MiR-99-b-5pinducesautophagy In Endometrial Cancer Cells By Targeting MTOR

Objectives :The mechanismand pathophysiological significance about EEC in the process of autophagy are not clear, autophagy is the second largest programmed way of cell death, m TOR is the core governor of autophagy, m TOR activity enhanced in the process of EEC. Hsa-mi R-99b-5p is already confirmed a strong targeting m TOR mi R by bioinformatics and experiment, its expression level in the process of EEC showed synchronization with the EEC autophagy, is gradually weakened, so this mi R may induce the EEC autophagy death by control m TOR in EEC autophagy.Confirm the mi R-99b-5p can be targeted on the m TOR to induce ishikawa cells autophagy and Analyze the time and dose effect of the control to autophagy,As the study prompted endometrial cancer cells autophagy mechanism and provides a new theoretical basis to treat endometrial cancer by means of regulation of autophagy.Methods:First,take a bioinformatics analysis about the combination of m TOR and mi R99b-5p, the cultivation of the endometrial cancer cells, and then analyzed autophagy related protein m RNA and protein level change of endometrial cancer cells under different concentrations and different time by the inductivity of mi R99b-5p.Set the same concentration mi R99b-5p in different time of transfection and observe effect on endometrial cancer cells, rows again protein imprinting method to detect autophagy marker protein LC3 and the expression of beclin1, RT-PCR detection of autophagy genes beclin1 m RNA expression level.Third, Set different control groups to observe cell morphology LC3 cell immunofluorescence and MDC autophagy bubble dyeing method detecting the expression of autophagy marker when given utophagy revulsant rapamycin, autophagy specific inhibitor 3ma and CQ together with mi R99b-5p after incubation, systematically analysis mi R99b-5p induced the occurrence of autophagy happened in endometrial cancer cell.Western blot test signaling pathways of the key protein m TOR and expression of LC3.Cellular immune fluorescence detecting the amount of the expression of autophagy proteins MAP1- LC3, MDC staining method to detect the quantity of autophagy bubble.With SPSS13.0 software analyze the testing data and mapping the results.Results: Bioinformatics analysis results showed that m TOR is one of thetarget gene of mi R-99b-5p,and the contact is conservative.Mi R-99b-5p dose and time dependent to induce the autophagy of ishikawa cells, when it at 80 nm and the action time is 6h, induction effect on autophagy of mi R-99b-5p is most obvious, ratio of its LC3â…¡and LC3â… increases, promote beclin 1 protein and m RNA expression,cell number of autophagy bubble and autophagosome were significantly increased, respectively use 3MA and CQ inhibit autophagy process,the effection of mi R-99b-5p cells induce ishikawa autophagy are weakened.Conclusions: mi R-99b-5p targeted m TOR which is the key protein of autophagy regulating, mi R99b-5p increase LC3 and beclin1 expression in the concentration and time dependence;Inhibition of autophagy can reduce the induction effect of mi R99b-5p for ishikawa cells apoptosis.