17β-estradiol Regulates The Expression Of MiR-155and TP53INP1in MCF-7Breast Cancer Cells And Its Significance

Objective1. To investigate the effects of miR-155on cellproliferation, cell cycle and cell apoptosis of human breast cancer MCF-7cells and try to explore the mechanisms of miR-155in this process; and toverify whether TP53INP1is a target of miR-155.2. To explore whethermiR-155involved in E2regulated expression of estrogen responsive genes.Methods1. The expression level of ERα in human breast cancerMCF-7and MDA-MB-231cells was evaluated by western blot; differentialexpression of miR-155between MCF-7and MDA-MB-231cells wasdetected by Real-time quantitative PCR; and ERα (+) MCF-7cells wereused as the research model.2. The effect of E2on the proliferation ofMCF-7cells was investigated with CCK-8assay so as to determining theappropriate concentration of E2for the future experiments.3. Transienttransfection of MCF-7cells with miR-155m (or miR-155m NC) andmiR-155i (or miR-155i NC) using lipfectamineTM2000.4. Luciferase reportassay was performed to indicate the direct target of miR-155.5. Theexpression levels of miR-155and TP53INP1mRNA were detected byReal-time quantitative PCR after differential treatment.6. Cell proliferationof MCF-7cells was evaluated by CCK-8assay after differential treatment.7. Cell cycle and cell apoptosis rate of MCF-7cells were evaluated by FCMassay after differential treatment.8. The protein expression levels of TP53INP1, cleaved-caspase-3,-8,-9and p21in MCF-7cells wereevaluated by western blot.9. The protein expression levels of p21andcleaved-caspase-3in MCF-7cells were evaluated by immunofluorescence.Results1. The expression of ERα in human breast cancer MCF-7cellsis positive; we chose the appropriate concentration of E2with100nM forthe future experiments; and the expression of miR-155is significantlyhigher in MCF-7cells compared with MDA-MB-231cells.2. Luciferasereporter reveales that TP53INP1is targeted by miR-155.3. MiR-155negatively regulates TP53INP1mRNA expression and the proteinexpression of TP53INP1, cleaved-caspase-3,-8,-9and p21; andoverexpression of miR-155promotes cell proliferation and suppress cellapoptosis, whereas abrogating expression of miR-155suppresses cellproliferation and promotes cell apoptosis of MCF-7cells.4. E2up-regulatesmiR-155expression and negatively regulates TP53INP1mRNA expressionand the protein expression of TP53INP1, caspase-3,-8,-9and p21,whereas transfection with miR-155i gave the opposite results.Conclusions1. MiR-155is significantly overexpressed in MCF-7cells with ERα (+).2. TP53INP1is the direct target of miR-155; andmiR-155contributes to cell proliferation of MCF-7cells possibly throughnegatively regulating TP53INP1, caspase-3,-8,-9and p21.3. E2promotesbreast cancer development and progression possibly through up-regulatingmiR-155expression and negatively regulates TP53INP1, caspase-3,-8,-9and p21.