarker or target in the preparation of products for diagnosis, monitoring severity assessment, efficacy assessment, prognosis assessment, prevention and / or treatment of tumors.

[0008] Preferably, the tumor includes but is not limited to liver cancer, colon cancer, breast cancer, pancreatic cancer, esophageal cancer, melanoma, non-small cell lung cancer, adrenocortical carcinoma (ACC), diffuse large B-cell lymphoma (DLBC), prostate cancer (PRAD), rectal cancer (READ) and endometrial carcinoma (UCEC).

[0009] Preferably, the diagnosis, monitoring, severity assessment, efficacy assessment or prognosis assessment of the tumor includes detecting the expression level of MYO1F, the presence or absence of Myo1f and / or the expression level of Myo1f mRNA.

[0010] Preferably, the detecting of Myo1f mRNA and / or MYO1F expression level comprises using a reagent.

[0011] Furthermore, the reagents may include but are not limited to reagents required for PCR, reagents required for RPA, reagents required for LAMP, reagents required for ERA, reagents required for RCA, reagents required for Western blot, reagents required for immunohistochemistry, reagents required for sequencing, reagents required for liquid phase or reagents required for mass spectrometry, etc.

[0012] Preferably, when the expression level of Myo1f mRNA and / or MYO1F is significantly lower than a threshold, it indicates that the individual is a tumor patient, wherein the threshold is obtained in an early stage through experiments.

[0013] Preferably, the prevention or diagnosis of tumors includes upregulating the expression level of MYO1F and / or the expression level of Myo1f mRNA.

[0014] Preferably, the MYO1F and / or Myo1f are MYO1F and / or Myo1f in MDSCs, and further, the MDSCs are PMN-MDSCs.

[0015] Preferably, the product comprises at least one of a reagent for detecting the presence of Myo1f, detecting the mRNA expression level of Myo1f, detecting the expression level of MYO1F, up-regulating the mRNA expression level of Myo1f, and up-regulating the expression level of MYO1F.

[0016] In a specific embodiment of the present invention, the product includes a drug, or a kit, test paper, mass spectrometer or biochip for tumor diagnosis, monitoring, severity assessment, efficacy assessment and / or prognosis assessment.

[0017] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0018] Preferably, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a diluent, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a solvent, a pH adjuster, a buffer, an antioxidant, a metal ion chelator, an antibacterial agent or an isotonicity adjuster.

[0019] Preferably, the drug can be in the form of a suspension, powder, granule, tablet, aqueous solution, cream, gel or emulsion. The various dosage forms of the drug can be prepared according to conventional production methods in the pharmaceutical field.

[0020] Preferably, the pharmaceutical preparation is a unit dose preparation.

[0021] Preferably, the active ingredient contained in the drug (the agent for increasing the expression of Myo1f mRNA and / or increasing the expression of MYO1F) is calculated by volume or mass ratio to include any value between 0.001% and 99.9%, for example, 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.1, 99.5, 99.9%, etc.

[0022] According to the requirements of the specific implementation, the medicine may also contain other suitable therapeutic agents.

[0023] The drug is suitable for parenteral administration such as, for example, preparations administered by intravenous, intramuscular, intradermal and subcutaneous routes include aqueous and non-aqueous isotonic sterile injections, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the recipient's blood, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers or preservatives. The preparation may be present in unit dose or multi-dose sealed containers such as ampoules and vials. Solutions and suspensions for injection may be prepared from sterile powders, granules and tablets of the types previously described.

[0024] Preferably, the administration method of the drug includes but is not limited to intravenous infusion, local administration, intraperitoneal administration or intrathecal administration.

[0025] Preferably, the kit, test paper, mass spectrometer or biochip specifically detects the mRNA expression level of Myo1f and / or the expression level of MYO1F, and preferably comprises a reagent for detecting the mRNA expression level of Myo1f and / or the expression level of MYO1F.

[0026] Preferably, the kit is selected from an immunomagnetic bead detection kit, an agglutination detection kit, a liquid phase chip detection kit, an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a mass spectrometry detection kit.

[0027] The kit also includes a diluent, a cleaning solution, a buffer, a substrate and / or a stop solution.

[0028] Preferably, the MYO1F is used to diagnose or treat tumors by immune checkpoint blockade.

[0029] Furthermore, MYO1F inhibits the expansion of MDSCs.

[0030] Preferably, the MYO1F inhibits STAT3 phosphorylation.

[0031] Furthermore, MYO1F binds to TRIM21, causing ubiquitin modification of downstream TRIM21 target proteins, thereby inhibiting STAT3 phosphorylation.

[0032] Preferably, TGF-β1 regulates the transcription and expression of Myo1f by affecting its DNA methylation modification.

[0033] Furthermore, the TGF-β1 reduces the transcription level of Myo1f.

[0034] More preferably, the TGF-β1 blocks the binding of SPI1 to the Myo1f intron through DNA methylation, thereby inhibiting the transcriptional expression of Myo1f.

[0035] Preferably, SPI binds to one or more of the 1st intron, 8th intron, 21st intron or 27th intron of Myo1f.

[0036] In a second aspect, the present invention provides use of an agent targeting MYO1F or Myo1f in MDSCs in the preparation of a drug for treating tumors, wherein the agent causes Myo1f, Myo1f mRNA and / or MYO1F in MDSCs to overexpress or increases the activity of MYO1F.

[0037] Preferably, the reagent is selected from:

[0038] 1) a viral vector, wherein the viral vector overexpresses MYO1F;

[0039] 2) small molecule drugs that increase Myo1f, Myo1f mRNA, and / or MYO1F expression in MDSCs;

[0040] 3) Small molecule drugs or antibodies that enhance MYO1F activity.

