The Roles Of TESTIN Gene In Endometrial Cancer And Mechanism

Part I The expression of TESTIN gene in endometrial cancerObjective: To explore the expression of TESTIN gene in endometrial cancer tissues.Methods: Real time PCR and immunohistochemical staining assay were used to determine the m RNA and protein 1evel of TESTIN in the tumor tissues.The relationship between TESTIN expression and clinico-pathological characteristics was analyzed. Methylation-specific PCR was performed to test the promoter status of TESTIN.Results: Compared to the non-cancerous tissues, more than 75 %(52/68) of endometrial cancer tissues presented loss of TESTIN protein(p<0.01),furthermore,this reduction was much more significant in the subgroup of endometrioid adenocarcinoma,but it was rarely observed in the subgroup of serous papillary adenoearcinoma(χ2=13.34,p=0.001).As the results of MSP shown, the promoter of TESTIN was hypermethylated in 44.1%(30/68) of endometrial cancer cases, while only 3.6%(1/28) of the non-cancerous tissues showed the hypermethylated TESTIN(p<0.001). Moreover, it was found that hypermethylation of TESTIN was significantly correlated to deep myometrial invasion(p=0.007) and lymphatic node metastasis(p=0.03). The downregulation of TESTIN in endometrial cancer was correlated to its hypermethylated promoter(p=0.021), which could be restored by the treatment of 5-aza-d C.Conclusions: TESTIN was significantly downregulated by methylation in endometrial cancer. The loss of TESTIN was mainly caused by the promoter hypermethylation.PartⅡ The functions of TESTIN in endometrial cancer developmentand its mechanismsObjective: To explore the mechanisms of TESTIN in the invasion of endometrial cancer.Methods: The constructed TESTIN-overexpressing plasmids(pc DNA3.1-TESTIN) and sh RNA-TESTIN were transfected respectively into AN3 CA and Ishikawa cells. Real time PCR and western blot were performed to confirm the alterations of TESTIN. The effects of TESTIN in endometrial cancer cells were detected by a battery of experiments, in vitro, such as MTT assay, cell colony formation assay, transwell assay, real time PCR and western blot. The morphological changes of the cells were examined under optical microscope and recorded. The detection of EMT related markers(E-cadherin, Snail, vimentin and N-cadherin) were carried out by western blot and real time PCR. The nude mice model bearing endometrial cancer xenografts were established to investigate the in vivo effects of TESTIN on the tumor growth.Results: In vitro, the proliferation and the invasion of AN3 CA cells were inhibited by the upregulatoin of the expression of TESTIN gene. The expression of E-cadherin was increased while the expressions of N-cadherin, vimentin and Snail were decreased by the upregulation of TESTIN gene; accordinglhy, the proliferation and the invasion of Ishikawa cells were increased by knockdown of TESTIN gene. The expression of E-cadherin was decreased while the expressions of N-cadherin, vimentin and Snail were increased. The tumor xenografts growth assay showed that TESTIN could significantly suppress the tumor growth and inhibited the EMT procedure.Conclusions: The proliferation, viability and invasion of endometrial cancer are inhibited by TESTIN gene, which acts as tumor suppressor and also prevented the EMT procedure. And the EMT procedure is processed by hypermethylation of TESTIN gene.