MicroRNA-19a Regulates Apoptosis And Invasion By Regulating PTEN/AKT/pAKT Pathway In Myeloma Cells

Objective: 1. To investigate the expression of miR-19 a, one of the oncogenic miR-17-92 cluster, in mutiple myeloma cell lines Lp-1 and U266 in vitro. 2. To detect the role of miR-19 a upregulation on biological behavior, such as proliferation, migration and apoptosis of myeloma cell lines Lp-1 and U266 by transfected with miR-19 a mimics. 3. To explore the expression of miR-19 a in myeloma and the relation between PTEN/AKT/p AKT signaling pathway and apoptosis/invason by transfection of miR-19 a mimics.Methods: 1. Reverse transcription-PCR was applied to examine the expression of miR-19 a in mutiple myeloma cell lines Lp-1 and U266 in vitro. 2. CCK8 method was used to test the influence of upregulation miR-19 a on the proliferation of mutiple myeloma cell lines Lp-1 and U266 in vitro. 3. Transwell migration test was adopted to determin the influence of upregulation miR-19 a on the migration of mutiple myeloma cell lines Lp-1 and U266 in vitro. 4. Anexin V-FITC/PI flow cytometry was employed to assay the apoptosis rates of pre-and post-transfection of miR-19 a in mutiple myeloma cell lines Lp-1 and U266 in vitro. 5. The level of apoptosis and drug resistance related gene bcl-2 and mdr-1 was detected with real time fluorescence quantitive(RT-PCR) after transfected with miR-19 a. 6. Western Blot was performed to study the level of apoptosis and drug resistance related protein BCL-2 and MDR-1 after transfected with miR-19 a. And futhermore, to analysis the regulatory effect of PTEN/AKT/p AKT signaling pathway on myeloma cells.Results:1. The expression of miR-19 a was higher in mutiple myeloma cell lines Lp-1 and U266; Compared with cells transfected with a specific miR-19 a NC, those samples transfected with miR-19 a mimic displayed significantly higher expression of miR-19a(p<0.05), which means higher transfection efficiency. 2. CCK8 method found that upregulation miR-19 a could promote the proliferation of mutiple myeloma cell lines Lp-1 and U266 in vitro. 3. Transwell migration test revealed that upregulation miR-19 a could promote migration ability of mutiple myeloma cell lines Lp-1 and U266 in vitro. 4. Anexin V-FITC/PI flow cytometry displayed that the apoptosis rates was decreased after transfection of miR-19 a mimics in mutiple myeloma cell lines Lp-1 and U266 in vitro. 5. RT-PCR assay revealed that both the level of apoptosis and drug resistance key regulatory related gene bcl-2, mdr-1 were overexpressed after upregulation miR-19 a. That is, miR-19 a antagonized myeloma cells drug resistance through the perturbation of bcl-2 and mdr1. 6. Western Blot was performed to study the level of apoptosis and drug resistance key regulatory related protein BCL-2 and MDR-1 after transfected with miR-19 a. And futhermore, to analysis the regulatory effect of PTEN/AKT/p AKT signaling pathway on myeloma cells. 7. Down-expression of PTEN could significantly attenuate the inhibitory effects of knockdown miR-19 a on the proliferation of myeloma cells.Conclusion: Cumulative evidence indicates PTEN/AKT/p AKT signaling pathway is closely related to cancer proliferation, apotosis, migration, invasion, drug resistance and tumor immune escape. Therefore, dysregulation of this pathway is important in the pathophysiology of human malignancies. Thus blocking PTEN/AKT/p AKT signaling pathway by targeting some critical protein molecules, ultimately can protect the tumor cells from unlimited proliferation, invasion and drug resistance. The expression of miR-19 a was higher in mutiple myeloma cell lines and miR-19 a may regulate the biological behavior through inhibition of PTEN; Meanwhile, it may activate the PTEN/AKT/p AKT signaling pathway and futherly promote the proliferation, migration and apoptosis of myeloma, which is responsible for the initiation and development of cancer. Our results suggest that miR-19 a acted as an oncomiR by targeting PTEN in myeloma. This novel miR-19a/PTEN/AKT axis sheds new light on the mechanisms underlying apoptosis and invision and possibly provide future therapeutic targets for the treatment of myeloma.