Effects And Molecular Mechanism Of MiR-199a-3p On The Cell Cycle And Migration Of Esophageal Squamous Cell Carcinoma

Objective1. To explore the effects and molecular mechanism of miR-199a-3p on cell cycle and migration of esophageal squamous cell carcinoma (ESCC).2. To explore the effects of PI3K/Akt/mTOR signaling pathway inhibitors on cell cycle and cell migration after down-regulating the expression of Rictor in ESCC.Methods1. The expression level of miR-199a-3p in different esophageal squamous cell carcinoma. The total RNAs were extracted from ECa109, EC9706, KYSE450,KYSE790 and TE1 and were transcripted reversely to cDNA, respectively. Finally,the expression levels of miR-199a-3p in them were detected by qRT-PCR.2. The effects of miR-199a-3p on cell cycle and migration of esophageal squamous cell carcinoma. After miR-199a-3p mimics and miR-199a-3p mimics negative control were transfected into ECa109, EC9706 and KYSE450, the transfection efficiency was detected by fluorescence microscope after being transfected for 6h.After cells transfected with miR-199a-3p mimics for 48 h, the expression level of miR-199a-3p was detected by qRT-PCR, and the cell cycle and cell migration were investigated by flow cytometry, wound healing assay and transwell assay,respectively.3. The effect of miR-199a-3p on the relative proteins of mTORC2 signaling pathway in esophageal squamous cell carcinoma. After miR-199a-3p mimics and miR-199a-3p mimics negative control were transfected into ECa109, EC9706 and KYSE450 for 72h, the total proteins were extracted respectively, and the exprssion of relative proteins in mTORC2 signaling pathway such as mTOR, Rictor,p-Akt(S473), PI3K and p-Akt(T308) were detected by Western blot.4. The effects of PI3K/Akt/mTOR signaling pathway inhibitors on cell cycle and cell migration after down-regulating the expression of Rictor in ESCC. After the ECa109 cells which stably expressed the Rictor-shRNA vector and the control cells were treated with RAD001, LY294002 and PP242 respectively, the cell cycle and cell migratory ability were investigated by flow cytometry and transwell assay,respectively.Results1. The results of qRT-PCR showed that miR-199a-3p expressed in as the five ESCC cell lines ECa109, EC9706, KYSE450, KYSE790 and TE1, while the expression level of miR-199a-3p had no statistical difference in them.2. After ECa109, EC9706 and KYSE450 cells were transfected with miR-199a-3p mimics, the green fluorescence in cells under fluorescent microscope was intenser,which indicated that miR-199a-3p mimics was transfected successfully into cells.Compared to control cells or cells transfected with negative control, the expression levels of miR-199a-3p were increased significantly, the number of cells in G1 phase was higher, the relative rate of cell migration and the number of migratory cells were lower in cells transfected with miR-199a-3p mimics.3. After the expression level of miR-199a-3p in ECa109, EC9706 and KYSE450 was improved, the expression levels of mTOR and Rictor were obviously reduced, while the expression levels of PI3K, p-Akt(T308) and p-Akt(S473) were obviously increased.4. After down-regulating the expression of Rictor in ECa109 cells, compared to the untransfected cells, LY294002 and PP242 retarded more numbers of cells in G2 phase and the inhibitory effects of RAD001,LY294002 and PP242 on cell migration were enhanced significantly.Conclusion1. miR-199a-3p can block the cell cycle progression and suppress the migration through regulating the mTORC2 signaling pathway in ESCC.2. The down-regulation of Rictor expression can enhance the inhibitory effects of PI3K/Akt/mTOR signaling pathway inhibitors on cell cycle and cell migration in ESCC.