[0041] Preferably, the tumor includes, but is not limited to, liver cancer, colon cancer, breast cancer, pancreatic cancer, esophageal cancer, adrenocortical carcinoma, diffuse large B-cell lymphoma, prostate cancer, rectal cancer and endometrial cancer.

[0042] In a third aspect, the present invention provides a method for inhibiting the infiltration of MDSCs in a tumor, the method comprising treating MDSCs with an agent, wherein the agent causes Myo1f overexpression, Myo1f mRNA overexpression or increases the activity of MYO1F in MDSCs.

[0043] Further, the reagent is selected from:

[0044] 1) a viral vector, wherein the viral vector expresses MYO1F;

[0045] 2) Small molecule drugs that increase the expression of Myo1f and / or Myo1f mRNA in MDSCs;

[0046] 3) Small molecule drugs or antibodies that enhance MYO1F activity in MDSCs

[0047] Furthermore, the reagent is a viral vector, and the viral vector expresses MYO1F. More preferably, the viral vector is a retroviral vector.

[0048] In a fourth aspect, the present invention provides a biomarker or target for a tumor, wherein the biomarker or target is Myo1f and / or MYO1F.

[0049] In a fifth aspect, the present invention provides a method for screening drugs for preventing or treating tumors, the method comprising treating a sample with the drug to be tested and then detecting the mRNA and / or MYO1F expression level of Myo1f.

[0050] In a sixth aspect, the present invention provides a method for constructing a tumor animal model, wherein the construction method comprises upregulating the expression level of MYO1F and / or the expression level of Myo1f mRNA.

[0051] In a seventh aspect, the present invention provides a method for diagnosing, monitoring, assessing severity, evaluating efficacy and / or evaluating prognosis of a tumor.

[0052] Preferably, the method comprises detecting the presence of Myo1f or the expression level of its mRNA and / or MYO1F.

[0053] When the expression level of MYO1F is below the threshold, it indicates disease or high risk.

[0054] In an eighth aspect, the present invention provides a method for preventing and / or treating tumors.

[0055] Preferably, the method comprises upregulating the expression level of Myo1f mRNA and / or MYO1F.

[0056] Beneficial effects of the present invention:

[0057] The present invention reveals that by upregulating the mRNA of Myo1f or the expression of MYO1F, the proliferation and migration and infiltration of MDSCs can be inhibited, thereby blocking the immune checkpoints of the tumor. Using MYO1F and Myo1f as new targets for tumor immunotherapy expands the ideas of tumor diagnosis and treatment, and lays a solid technical foundation for solving the difficult problems of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 It is a schematic diagram of the present invention, wherein A indicates that MDSCs expansion leads to low ICB response, and B indicates the molecular mechanism by which Myo1f regulates MDSCs expansion and function;

[0059] Figure 2 The figure shows that the Myo1f expression level is closely related to the ICB response, where A represents the expression level of Myo1f in tumor and adjacent cancer tissues of clinical samples, ACC (Adrenocortical carcinoma), DLBC (Lymphoid NeoplasmDiffuse Large B-cell Lymphoma), PRAD (Prostate adenocarcinoma), READ (Rectumadenocarcinoma), and UCEC (Uterine Corpus Endometrial Carcinoma); B represents the Myo1f gene expression level and the corresponding survival rate; C represents the correlation between the clinical ICB treatment response and the expression levels of Myo1f and Myo1e. #1: melanoma, PD-1 and CTLA-4 antibody combination; #2: melanoma, PD-1 antibody; #3: non-small cell lung cancer, PD-1 antibody; #4: melanoma, renal adenocarcinoma, PD-1 and CTLA-4 antibody combination; #5-#11: melanoma; PD-1, PD-L1 and CTLA-4 antibody combination and CTLA-4 antibody alone; D shows the effect of Myo1f gene knockout on the therapeutic effect of ICB in mice;

[0060] Figure 3The figure shows the effect of Myo1f deficiency on tumor progression, where A represents the tumor tissue and spleen size and tumor and spleen weight data of B16F10 tumor-bearing mice; B represents the survival curve of B16F10 tumor-bearing mice; C represents the tumor tissue and spleen size and tumor and spleen weight data of B16F10 tumor-bearing mice; D represents the survival curve of MC38 tumor-bearing mice;

[0061] Figure 4 The figure shows Myo1f-mediated MDSCs expansion, where A shows flow cytometry detection of WT and Myo1f-inoculated B16F10 cells. - / - MDSCs (CD11b + Gr1 + ) and their PMN-MDSCs (Ly6G + Ly6C - ) and M-MDSCs (Ly6G - Ly6C + ) The proportion of subpopulations; B represents flow cytometry detection of WT and Myo1f - / - T cells infiltrating in tumor tissues of tumor-bearing mice; C indicates flow cytometry detection of WT and Myo1f - / - MDSCs in peripheral blood and spleen of tumor-bearing mice; D indicates flow cytometry detection of bone marrow MDSCs (CD11b + Gr1 + ) and non-MDSCs (CD11b - Gr1 - ) Ki67 expression level in ;

[0062] Figure 5 The figure shows that Myo1f deficiency mediates high expression of PD-L1 in MDSCs, where A represents flow cytometry detection of PD-L1 expression levels on bone marrow MDSCs; B represents flow cytometry detection of PD-L1 expression levels in MDSCs with Myo1f knockdown; C represents flow cytometry sorting of tumor-infiltrating MDSCs; D represents WT and Myo1f - / - The sorted MDSCs were co-cultured with T cells, and the CD8+ T proliferation signal CellTrace Violet was detected by flow cytometry, and the IFN-γ secretion level was detected by ELISA;

