The Study On The Role And Molecular Mechanism Of TC2N In Breast Cancer

Background:As the number of cancer patients is increasing globally,the cancer burden of our country is also increasing.In our country,about 2 million patients died from cancer,and about 3million new cancer patients were diagnosed each year.Breast cancer is one of the most common cancers and also is a serious public health problem for women.The incidence of new breast cancer in Chinese women accounts for 12.2% of the world,and the mortality rate accounts for 9.6%.Although the diagnosis and treatment of breast cancer has been improved,the treatment effect and prognosis of some patients are still not ideal.Therefore,it is very important to explore the potential mechanism of the development of breast cancer and the new markers for diagnosis and prognosis to improve the survival of breast cancer patients.Our research group found TC2N may be involved in the development of breast cancer through genomic expression screening.For the first time,we found that TC2N protein was highly expressed in breast cancer tissues through IHC,but the function of this gene was still unknown,so we conducted in-depth research on the role and molecular mechanism of TC2N gene in the development of breast cancer,and clarified its clinical significance in breast cancer.Contents:1.Systematically analysis of the expression and clinical significance of TC2N in breast cancerTC2N mRNA expression in breast cancer and normal breast tissue was analyzed by Oncomine database.Immunohistochemical staining(IHC)was used to verify the expression of TC2N protein in breast cancer and normal breast tissue adjacent to the cancer.2.Evaluation of the biological function of TC2N in breast cancer cellsEffects of stable differential expression of TC2N on proliferation and clone formation of breast cancer cells were analyzed by MTS method and clone formation experiment in vitro.Nude mice were used to analyze the capability of tumor growth induced by TC2N in vivo.3.Investigation of the biological functional molecular mechanism of TC2N in breast cancer cellsThrough bioinformatics analysis,TC2N was involved in the signaling pathway regulating the proliferation of breast cancer cells,which was verified by western blot assay(WB).The specific molecular mechanism of TC2N’s involvement in PI3K/AKT signaling pathway was elucidated by co-immunoprecipitation(Co-IP)and WB.Results:1.Analysis of the correlation between TC2N high expression and clinical stage and prognosis of breast cancer patients(1)Firstly,we found the expression of TC2N m RNA in breast cancer tissues was significantly higher than that in normal breast tissues through Oncomine database analysis.Then,we used tissue chips containing 75 breast cancer samples and 75 matched normal breast tissues to analyze the expression of TC2N protein.The results showed that the expression of TC2N protein in breast cancer tissues was significantly higher than that in normal breast tissues.(2)Analysis of the clinical parameters of 75 breast cancer patients found that TC2N protein expression was highly correlated with clinical stage(P<0.001),tumor size(P=0.021),lymph node metastasis(P<0.001)and HER-2 receptor expression(P=0.039).(3)Kaplan-meier univariate analysis and multivariate COX proportional risk regression were used to further evaluate the effect of TC2N on the clinical prognosis of breast cancer.The results showed that the breast cancer patients with high TC2N expression had a good prognosis,and TC2N expression was an independent prognostic factor for the survival of breast cancer patients.2.TC2N inhibits the proliferation of breast cancer cells in vitro and in vivo(1)Firstly,we enriched the gene through TCGA database and GO analysis and found that the TC2N gene may be involved in the proliferation and cell survival of breast cancer cells.Therefore,we successfully constructed a breast cancer cell line with stable differential expression of TC2N by using plasmid transfection technique.(2)In vitro,we evaluated the proliferation of breast cancer cells expressing TC2N gene stably by MTS method and clone formation experiment and found that the proliferation andclone formation ability of TC2N overexpressed breast cancer cells were significantly inhibited,while the proliferation and clone formation ability of TC2N low-expressed breast cancer cells were significantly enhanced.(3)In order to verify the effect of TC2N gene on the proliferation of breast cancer cells,we conducted in vivo experiments using the xenograft model of nude mice,and found that the TC2N overexpression can inhibit the growth of breast cancer cells in nude mice,suggesting that TC2N gene has a strong anti-tumor effect in breast cancer.3.TC2N regulates the proliferation of breast cancer cells by participating in the PI3K/AKT signaling pathway(1)First,we enriched the signaling pathway of TC2N overexpressed gene by GO analysis,and found that TC2N gene might inhibit the proliferation of breast cancer cells by regulating the PI3K/AKT signaling pathway.Then,we analyzed the protein of related to the PI3K/AKT pathway in the TC2N overexpression breast cancer cells,and found that the overexpression of TC2N significantly inhibited the phosphorylation of p55 and AKT,as well as inhibit the downstream activation of AKT molecules and the activation of downstream AKT inhibitory molecules.(2)Through proteomics and bioinformatics analysis,it was found that ALK and EBP1 may be involved in the regulation of AKT phosphorylation of TC2N in breast cancer cells.Therefore,we found that interference with ALK could significantly reduce the p55 phosphorylation of TC2N low-expression breast cancer cells,while the phosphorylation of AKT was partially reduced through WB.Co-IP showed that ALK could interact with TC2N,and the interaction between EBP1 and AKT was inhibited by TC2N.The above results showed that TC2N inhibited the phosphorylation of p55 by competing with p55 to bind ALK,and TC2N inhibited the phosphorylation of AKT by competing with EBP1 to bind AKT.(3)WB showed that PI3K inhibitor can partially reduce the phosphorylation of AKT in TC2N low expression breast cancer cells,the MTS and clone formation experiment were used to verify that PI3K inhibitor can partially reduce the proliferation ability and clone formation ability of TC2N low-expression breast cancer cells,suggesting that TC2N inhibits AKT phosphorylation of breast cancer cells through a PI3K independent signaling pathway.(4)In order to clarify that the PI3K/AKT signaling pathway is a key mediator of TC2N in regulating the proliferation of breast cancer cells,we found that the AKT phosphorylationof TC2N low-expression breast cancer cells significantly decreased after blocking the PI3K/AKT pathway by RNA interference.Meanwhile,it was found that the proliferation and clone formation ability of TC2N low-expression breast cancer cells were significantly inhibited after blocking PI3K/AKT pathway by MTS and clone formation experiment.Indeed,suppression of PI3K-AKT signaling strongly reversed the stimulatory effect of TC2N knockdown on cell proliferation.Conclusion:We proved for the first time that TC2N plays an inhibitory role in the development of breast cancer.Its overexpression blocks the PI3K/AKT signaling pathway by inhibiting p55γand AKT phosphorylation,thus preventing the proliferation and growth of breast cancer cells.