Mechanism Study On METTL3 Inhibiting The Malignant Progression Of Triple-negative Breast Cancer And Negatively Regulating PD-L1 Expression

Background:Triple-negative breast cancer(TNBC)is a relatively poor prognosis of breast cancer.It is not only sensitive to the common endocrine therapy,but also lacks effective therapeutic targets.The main adjuvant treatment of TNBC is chemotherapy,but it is prone to drug resistance and lead to treatment failure.Therefore,screening and identifying new therapeutic targets of TNBC and deeply studying the molecular mechanism of its occurrence and development have important clinical and scientific research value.Recently,the expression of PD-L1 programmed death ligand 1(PDL1)in TNBC patients is higher than that in other subtypes of breast cancer.The combination of programmed cell death protein 1(PD-1)and PD-L1 can inhibit the activation of tumor infiltrating lymphocytes(TILs)and make TNBC cells survive through immune escape.Therefore,the treatment of anti-PD-1/PD-L1 immune checkpoint inhibitors(ICIs)is one of the research hotspots with broad application prospects of TNBC,however,the treatment response of patients is heterogeneous,and the regulation mechanism of PD-1/ PD-L1 needs to be further studied.At present,studies have found that epigenetics plays an important role in tumor immunotherapy,N6 methyladenosine mRNA modification(m6A)is one of the most abundant RNA modifications,which is usually enriched near 5 ′UTR.It can regulate cell proliferation,migration and apoptosis and participate in cancer progression.At present,studies have found that epigenetics plays a certain role in tumor immunotherapy.The m6A modification of RNA is a modification method of epigenetics,in which methyltransferase like 3(METTL3)is an component of m6A modification of m6A.METTL3 modification may play an important role in the immune escape of PD-1/ PD-L1 by enhancing the survival of tumor cells,which may become a potential target of immunotherapy,but there is still a lack of relevant research.Purpose:1.To explore the role of m6A modification in triple-negative breast cancer;2.To explore the relationship between METTL3 and c-myc and PD-L1 in triple-negative breast cancer and its mechanism;3.To explore the role of c-myc and its inhibitors in triple-negative breast cancer.Methods:1.Bioinformatics analysis was performed to investigate the prognostic impact of m6A related molecules on patients of triple-negative breast cancer.The silence and stable over-expression of METTL3 were constructed in triple-negative breast cancer cell lines(MDA-MB-231),and the infection efficiency was confirmed through q-PCR and Western Blotting.Cell proliferation,colony formation,Transwell,scratch test,flow cytometry and animal experiments were used to clarify the regulatory effects of METTL3 on the proliferation,apoptosis and migration of triplenegative breast cancer cells.2.Bioinformatics analysis was used to analyze the correlation between METTL3 and PD-L1,METTL3 and c-myc,c-myc and PD-L1 in triple-negative breast cancer.The regulatory relationship between METTL3,c-myc and PD-L1 in triple-negative breast cancer cell line MDA-MB-231 was verified through q-PCR and Western Blotting experiments.3.Cell proliferation,colony formation,Transwell,scratch test and flow cytometry were used to investigate the effects of c-myc and its inhibitors on proliferation,apoptosis and migration,and cell cycle of triplenegative breast cancer cells.4.Western blotting experiment and m6A-IPqPCR experiment verified that METTL3 regulated c-myc in m6A-FBXW7 mode.Results:1.METTL3 is associated with good prognosis in patients of triplenegative breast cancer.The proliferation,clone formation and migration ability of triple-negative breast cancer cell lines with low expression of METTL3 were increased.Conversely,the proliferation,colony formation and migration ability of triple-negative breast cancer cell lines with high expression of METTL3 were decreased.Animal experiments showed that the expression of METTL3 in triple-negative breast cancer cells decreased in tumor formation and proliferation.2.In triple-negative breast cancer cell lines,METTL3 negatively regulates c-myc through negatively regulating PD-L1.It has also been verified in animal experiments and clinical samples.3.The proliferation,colony formation and migration ability of triplenegative breast cancer cell lines with low expression of c-myc were decreased.c-myc inhibitor inhibited the proliferation,clone formation and migration ability of triple-negative breast cancer cell lines.4.In triple-negative breast cancer cell lines,METTL3 regulates FBXW7 expression by regulating m6A modification of FBXW7 site,thus negatively regulating c-myc.Conclusion:In triple-negative breast cancer,low expression of METTL3 promotes proliferation and migration of tumor cells,and regulates FBXW7 through m6A dependent pathway,and then reduces c-myc degradation by inhibiting proteasome pathway,stabilizes the expression of c-myc protein,and finally the expression level of PD-L1 was up-regulated,which enhanced immune escape of tumor cells.Inhibiting the expression of c-myc will not only inhibit the proliferation and migration of tumor cells,but also down regulate the expression of PD-L1 and reduce immune escape,which provides an experimental basis for the development of new drugs for TNBC clinical treatment or the clinical research of drug application.