[0063] Figure 6 The figure shows the effect of Myo1f on tumor progression by regulating MDSCs, where A is the flow chart of bone marrow adoptive therapy; B is the size of melanoma and tumor growth curve after MDSCs adoptive therapy; C is the flow cytometry detection of tumor infiltrating CD8 + T cell activation signal; D shows the melanoma size and growth curve at the end point of SX682-treated tumor-bearing mice; E shows the flow cytometry detection of MDSCs infiltration in tumors of SX682-treated tumor-bearing mice;

[0064] Figure 7 The figure shows the binding of MYO1F and TRIM21, where A indicates the TRIM21 information in the mass spectrometry results of MYO1F; B indicates exogenous overexpression of Flag-TRIM21 and HIS-MYO1F, and the detection of IPed His-MYO1F by the Flag co-IP method, and gel beads are added to exclude non-specific binding of gel beads; C indicates MDSCs cells are IPed with endogenous TRIM21 using TRIM21 antibody, and Western Blot is used to detect IPed MYO1F;

[0065] Figure 8 The figure shows the binding of MYO1F and TRIM21, where A indicates the co-localization of MYO1F and TRIM21 in MDSCs detected by confocal microscopy; B indicates the binding of MYO1F and TRIM21 detected by endogenous IP in MDSCs cells; C indicates the binding of Flag-MYO1F and His-TRIM21 detected by co-IP after overexpression of Flag-MYO1F and His-TRIM21 in vitro;

[0066] Fig. 9 The figure shows that TGF-β1 in the tumor microenvironment regulates Myo1f intron and SPI1-mediated Myo1f downregulation through DNA methylation modification, where A represents tumor modeling by subcutaneous implantation of MC38 cells; B represents flow cytometry detection of bone marrow MDSCs proliferation; C represents flow cytometry detection of MYO1F expression levels in MDSCs and non-MDSCs cells in the bone marrow; D represents Western Blot detection of MYO1F protein levels in MDSCs cells induced in vitro by cell line supernatant medium; E represents qPCR detection of Myo1f transcription levels in MDSCs cells stimulated by mixed medium of tumor cell line B16F10 and MC38; F represents ELISA detection of TGF-β1 levels in various cell lines; G represents the correlation between TGFB1 and Myo1f expression in more than 300 human tumor cell lines; H represents the methylation sequencing data of Myo1f gene in mouse liver cells in the MethBank database; I represents the magnified display of CG methylation modification of introns 1, 8, 21, and 27 of the Myo1f gene;

[0067] Fig.10The figure shows that tumor-derived TGF-β1 down-regulates Myo1f expression through a DNA methylation-related pathway, where A represents the MYO1F protein expression level after 24 hours of in vitro induction of MDSCs by multiple cytokines, Medium represents the supernatant of tumor cell lines B16F10 and MC38, 1 represents IL-6, 2 represents IL-10, 3 represents CSF, 4 represents GM-CSF, 5 represents MCSF, 6 represents VEGF, 7 represents KC, 8 represents MCP1, 9 represents MIG, 11 represents IL1b, 12 represents MIP, and 13 represents IL-4; B represents the effect of concentration gradient TGF-β1-induced MDSCs on the MYO1F protein expression level; C represents flow cytometry detection of TGF-β1-induced MDS Cs shows the effect on Myo1f transcriptional expression; D shows the effect of TGF-β1-induced CFSE-labeled MDSCs proliferation detected by flow cytometry; E shows the effect of TGF-β1-induced MDSCs on Dnmt1 transcription; F shows the effect of TGF-β1-induced MDSCs on DNMT1 enzyme activity; G shows the effect of DNMTs inhibitor SGI on MYO1F protein expression in TGF-β1-induced MDSCs; H shows the effect of DNMTs inhibitor SGI on Myo1f gene transcription in TGF-β1-induced MDSCs. DETAILED DESCRIPTION

[0068] The present invention is further described below in conjunction with the accompanying drawings and specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed according to conventional conditions in the art or according to conditions recommended by the manufacturer. Unless otherwise specified, all conventional methods are used. Unless otherwise defined, the professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art.

[0069] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0070] The "prognosis assessment" mentioned in the present invention refers to predicting the possible course and outcome of a disease, including determining specific consequences of the disease (such as recovery, the appearance or disappearance of certain symptoms, signs, complications and other abnormalities, and death).

[0071] The "evaluation of therapeutic efficacy" described in the present invention refers to the evaluation of a patient's response to treatment.

[0072] The "monitoring" mentioned in the present invention refers to observing the occurrence and development of a disease.

[0073] The term "prevention" as used in the present invention refers to an individual taking specific measures to prevent the occurrence of a disease before the disease is diagnosed or develops.

[0074] The term "diagnosis" in the present invention refers to finding out whether a patient has a disease or condition in the past, at the time of diagnosis, or in the future, or to finding out the progression or possible future progression of a disease.

[0075] The term "pharmaceutically acceptable" as used herein refers to a substance that neither significantly irritates an organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0076] Some of the sources of materials and experimental methods involved in this application are as follows:

[0077] 1. C57BL / 6WT and Myo1f - / - Construction of mouse melanoma cell B16F10 and MC38 tumor models.

