The Expression And Regulating Mechanism Of TPM1in Cholangiocarcinoma

[Background] TPM1is a key gene involved in the regulation of cytoskeletal protein,some studies have shown that TPM1plays a role of tumor suppressor genesimportant in a variety of tumors, but the expression of TPM1in cholangiocarcinomahave not yet no study, this research tries to investigate the expression and regulatingmechanism of TPM1in cholangiocarcinoma,to reveal the abnormal signal pathwayincluding RAS/PI3K/AKT and RAS/MEK/ERK, as well as the epigeneticmechanism including DNA methylation, histone acetylation, and abnormalmicroRNA-21are involved in regulation of TPM1,in order to clarify the molecularregulating mechanism of TPM1in cholangiocarcinoma, to provide new moleculartargets for the treatment of cholangiocarcinoma.[Methods]1.Collect30cases of intrahepatic cholangiocarcinoma specimens,analysethe expression of TPM1in cholangiocarcinoma cancerous and adjacent non-cancerous tissue by Immunohistochemistry， and cultivate the intrahepaticcholangiocarcinoma cell HuCCT1, extrahepatic cholangiocarcinoma cell QBC939, the normal bile duct epithelial cell HIBEC,and investigate the expression of TPM1by western blot、quantitative PCR.2.Investigate the expression alteration of TPM1by western blot and quantitative PCR after the cholangiocarcinoma cells are treatedwith RAS specific inhibitor manumycin A、PI3K/AKT inhibitor LY294002、MEK\/ERK inhibitor U0126、 DNA methyltransferase inhibitor DAC、 histonedeacetylase inhibitor TSA,and downregulating microrna-21by constructing thelentivirus vector.3. Investigate the changes of proliferation, apoptosis and migrationafter the cholangiocarcinoma cells are treated with RAS specific inhibitormanumycin A、PI3K/AKT inhibitor LY294002、MEK\/ERK inhibitor U0126、DNAmethyltransferase inhibitor DAC、histone deacetylase inhibitor TSA.[Results]1.The results of immunohistochemistry in30cases of cholangiocarcinomacancerous tissue showed that5cases were negative,25cases were weakly positive,30cases of adjacent non-cancerous tissue showed that30cases were all weaklypositive. The expression of TPM1were obviously decreased in HuCCT1,andupregulated in QBC939,compared with HIBEC, according to the western blot andquantitative PCR results.2. TPM1expression were changed in protein level andmRNA level after the cholangiocarcinoma cells are treated with RAS specificinhibitor manumycin A、PI3K/AKT inhibitor LY294002、MEK\/ERK inhibitorU0126、DNA methyltransferase inhibitor DAC、histone deacetylase inhibitor TSAand knocking down microrna-21.3.The cholangiocarcinoma cell proliferation abilitywas decreased, the apoptosis rate was significantly increased,and migration abilitywere inhibited after the cholangiocarcinoma cells are treated with manumycin A、LY294002、U0126、DAC、TSA.[Conclusion] This study confirmed that TPM1was downregulated in30cases ofcholangiocarcinoma cancerous tissue,however,there was no difference betweencancerous tissue and adjacent non-cancerous tissue through statistical analysis,partly due to the small amount of samples. The expression of TPM1was significantlydecreased in HuCCT1,and upregulated in QBC939, suggesting that TPM1would bedownregulated in intraheptic cholangiocarcinoma cell and upregulated in extrahepticcholangiocarcinoma cell,and we confirm that mutation in the RAS gene, and theabnormal signal pathway of RAS/PI3K/AKT, RAS/MEK/ERK, and epigeneticmechanisms including DNA methylation, histone deacetylation, abnormal expressionof microrna-21were involved in the regulation of TPM1expression.And there wasan obvious anti-cancer effect of manumycin A、LY294002、U0126、DAC、TSA.