Research On The Influence And Its Mechanism Of Synthetic MiR-145Mimic In Multiple Myeloma

Objective：To research roles of miR-145in multiple myeloma cell proliferation,apoptosis, and invasion function in vitro and in vivo, and to elucidate itspossible mechanisms.Methods:RT-qPCR was performed to examine the the expression of miR-145in the H929, MM1S and RPMI-8226MM cell lines. MTT analysis, BrdUmethod and cell clone formation experiments were used to study theeffects of miR-145upregulation on multiple myeloma cell proliferationability and cell clone formation; the FACScan flow cytometry assays,together with Western blot were used to analysis the expression ofcell-cycle relevant proteins, such as cyclin D1and p21; TUNEL, ELISAand Western blot were performed to detect the influence of miR-145cellapoptosis to the MM, such as the effects of miR-145on caspase-3,-8and-9activity; Transwell method analysis, ELISA, together with Westernblot were used in analysing the influence of Transwell invasion andmigration of miR-145into MM cells, the secretion of VEGF andexpression of MMP-2and MMP-9, respectively. Western blot was performed to detect PI3K/AKT signaling pathway related proteins forfurther researching the mechanism of miR-145in MM inhibition; Wedetermined whether miR-145was able to inhibit tumor growth in theNOD-SCID tumor model, RT-qPCR and ELISA were used to examinedthe miR-145level and VEGF secretion in NOD-SCID tumor tissues,respectively.Results:miR-145is decreased in MM cell lines, and the plasma level ofmiR-145was signifcantly downregulated in patients with MM comparedwith healthy control subjects. The miR-145overexpression markedlyinhibited cell proliferation and colony formation in H929cells. We alsoanalyzed the effects of miR-145on the expression of cell-cycle relevantproteins, such as cyclin D1and p21, as a result, comparing to cellstransfected with the negative control, p21expression was signifcantlyincreased, while cyclin D1expression was signifcantly decreased in cellstransfected with miR-145mimic. Caspase-3,-8and-9activity in cellstransfected with miR-145mimics was signifcantly increased comparedwith those cells transfected with the negative control. It suggested that theoverexpression of miR-145induces cell apoptosis in H929cells. miR-145inhibits cell migration and invasion in H929cells. The results showedthat overexpression of miR-145mimic signifcantly inhibited VEGFsecretion in H929cells. miR-145mimic inhibited the phosphorylation of PI3K and AKT. miR-145inhibites tumor growth in vivo as well.In this study, we have shown that miR-145expression wasdownregulated in MM plasma and MM cell lines, and that the enforcedexpression of miR-145by miR-145mimic inhibited cell proliferation,migration, and invasion of H929cells. We also found that the enforcedexpression of miR-145suppressed tumor growth of MM in a NOD-SCIDmouse model. In addition, our results demonstrated that the enforcedexpression of miR-145in H929cells profoundly decreased levels ofp-AKT and p-PI3K, which may contributed to the inhibition of MM cellproliferation and survival to some extent.Conclusion:Our study identifes that miR-145may be a tumor suppressor in theprogression of MM. miR-145inhibites cell proliferation, migration andinvasion, induces cell apoptosis, and suppresses tumor growth at least inpart by inhibiting the PI3K/AKT signaling pathway. Our findings suggestthat miR-145is a potential therapeutic target for MM treatment. To thebest of our knowledge, this is the frst experimental evidence of antitumoractivity of miR-145in the treatment for MM in vitro and in vivo.