[0078] 2. Construction of bone marrow ablation model: 6-7 weeks old C57BL / 6 background CD45.2 + The recipient mice were subcutaneously inoculated with 2×10^5 B16F10 cells / mouse 12 days before adoptive transfer. The mice with tumors that were too large or too small were eliminated, and the mice with tumors that were uniform were randomly grouped. The tumor volume was controlled at 100-150mm at the beginning of the experiment. 3 . The mice were irradiated with 2 Gy × 3 times for myeloablation (using a tumor lead shielding module) one day before adoptive transfer, and the SPF environment was stabilized for one day.

[0079] 3. Bone marrow adoptive transfer method: Femurs and tibias were taken from 6-7 week old C57BL / 6 background mice 12 days after tumor inoculation. After rinsing with pre-cooled 1×PBS, they were filtered with a 40μm filter, centrifuged at 400g for 5 minutes, red blood cells were lysed, and washed twice with pre-cooled 1×PBS. The cells were sorted using a mouse MDSCs negative selection isolation kit. After sorting, the cells were washed twice with pre-cooled 1×PBS and added to PBS to prepare a 1×10^7 cell / mL suspension. Orbital vein injection or tail vein injection was performed alternately every 7 days, 1×10^6 cells / mouse. Samples were collected from recipient mice according to tumor progression during 24 days. The specific experimental groupings are as follows:

[0080] (1) Tumor-bearing WT-donor CD45.1 + Bone marrow MDSCs → receptor CD45.2 + Tumor-bearing mice;

[0081] (2) Tumor-bearing Myo1f - / - Donor CD45.1 + Bone marrow MDSCs → receptor CD45.2 + Tumor-bearing mice;

[0082] (3) Donor CD45.1 bearing Myo1ff / fΔS100A8 + Bone marrow MDSCs → receptor CD45.2+ Tumor-bearing mice;

[0083] (4) Tumor-bearing WT-donor CD45.1 + Tumor microenvironment MDSCs → receptor CD45.2 + Tumor-bearing mice;

[0084] (5) Tumor-bearing Myo1f - / - Donor CD45.1 + Tumor microenvironment MDSCs → receptor CD45.2 + Tumor-bearing mice;

[0085] (6) Donor CD45.1 bearing Myo1ff / fΔS100A8 + Tumor microenvironment MDSCs → receptor CD45.2 + Tumor-bearing mice.

[0086] 4. Effect of Myo1f deficiency on PMN-MDSCs expansion in ICB response

[0087] 4.1 In vivo tumor growth functional verification

[0088] Construct WT and Myo1f - / - , Myo1ff / fΔS100A8 mouse melanoma and colon cancer models were established, and the tumor growth curve was monitored. Samples were collected after 14 days, and the infiltration of MDSCs and the phosphorylation level of STAT3 in the bone marrow and tumor tissue were detected by flow cytometry. The bone marrow was stained with immunofluorescence to detect the proliferation and inhibitory function of MDSCs from the perspective of visual imaging.

[0089] 4.2 Detection of the effect of Myo1f deficiency on the proliferation and immunosuppressive ability of PMN-MDSCs

[0090] Using Myo1f-deficient MDSCs cells under different conditions, siRNA / shRNA technology was used to construct Myo1f-knockdown MDSCs cells, and the normal and tumor-bearing WT and Myo1f- - / - MDSCs cells from Myo1ff / fΔS100A8 mice were co-incubated with CSFE-labeled T cells at different time points, and flow cytometry was used to detect T cell effector indicators (such as IFN-γ, TNF-α, CD69, etc.) and CSFE proliferation indicators, and MDSCs Ki67, STAT3, PD-L1 and other indicators; Western Blot was used to detect the phosphorylation level of STAT3; ELISA was used to detect the expression levels of inflammatory factors such as IFN-γ, TNF-α, IL-2, TGF-β1 in the incubation supernatant.

[0091] 4.3 Detection of the effect of Myo1f deficiency on PMN-MDSCs migration ability

[0092] (1) Adoptive transfer of PMN-MDSCs

[0093] Construct CD45.2 + Background irradiation myeloablation mouse recipient model, based on which B16F10 or MC38 cells were inoculated to induce tumor formation; CD45.1 was sorted by flow cytometry + Background WT and Myo1f - / - PMN-MDSCs of Myo1ff / fΔS100A8 mice or directly isolated bone marrow cells were adopted to myeloablated tumor-bearing mice to monitor tumor growth curves. Flow cytometry was used to detect the number and proportion of bone marrow and tumor-infiltrating MDSCs and functional indicators such as Ki67, STAT3, and PD-L1. ELISA was used to detect changes in inflammatory factors: tumor tissue was collected, cryogenically ground and centrifuged at high speed, and the supernatant was obtained. ELISA was used to detect the expression levels of inflammatory factors such as IFN-γ, TNF-α, IL-2, and TGF-β1.

[0094] (2) Treatment of mouse subcutaneous tumor model with MDSCs migration inhibitor

[0095] WT and Myo1f - / - After B16F10 or MC38 tumors were inoculated in Myo1ff / fΔS100A8 mice, the CXCR1 / 2 inhibitor SX-682 was orally administered to inhibit MDSCs migration, the tumor growth curve was monitored, and the infiltration of MDSCs in the blood and tumors was detected by flow cytometry.

[0096] 4.4 Effect of Myo1f on PMN-MDSCs proliferation and ICB response

[0097] WT and Myo1f - / - After B16F10 or MC38 tumors were inoculated in , Myo1ff / fΔS100A8 mice, ICB treatments such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 were performed. The tumor growth curve was monitored, and the number and proportion of bone marrow and tumor-infiltrating MDSCs and functional indicators such as Ki67, STAT3, and PD-L1 were detected by flow cytometry.

