A Mechanism Study On MiR-137-regulated Improvement Of Dexamethasone Sensitivity And Autophagic Activity In Multiple Myeloma Cells

Part I. miR-137decreases the phosphorylation of AKT and improves the drug sensitivity of multiple myeloma cells to dexamethasone via targeting MITFAim:To detect the expression of miR-137in multiple myeloma RPMI8226cells, U266cells and CD138+bone marrow mononuclear cells of patients with or without multiple myeloma. To investigate the affect of miR-137on drug sensitivity of multiple myeloma cells to dexamethasone and explore the underlying mechanism.Methods:CD138+bone marrow mononuclear cells of patients were separated by magnetic beds separation method; Taqman real-time PCR was used to detect the expression of miR-137; dual luciferase reporter gene analysis was used to verify the target of miR-137; lentivirus transfection was carried out for over-expression of miR-137, MITF, and MITF-shRNA, respectively; the mRNA level of MITF, c-MET, P53was assessed with real-time PCR; western blot or immunofluorescence were used to examine the expression of MITF, c-MET, AKT, P53, p-AKT and its substrate protein; drug sensitivity of multiple myeloma cells to dexamethasone was evaluated with MTT assay and double-labeled flow cytometry apoptosis assay.Results:miR-137was not detected in both RPMI8226cells and U266cells, and its expression in CD138+bone marrow mononuclear cells of patients with multiple myeloma significantly decreased compared with the ones of patients without multiple myeloma. Dual luciferase reporter gene analysis indicated that MITF is a target of miR-137. After the over-expression of miR-137or transfection of MITF-shRNA using lentivirus, the expression of AKT(pan) existed no significant change, at the same time, the expression of MITF, c-MET, p-AKT and its phosphorylated substrate protein apparently decreased, while the P53expression increased. In addition, over-expression of miR-137or MITF-shRNA significantly improved the36-hours inhibition rate and apoptosis rate by dexamethasone in multiple myeloma cells. Moreover, over-expression of MITF could rescue the biological effect of miR-137in multiple myeloma cells.Conclusion:miR-137improved the drug sensitivity of multiple myeloma cells to dexamethasone mainly through the mechanism of decreasing the expression of c-MET and further decreasing the phosphorylation of AKT via targeting MITF. Part II. miR-137decreases the phosphorylation of AKT and improves the autophagic activity in multiple myeloma RPMI8226cells via targeting MITFAim:To investigate the affect of miR-137on autophagic activity of multiple myeloma RPMI8226cells and explore the underlying mechanism.Methods:Lentivirus transfection was carried out for over-expression of miR-137, MITF, and MITF-shRNA, respectively; western blot was used to examine the expression of MITF, c-MET, LC3Ⅱ, AKT, p-AKT and its phosphorylated substrate protein including p-mTOR and p-Beclinl; the binding of p-Beclinl with14-3-3protein and vimentin was analyzed using co-immunoprecipitation (co-IP).Results:After the over-expression of miR-137or transfection of MITF-shRNA using lentivirus, the expression of AKT(pan) existed no significant change, at the same time, the expression of MITF, c-MET, p-AKT and its phosphorylated substrate protein including p-mTOR and p-Beclinl apparently decreased. In addition, over-expression of miR-137or MITF-shRNA promoted both the basic and starvation-induced autophagic activity in RPMI8226cells. Moreover, over-expression of MITF could rescue the biological effect of miR-137in multiple myeloma cells.Conclusion:miR-137promoted the autophagy of RPMI8226cells mainly through the mechanism of decreasing the expression of c-MET and further decreasing the phosphorylation of AKT via targeting MITF.