The Effect Of MicroRNA-375 On Myeloma Cell Survival By Targeting PDK1 And IGF-1R And The Underlying Molecular Mechanisms

Objective:1.The roles of tumor-suppressor gene of miR-375 in multiple myeloma cells were investigated through the effect of cell proliferation and apoptosis triggered by the transfection of miR-375 mimic into the myeloma cell lines(MM1.S and RPMI-8226).2.The underlying anti-myeloma mechanisms of miR-375 were explored via determining the level of PDK1 and IGF-1R in myeloma cells transfected with miR-375 mimic.Methods:1.In MM1.S and RPMI-8226 cells transfected with miR-375 mimic by Lipofectamine-mediated transfection,the rate of transfection was examined in fluorescence micrcscope and the level of miR-375 was measured by q RT-PCR to determine the transfection effect.2.The proliferative changes were investigated in MM1.S and PRMI-8226 cells with or without transfection of miR-375 mimic by CCK-8 method.3.The apoptosis rates were assayed in MM1.S and PRMI-8226 cells with or without transfection of miR-375 mimic by Annexin V-FITC/PI method.4.The cell cycle was tested in MM1.S and PRMI-8226 cells with or without transfection of miR-375 mimic using PI staining method by fiow cytometer.5.The m RNA levels of PDK1 and IGF-1R genes were measured in MM1.S and RPMI-8226 cells before and after transfection of miR-375 mimic by q RT-PCR.5.The epxression of PDK1 and IGF-1R protein were examined in MM1.S and RPMI-8226 cells before and after transfection of miR-375 mimic by Western-blot.Results:1.MiR-375 mimic transfection caused the significant increase of miR-375 m RNA level to 42.85 and 51.30 times in MM1.S and RPMI-8226 cells respectively,indicating the successful transfection of miR-375 into myeloma cells.2.Compared with the blank group and NC group,miR-375 mimic transfection attenuated the proliferative action of myeloma cells(i.e.MM1.S and RPMI-8226).3.When compared with the blank group and NC group,the cell apoptosis was promoted in MM1.S and RPMI-8226 cell transfected with miR-375 mimic.4.In comparison with the blank group and NC group,miR-375 mimic transfection substantially increased the induction of cell cycle arrest in G1 phase in MM1.S and RPMI-8226 cells.5.Compared with the blank group and NC group,the expression of PDK1 m RNA and protein in MM1.S and RPMI-8226 cells was decreased after transfection with miR-375 mimic.6.In comparison with the blank group and NC group,miR-375 mimic transfection in MM1.S and RPMI-8226 cells reduced the expression of IGF-1R m RNA and protein.Conclusions:1.MiR-375 functions as a tumor suppressor in multiple myeloma cells by inducing apoptosis,inhibiting proliferativity and arresting G1 phase of cell cycle.2.MiR-375 plays anti-myeloma activity in multiple myeloma cells via downregulation of PDK1 and IGF-1R.