[0098] 5. Molecular mechanism of MYO1F regulating STAT3 activation by competing with TRIM21

[0099] 5.1 Study on the authenticity of the binding between MYO1F and TRIM21

[0100] (1) Co-IP exogenous identification system: MYO1F and TRIM21 with His or Flag tags were co-transfected through 293T. After protein lysis, His or Flag beads were used to pull up the target protein, and the authenticity of the binding between the two was detected by the forward and reverse co-IP methods.

[0101] (2) FRET exogenous identification system: 293T cells overexpressed CFP, YFP, and CFP / YFP fusion proteins of MYO1F and TRIM21, and the authenticity of their binding was detected by spectral laser scanning confocal microscopy receptor photobleaching FRET technology.

[0102] (3) Co-IP MDSCs endogenous identification system: MDSCs cells were sorted and lysed to obtain protein supernatant. The authenticity of the binding between the two endogenous proteins was detected by anti-MYO1F or anti-TRIM21 forward and reverse co-IP methods.

[0103] (4) Immunofluorescence co-localization MDSCs endogenous identification system: MDSCs were immunofluorescently stained with MYO1F and TRIM21, and the co-localization information of the two was obtained by laser scanning confocal microscopy.

[0104] 5.2 Molecular cloning and identification of MYO1F and TRIM21 binding sites

[0105] (1) Using point mutagenesis technology, MYO1F or TRIM21 was domain-specifically deleted, and the mutants were used to construct eukaryotic and prokaryotic overexpression vectors.

[0106] (2) The co-IP and GST-pulldown methods were used to identify the binding of MYO1F mutants to the full-length TRIM21 sequence, and the TRIM21 binding region on MYO1F was screened out and named MYO1F-Domain.

[0107] (3) The co-IP and GST-pulldown methods were used to identify the binding of TRIM21 mutants to the full-length MYO1F sequence, and the MYO1F-binding region on TRIM21 was screened out and named TRIM21-Domain.

[0108] (4) The MYO1F-Domain in the full-length sequence was mutated to alanine in small fragments of 10 amino acids, and the co-IP method was used to detect the binding ability with the full-length sequence TRIM21.

[0109] (5) The TRIM21-Domain in the full-length sequence was mutated to alanine in small fragments of 10 amino acids, and the co-IP method was used to detect the binding ability with the full-length sequence MYO1F.

[0110] (6) Screening out small mutation fragments and further refining them into single amino acid to alanine mutations until one or more core amino acid binding sites are identified.

[0111] 5.3 Molecular cloning and identification of the binding site between TRIM21 and target protein X

[0112] (1) Technology and detection methods refer to step 6.2.

[0113] (2) Compare the binding region of TRIM21 with the target protein X, comprehensively analyze whether it is close to the MYO1F binding site, and evaluate the possibility of competitive binding.

[0114] 5.4 Effect of MYO1F competition with TRIM21 on ubiquitination of target protein X

[0115] (1) MYO1F concentration gradient overexpression system: 293T cells overexpressed TRIM21 and related mutants, target protein X, and Ubiquitin to construct a ubiquitin modification system. On this basis, MYO1F was gradiently overexpressed, and the co-IP method was used to detect the binding ability of MYO1F to TRIM21 and the ubiquitination degree of target protein X.

[0116] (2) MDSCs endogenous modification system: MDSCs knockdown of MYO1F or TRIM21 for endogenous target protein X ubiquitination co-IP; sorting of WT and Myo1f - / - Mouse bone marrow MDSCs, detecting target protein X ubiquitination.

[0117] 5.5 Effect of MYO1F competition with TRIM21 on affinity of target protein X

[0118] Microthermophoresis molecular interaction technology (MST) was used to detect affinity: prokaryotic expression and purification of MYO1F, TRIM21, and candidate target protein X were constructed. TRIM21 and target protein X were labeled and quantitatively co-incubated to form a binding, and then non-labeled MYO1F protein with gradient concentrations was added. MST was used to detect the affinity curve and affinity dissociation constant Kd value.

[0119] (1) Construct pET28a-His-TRIM21 and prokaryotic overexpression vectors, construct pET28a-target protein X gene and pET28a-MYO1F (or MYO1F-Domain, refer to research content 2.1), and commercially purify the protein for use.

[0120] (2) His-tagged TRIM21 and target protein X were dissolved in 1×PBS-T solution to prepare target protein X solution; the dye RED-tris-NTA 2 nd Generation dye was dissolved in 1×PBS-T to prepare dye solution.

[0121] (3) Place TRIM21 and target protein X solution in the first well of the PCR strip tube, add 1× PBS-T to the remaining wells for dilution, and dilute the target protein X solution into a 1:1 gradient by evenly pipetting. Set up a control well.

[0122] (4) Add an equal amount of dye solution to each well and incubate at room temperature for 30 minutes; use the matching glass capillary to draw the liquid from each well until the center of the capillary is filled without bubbles, arrange them regularly in the sample injection plate and put them on the machine, and measure the affinity with 40% LED / excitation power / 40% MST power parameters, and calculate the equilibrium dissociation constant Kd by fitting. Add an equal amount of MYO1F protein to each well (set a dose for each experimental group, and set a gradient experimental group) to evaluate the effect of MYO1F on the affinity of TRIM21 / target protein X.

[0123] 5.6MYO1F regulates target protein X and STAT3 signaling

[0124] (1) Cell system: Construct Myo1f knockdown or overexpression vectors, prepare virus-infected MDSCs cells, perform immunofluorescence staining on MDSCs cells, use confocal microscopy to detect the colocalization of target protein X and STAT3, and detect STAT3 phosphorylation by flow cytometry.

