Effects Of MiR-199a-3p On Cell Proliferationand Apoptosis In Esophageal Squamous Carcinoma And Its Molecular Mechanism

Aim 1.To investigate the effects of mi R-199a-3p on the proliferation and apoptosis of esophageal squamous cell cacinoma and the possible molecular mechanism.2.To investigate the effects of PI3K/AKT/m TOR signaling pathwayinhibitors on the proliferation and apoptosis of cells after down-regulated the expression of Rictorin esophageal squamous cell carcinoma(ESCC).Methods 1.The expression of mi R-199a-3p in the five ESCC cell line cells were detected by q RT-PCR(TE1 and EC9706 are poorlydifferentiated cell lines,ECa109,KYSE450 and KYSE790 arewell differentiated cell lines).2.After the synthesized mi R-199a-3p mimics were transfected into three ESCC cell linecells(ECa109 and EC9706 and KYSE450)by si RNA-MateTM,the expression of mi R-199a-3p in them were detected by q RT-PCR,and the effect of mi R-199a-3p on cell proliferation and apoptosis were detected by CCK-8 assays,colony formation assays and flow cytometry,respectively.3.After mi R-199a-3p mimics were transfected into three ESCC cell line cells,the protein and m RNA expressions of m TOR,Raptor and p70S6 K,the factors in the m TORC1 signaling pathway,anti-apoptotic protein Bcl-2 and autophagy relative protein p62 and LC3A/B were detected by western blots and q RT-PCR,respectively.4.The expressions of mi R-199a-3pin the three esophageal squamous cell cacinoma cell lines after treated with RAD001,the specific inhibitor of m TORC1,were detected by q RT-PCR.5.After the screened ECa109 cellsstable expressing Rictor-sh RNA and the untransfected cells were treated with PI3K/AKT/m TOR pathway inhibitors,the cell colony formation ability and apoptosis of cells were detected by colony formation assays and flow cytometry,respectively.Results 1.There were mi R-199a-3p expression in the five ESCC cell linecells,while the expression level of mi R-199a-3p in them had no significant differences,which indicated that the expression level of mi R-199a-3p had nocorrelation with cell differentiation degree.2.Compared to control cells and cells transfected with mi R-199a-3p mimics negative,the expression levels of mi R-199a-3p were significantly increased,the cell colony-formation abilities were significantly decreased,cell proliferationsbecame slowand the numbers of cell apoptosis were increased in the three cell line cells transfected with mi R-199a-3p mimics.3.Compared to control group and mimics negative group,the expression levels of protein and m RNA of m TOR,Raptor and p70S6 K were significantly decreased,the expressions of Bcl-2 and p62 were obviously decreased,while the expressions of LC3A/B were distinctly increased in the three cell line cells transfected with mi R-199a-3p mimics.4.After treated with RAD001,the expressions of mi R-199a-3pin the three ESCC cell linecells had no distinctly difference compared to untreated cells.5.After cells with down-regulated the expression of Rictor treated with the inhibitors of PI3K/AKT/m TOR pathway,the number of cell clones were obviously decreasedand the number of cell apoptosis was increasedin ECa109 cells compared to control group.Conclusions 1.mi R-199a-3p could inhibitsignificantly cell proliferation and induce cell apoptosis in ESCCand its molecular mechanism may be related with m TORC1/p70S6 K signaling pathway and cell autophagy.2.Down-regulated theexpression of Rictor could enhance the effect of PI3K/AKT/m TOR pathway inhibitors on cell proliferation and apoptosis in ESCC.