The Expression Of TNK2 Gene Splice Variant 1 Affects Breast Cancer Progression

Breast cancer is a malignant tumor that occured in the epithelial tissue of the breast.In the crowd,99% of the patients were female,and the incidence is increasing year by year.Synthesized the molecular expression and clinical characterization of breast cancer in the international breast cancer conference,breast cancer would be divided into 5 subtypes to facilitate clinical diagnosis and basic research.The occurrence,development,metastasis,infiltration and prognosis of breast cancer is associated with the mutation or abnormal expression of a variety of oncogenes.At the molecular leveltargeting specific oncogenes or tumor suppressor genes.,it is the most important target to research,diagnose and treatment the breast cancercurrently.Because of the non-receptor tyrosine kinase activity,TNK2 gene has received much attention in early screening,targeted therapy and prognosis evaluation of breast cancer.Therefore,it wasselected as the target gene in this study.In order to more accurate understanding the effect of gene expression changes on the development of breast cancer,this study focuses on the regulation mechanism of gene alternative splicing that more refined gene expression.By comparing the expression changes of whole gene and the individual splice variant of TNK2 gene,we analyzed the biological roles of TNK2 splice variants in the development of breast cancer.The main results of the study are as follows:1.Synthesizingthespecific primers of TNK2 gene splicing variant,and amplifying the TNK2 gene splicing variant in normal breast epithelial cells and four different phenotypes of breast cancer cell lines byRT-PCR method to comparethe expression.It was found that the splice vaiant 1 of TNK2(Variant 1)was highly expressed in MDA-MB-231 and T-47 D cells of breast cancer cell lines,so MDA-MB-231 and T-47 D cells were determined to be used for further study.2.Synthesizingthe siRNA which was specifically knock-down of the Variant 1 and total of TNK2 gene,and transfecting into MDA-MB-231 and T-47 D breast cancer cell lines,thenamplifying the mRNA of TNK2 gene by RT-PCR method after collecting of cells to comparethe expressionto ensurethat siRNAs can effectively downregulate the expression levels of total TNK2 and variant 1.3.Using MTT cell viability assay,BrdU incorporation,flow cytometry and Western blot,it was confirmed that the proliferation of breast cancer cells was inhibited and the cell cycle was arrested in G1/S phase after the expression of Variant 1 and total TNK2 of siRNA interference;using the scratch test,Transwell chamber method and Western blot method,it was confirmed when the expression of Variant 1 and total TNK2 was down-regulated,the migration and invasion ability of breast cancer cells was significantly inhibited,which may be related to the inhibition of the expression of EMT related to the migration and invasion;using plate clone test and soft agar colony forming assay,it was confirmedwhen the inhibition of TNK2 gene expression of Variant 1 and total TNK2,breast cancer cells in vitro tumorigenic ability was significantly inhibited.4.Using DAPI immunofluorescence,flow cytometry and Western blot method to detect cell apoptosis,the results showed that the expression of Variant 1 and total TNK2 was decreased,but the apoptosis of breast cancer cells was not induced.SiRNAs interfering with the expression of TNK2 Variant 1 on the cell proliferation,migration and clonal formationof breast cancer cells was stronger than or similar to the effect of interfering with total TNK2 expression,indicating that the regulation of TNK2 on the biological characteristics of breast cancers is mainly mediated by the TNK2 gene Variant 1.