[0125] (2) In vivo experimental system: WT and Myo1f - / - Mouse and WT with Myo1f - / - The co-localization of target protein X and STAT3 was detected by immunofluorescence confocal microscopy in the bone marrow of tumor-bearing mice, and STAT3 phosphorylation was detected by flow cytometry.

[0126] 6. Molecular mechanism of TGF-β1 regulating MYO1F transcriptional expression through epigenetic modification and introns during tumor progression

[0127] 6.1 SPI1-CHIPseq detection of MDSCs

[0128] Femurs of MC38 tumor-bearing and non-tumor-bearing mice were taken, and the two ends were removed and the bone marrow was washed with pre-cooled PBS; centrifuged at 1000rpm×3min, and the supernatant was removed; 5mL of lysate was added and lysed at room temperature for 5min; centrifuged at 1000rpm×3min, and the supernatant was removed and then stained with dead and live dyes, CD11b, and Gr1. CD11b was sorted by flow cytometry. + Gr1 + For MDSCs subpopulation, the cells were lysed to obtain protein supernatant, and then endogenous IP was performed using SPI1 antibody. The protein obtained by IP was detected by mass spectrometry to analyze the tumor-bearing effect and screen potential binding sites of SPI1.

[0129] 6.2 Dual luciferase reporter system to detect the binding interaction between SPI1 and Myo1f intron

[0130] (1) Based on the SPI1-CHIPseq results and related literature, we designed and cloned the 500 bp around the SPI1 binding peak into the pGL3-Basic vector, and knocked down or overexpressed Spi1 in MDSCs cells to detect the promoter activity.

[0131] (2) Clone the 500 bp around the SPI1 binding peak into the pGL3-Promoter vector, and knock down or overexpress Spi1 in MDSCs cells to detect enhanced activity.

[0132] (3) Screening the target fragment, mutating the core binding sequence, and then testing the activation activity and enhancement activity to confirm whether the fragment is the SPI1 binding sequence.

[0133] 6.3 Detection of the binding ability of intronic DNA to SPI1 by EMSA

[0134] EMSA experiment is a special gel electrophoresis technique for analyzing the interaction between DNA and protein in vitro. Its principle is that after the protein binds to the nucleic acid molecule, its relative molecular mass will be greatly increased, and the mobility of nucleic acid in gel electrophoresis is proportional to its relative molecular mass. Therefore, the nucleic acid fragments that are not bound to the protein migrate quickly, while the nucleic acid that forms a complex with the protein migrates slowly.

[0135] (1) Sample preparation: Nuclear proteins of MDSCs treated and untreated with TGF-β1 were extracted using a nuclear extraction kit. The total protein amount was determined by BCA colorimetry. SPI1 protein was quantified by Western Blot combined with SPI1. Equal amounts of protein were added in the experiment.

[0136] (2) Probe preparation: Based on the SPI1 binding position information and Myo1f intron binding site data from the previous SPI1-CHIPseq, one double-stranded DNA probe and one single-stranded DNA probe were designed near the DNA site where SPI1 binds Myo1f. The probes were synthesized and biotin-labeled, and then purified.

[0137] (3) Forming a protein-probe complex: In a 0.5 ml centrifuge tube, mix the following components in order: protein sample (2-5 μg) × μL; poly d(IC) 1 μL; Binding Buffer 2 μL; Nuclease-Free ddH2O × μL; total volume 9 μL; after ice bathing for 5 minutes, add 1 μL of probe; incubate at room temperature (20-23°C) in a PCR instrument for 30 minutes.

[0138] (4) Prepare 6.5% non-denaturing polyacrylamide gel, spot electrophoresis, transfer to membrane, and expose for imaging using a chemiluminescence imaging system.

[0139] 6.4 Identification of intron methylation modification of MDSCs by TGF-β1

[0140] (1) MDSCs cells were treated with TGF-β1 in vitro for 0, 12, 24, and 48 hours. Samples were collected at each time point and the genome of each sample was extracted using a total DNA extraction kit.

[0141] (2) Take 5 μg of genomic DNA and dilute it to 50 μL with sterile water. Add NaOH (final concentration is 0.2 M) and denature it in a 42°C water bath for 30 min. Add 30 μL of 10 mM hydroquinone and 520 μL of 3 M sodium bisulfite (pH = 5.0) to the mixture and mix by inversion. Add 200 μL of paraffin oil to prevent water evaporation and limit oxidation. Incubate in a 50°C water bath in the dark for 16 hrs.

[0142] (3) The modified DNA was purified by column purification using a DNA purification kit, recovered and dissolved in 50 μl of sterile double distilled water, and the concentration was measured.

[0143] (4) Design BSP-PCR primers at the 300-500 bp position near the SPI1 intron binding site and perform amplification. Obtain the PCR amplification product and perform agarose gel electrophoresis and gel recovery to obtain the purified fragment for sequencing.

[0144] (5) Bisulfite sequencing PCR (BSP) sequencing was used to analyze the methylation sites of TGF-β1 in the key regulatory intron region of MYO1F.

[0145] 6.5 Verification of TGF-β1-induced methylation modification sites in MDSCs

[0146] (1) MDSCs were sorted and the methylation of MDSCs was intervened by inhibitors (such as SGI-1027, a DNA methyltransferase inhibitor). The methylation effect of TGF-β1 on Myo1f was verified by EMSA and luciferase reporter assay.

