The Relationship Between The Expression Level Of Rictor And Cell Sensitivity To MTOR Inhibitors And Its Molecular Mechanism

Objective To investigate the expressions and clinical significance of Rictor and p-Akt in human ESCC,and to analyze the relationship between the expression level of Rictor and cell sensitivity to m TOR inhibitors(everolimus and PP242)and its molecular mechanism.Methods Part one The expressions and clinical significance of Rictor and p-Akt in human ESCC The expressions of Rictor and p-Akt in 150 ESCC tissues and normal esophageal tissues were detected by Immunohistochemistry,and the clinical significances of them in ESCC were analyed by Chi-square test and Spearman's rank correlation analysis.Part two The changes of cell sensitivity to m TOR inhibitors(everolimus and PP242)after knocking down the expression of Rictor 1.Rictor-sh RNA and Control-sh RNA were transfected in ECa109 cell lines by Lipo3000 TM and ECa109 cell lines stably expressing Rictor-sh RNA and Control-sh RNA were screened using Puromycin(ECa109-Rictor-sh RNA cells and ECa109-Control-sh RNA cells).The expression of Rictor in them were detected by Western blot.2.The effects of m TOR inhibitors(everolimus and PP242)on proliferation of ECa109 and EC9706 cells were detected by CCK-8 assay,and the IC50 of m TOR inhibitors(everolimus and PP242)to ECa109 or EC9706 cells for 24 h and 48 h were calculated,respectively.3.The xenografts of nude mice from ECa109-Rictor-sh RNA cells and ECa109-Control-sh RNA cells were established for detecting the effects of m TOR inhibitors(everolimus and PP242)on growth of tumor with different level of Rictor.4.Cell poptosis of xenograft tissue was investigated by H&E staining and TUNEL methods.Part three The molecular mechanism of changes in cell sensitivity to m TOR inhibitors(everolimus and PP242)after knocking down the expression of RictorAfter cell with different level of Rictor were treated with m TOR inhibitors(everolimus and PP242)in vitro and in vivo,the expression levels of key factors in PI3K/Akt/m TOR pathway were investigated by Western blot.Results Part one There were positive expressions of Rictor and p-Akt in human ESCC and had asignificantly clinical significance The positive rates of Rictor and p-Akt expression in ESCC tissues were higher than that in normal esophageal tissues and both of them had significant correlation with TNM classification of ESCC patients.There were a positive correlation between the expression of p-Akt and Rictor in ESCC tissues.Part two The sensitivity of cell to m TOR inhibitors(everolimus and PP242)was improved after knocking down the expression of Rictor 1.ECa109-Rictor-sh RNA and ECa109-control-sh RNA cells were obtained succesfully.The results of Western blot showed that the expression of Rictor in ECa109-Rictor-sh RNA cells was decreased significantly,compared with ECa109-Control-sh RNA cells.2.The results of CCK-8 assay showed that ECa109 and EC9706 cell viability rate decreased significantly in a dose and time dependent manner.The IC50 of everolimus on ECa109 cells for 24 h and 48 h is 52.18±1.72 ?M and 19.04±1.28 ?M,respectively,and the IC50 of everolimus on EC9706 cells for 24 h and 48 h is 52.07±1.72 ?M and 17.07±1.23 ?M,respectively.The IC50 of PP242 on ECa109 cells for 24 h and 48 h is 11.28±1.05 ?M and 4.07±1.61 ?M,respectively,and the IC50 of PP242 on EC9706 cells for 24 h and 48 h is 13.84±1.14 ?M and 3.46±0.54 ?M,respectively.3.As showed in the results of xenograft tumors experiment,compared to the Control-sh RNA group,tumor growth became slow in Rictor-sh RNA group and Control-sh RNA + m TOR inhibitors(everolimus and PP242)group,and tumor growth was slowest in Rictor-sh RNA + m TOR inhibitors(everolimus and PP242)group.Compared with Control-sh RNA + m TOR inhibitors(everolimus and PP242)group,tumor growth became slow in Rictor-sh RNA + m TOR inhibitors(everolimus and PP242)group.4.The results of H&E staining showed that compared with Control-sh RNA group,the phenomenon of cell shrinkage and cell nucleation shrinkage was more pronounced in Rictor-sh RNA + m TOR inhibitors(everolimus and PP242)group.The results of TUNEL assay showed that compared with Control-sh RNA group,the cell apoptosis rates of Rictor-sh RNA group and m TOR inhibitors(everolimus and PP242)group increased significantly,the cell apoptosis rates of Rictor-sh RNA + m TOR inhibitors(everolimus and PP242)group was highest.Part three The molecular mechanism of improving cell sensitivity to m TOR inhibitors(everolimus and PP242)after knocking down the expression of Rictor The in vitro and in vivo results of Western blot showed that compared with Control-sh RNA group,the protein expression of p-Akt(Ser473)and p-PRAS40(Thr246)were decreased significantly in Rictor-sh RNA group and PP242 group,and the protein expression of p-Akt(Ser473)and p-PRAS40(Thr246)were increased significantly in everolimus group.Compared with everolimus group,the protein expression of Rictor,p-Akt(Ser473)and p-PRAS40(Thr246)were decreased significantly in Rictor-sh RNA+everolimus group.Compared with PP242 group,the protein expression of Rictor,p-Akt(Ser473)and p-PRAS40(Thr246)were decreased significantly in Rictor-sh RNA+ PP242 group.Conclusios The Rictor and p-Akt are hyperactivated in human ESCC,which may play a crucial role in tumorigenesis of ESCC.Knocking down the expression of Rictor could improve cell sensitivity to m TOR inhibitors(everolimus and PP242)in esophageal squamous cell carcinoma in vitro and in vivo.