[0147] (2) We constructed a MYO1F methylation site mutation overexpression vector and prepared virus-infected MDSCs cells. We combined the binding information of SPI1 and MYO1F and used inhibitors (such as DNA methyltransferase inhibitor SGI-1027) to intervene in the methylation of MDSCs cells. We used EMSA experiments and SPI1-CHIPseq to detect the effect of TGF-β1 on the binding of SPI1 to introns in regulating the DNA methylation of the Myo1f gene. We also used q-PCR to detect the effect of TGF-β1 on the transcriptional regulation of the Myo1f gene.

[0148] according to Figure 1 The following experiments were performed according to the scheme in .

[0149] Example 1 Role of MYO1F in Tumor Progression and ICB Response

[0150] The TCGA (The Cancer Genome Atlas, TCGA) database was used to analyze the expression levels of My1f and My1e in clinical samples, and Myo1e, which is highly homologous to Myo1f, was used as a reference. The analysis results showed that Myo1f was significantly decreased in a variety of malignant tumors (such as colon adenocarcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, and pancreatic cancer). Figure 2 A), and Myo1f expression levels are negatively correlated with the survival rate of various clinical malignant tumors ( Figure 2 B). Further analysis of the ICB therapy transcriptome resource database ICBatlas (bioinfo.life.hust.edu.cn) revealed that the MYO1F level in patients with clinical ICB therapy resistance was significantly lower than that in patients with ICB response, while MYO1E, the isotype control of MYO1F, did not show a corresponding trend ( Figure 2 C), indicating that MYO1F may be a key functional molecule in ICB response. - / - The mice were treated with ICB and found that Myo1f deficiency in the mouse subcutaneous tumor model also severely weakened the anti-tumor immune effect of ICB in mice ( Figure 2 D).

[0151] C57BL / 6WT and Myo1f - / - The mice were treated with melanoma cell B16F10 and MC38 tumor models, and the tumor growth data showed that Myo1f - / - Mice have stronger tumor formation ability, accompanied by spleen enlargement and reduced survival rate ( Figure 3). After the two mouse melanoma cell B16F10 models were established, 10 mg / kg of anti-PD-1 or anti-CTLA-4 was administered every 2 days, and the tumor growth curve was monitored during the treatment. The treatment experiment showed that WT mice had a significant response to anti-PD-1 or anti-CTLA-4 treatment, and tumor growth was effectively inhibited, but Myo1f - / - Mice did not respond to these ICB therapies ( Figure 2 C).

[0152] Example 2 Myo1f deficiency leads to expansion of bone marrow PMN-MDSCs

[0153] WT and Myo1f - / - Flow cytometry of tumor-infiltrating immune cells in tumor-bearing mice revealed that the infiltration of MDSCs cells in the tumor increased significantly, and was mainly composed of PMN-MDSCs ( Figure 4 A). In addition, CD8 + The number and anti-tumor activity of T cells were significantly inhibited, suggesting that MDSCs + Immunosuppressive effects of T cells ( Figure 4 B) Myo1f - / - Splenomegaly in tumor-bearing mice also suggests the presence of MDSCs infiltration ( Figure 3 ). Flow cytometry staining of peripheral blood and spleen revealed that MDSCs were in an aggregated state ( Figure 4 C), suggesting that MDSCs infiltrate into tumor tissues through peripheral circulation. - / - The bone marrow of tumor-bearing mice showed a highly expanded state of MDSCs, and the expanded population was mainly PMN-MDSCs ( Figure 4 AC). Further detection of STAT3 phosphorylation revealed that Myo1f - / - In MDSCs cells of tumor-bearing mice, STAT3 activation was enhanced ( Figure 4 D). Ki67 flow cytometry also confirmed the signal of MDSCs expansion ( Figure 4 D) This indicates that Myo1f deficiency leads to the proliferation and accumulation of PMN-MDSCs in the bone marrow and their infiltration into the tumor microenvironment.

[0154] Example 3 Myo1f deficiency promotes high expression of PD-L1 in MDSCs and improves their immunosuppressive function

[0155] First, flow cytometry was used to detect Myo1f - / - The MDSCs in the bone marrow of tumor-bearing mice were expanded and highly expressed with PD-L1 ( Figure 5A), similar situations were also observed in tumor-infiltrating MDSCs. By knocking down Myo1f in MDSCs, it was found that Myo1f deficiency directly led to upregulation of PD-L1 expression ( Figure 5 B), further proving that Myo1f directly regulates PD-L1 expression in MDSCs cells. MDSCs in tumors were sorted by flow cytometry ( Figure 5 C), in vitro with CD8 + In co-culture with T cells, Myo1f-deficient MDSCs have a stronger inhibitory effect on T cells ( Figure 5 D). These results indicate that Myo1f deficiency has a significant immunosuppressive effect on MDSCs with high PD-L1 expression.

[0156] Example 4 Myo1f affects tumor progression by regulating MDSCs bone marrow expansion and tumor microenvironment migration

[0157] The effect of MDSCs on tumor growth was studied by adoptive MDSCs transfer in recipient tumor-bearing mice. - / - Mouse-derived MDSCs promote tumor progression ( Figure 6 A, 6B), flow cytometry was used to detect T cell activation markers, and the results showed that MDSCs exhibited a specific immunosuppressive effect on T cells ( Figure 6 C) The tumor-bearing mice were treated with SX-682, an inhibitor of MDSCs migration, to inhibit the migration of MDSCs into the tumor. The results showed that WT and Myo1f - / - The differences in tumor progression disappeared in the tumor-bearing mice ( Figure 6 D, 6E). Therefore, Myo1f affects tumor progression by regulating the number and infiltration of MDSCs.

[0158] Example 5 MYO1F binds to TRIM21 to regulate STAT3 phosphorylation

[0159] Flag-(Ctrl / MYO1F) was overexpressed in MDSCs cells, and MYO1F was immunoprecipitated with anti-Flag and then detected by mass spectrometry to obtain the binding information of MYO1F interacting protein TRIM21 ( Figure 7 A). Immunofluorescence confocal method detected the co-localization of the two ( Figure 8 A). Validation by exogenous overexpression in 293T cells and endogenous immunoprecipitation in MDSCs showed that MYO1F can bind to the E3 ubiquitin ligase TRIM21 ( Figure 8 B, 8C; Figure 7B, 7C). This indicates that MYO1F is a key molecule for MDSCs expansion and low ICB response. MYO1F competitively binds to TRIM21, causing changes in ubiquitin modification of downstream key target proteins, enhancing STAT3 signaling, and mediating MDSCs expansion.

[0160] Example 6 Tumor microenvironment TGF-β1 mediates MYO1F downregulation

[0161] MC38 or B16F10 cells were used for tumor formation experiments ( Fig. 9 A). Flow cytometry revealed that tumors can stimulate the proliferation of bone marrow MDSCs ( Fig. 9 B). Further detection of MYO1F expression in MDSCs and non-MDSCs in bone marrow myeloid cells revealed that Myo1f expression in non-MDSCs was not affected, while Myo1f expression in MDSCs was significantly reduced ( Fig. 9 C). MDSCs were induced with culture supernatant of MC38 or B16F10 cell lines, and it was found that the expression of Myo1f was significantly reduced at both transcriptional and protein levels ( Fig. 9 D, 9E). 25 tumor-secreted or microenvironment-enriched cytokines were used to induce MDSCs and detect their MYO1F protein levels. It was found that TGF-β1 was the main cytokine that down-regulated MYO1F ( Fig.10 A). Gradient induction experiments showed that TGF-β1 inhibited Myo1f transcription ( Fig.10 B, 10C), and mediate MDSCs expansion ( Fig.10 D). The TGF-β1 level of the cell line was further detected by ELISA method to verify the results of the induction experiment ( Fig. 9 F). Further analysis of the gene expression of TGFB1 and Myo1f in more than 300 human tumor cell lines revealed that high TGFB1 expression corresponds to low Myo1f expression ( Fig. 9 G), suggesting that TGF-β1 is negatively correlated with MYO1F levels in tumor cells. By detecting the level of DNA methyltransferase I (DNMT1), it was found that TGF-β1 treatment significantly upregulated the expression of Dnmt1 gene in MDSCs ( Fig.10 E) and DNMT1 enzyme activity ( Fig.10 F), and treatment with the DNMTs inhibitor SGI abolished the downregulation of Myo1f by TGF-β1 ( Fig.10 G, 10H). Further analysis of the MethBank database revealed that the entire Myo1f gene segment in mouse liver cells was methylated with CG, CHG, and CH ( Fig. 9 H). Further amplification of the 1st, 8th, 21st, and 27th intron regions of the Myo1f gene revealed that the intron regions also showed varying degrees of methylation modification ( Fig. 9 I). This indicates that TGF-β1 regulates the transcription and expression of Myo1f by affecting its DNA methylation modification.

[0162] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. The use of MYO1F protein or its encoding gene Myo1f as a biomarker or target in the preparation of products for diagnosis, monitoring severity assessment, efficacy assessment, prognosis assessment, prevention and / or treatment of tumors.

2. The use according to claim 1, characterized in that: The diagnosis, monitoring severity assessment, efficacy assessment or prognosis assessment of the tumor includes detecting the expression level of MYO1F, the presence or absence of Myo1f and / or the expression level of Myo1f mRNA.

3. The use according to any one of claims 1-2, characterized in that: The prevention or treatment of tumors includes upregulating the expression level of MYO1F and / or the expression level of Myo1f mRNA.

4. The use according to claim 1, characterized in that: The MYO1F protein or its encoding gene Myo1f is MYO1F and / or Myo1f in MDSCs; Preferably, the MDSCs are PMN-MDSCs.

5. The use according to any one of claims 1 to 4, characterized in that: The product includes at least one of a reagent for detecting the presence of Myo1f, detecting the mRNA expression level of Myo1f, detecting the expression level of MYO1F, up-regulating the mRNA expression level of Myo1f, and up-regulating the expression level of MYO1F.

6. Use of an agent targeting the MYO1F protein or its encoding gene Myo1f in MDSCs in the preparation of a drug for treating tumors, characterized in that: The reagent promotes the overexpression of Myo1f, Myo1f mRNA and / or MYO1F in MDSCs or increases the activity of MYO1F.

7. The use according to claim 6, characterized in that: The reagent is selected from: 1) a viral vector, wherein the viral vector overexpresses MYO1F; 2) Small molecule drugs that increase Myo1f, Myo1f mRNA and / or MYO1F expression in MDSCs; 3) Small molecule drugs or antibodies that can enhance MYO1F activity.

8. The use according to any one of claims 1 to 7, characterized in that: The tumors include liver cancer, colon cancer, breast cancer, pancreatic cancer, esophageal cancer, melanoma, non-small cell lung cancer, adrenocortical carcinoma, diffuse large B-cell lymphoma, prostate cancer, rectal cancer or endometrial cancer.

9. A method for inhibiting the infiltration of MDSCs in a tumor, characterized in that: The method comprises treating MDSCs with an agent, wherein the agent promotes Myo1f, Myo1f mRNA and / or MYO1F overexpression in MDSCs or increases the activity of MYO1F.

10. A method for constructing a tumor animal model, characterized in that: The construction method includes up-regulating the expression level of MYO1F and / or the expression level of Myo1f mRNA.

Images