Effects And Mechanisms Of Mir-221 In Osteosarcoma Cells

Part 1:Expression of miR-221 in osteosarcoma cellsObjective:1.To determine whether there was a consistent upregulation or downregulation in the process,the expression of miR-221 in different osteosarcoma cell lines compared with human osteoblast cell line(control)were investigated.2.To screen out suitable cell lines from various osteosarcoma cell lines for further studies.Methods:1.Human osteosarcoma cell lines(HOS,SaOS2,MG63,U-20S)and human osteoblast cell line(hFOB1.19)were cultured.2.The expression of miR-221 in these osteosarcoma cell lines and human osteoblast cell line were detected by using quantitative real-time PCR(qRT-PCR).Results:1.Relative value of miR-221 expression in hFOB1.19,HOS,SaOS2,MG63,U-20S cell lines were 0.03175910.004444,0.03771710.003754,0.05615110.003706,0.10402610.013724,and 0.14481210.010018,respectively.Relative value of miR-221 expression in osteosarcoma cells of each group were significantly different from that in the control group(P<0.05).2.The relative value of miR-221 expression in MG63 and U-20S cells were significantly different from that in the control group(P<0.01).The expression of miR-221 in MG63 and U-20S cells were markedly increased.Conclusion:The expression of miR-221 in different cell lines of osteosarcoma were significantly higher than that in osteoblast cell line.The expression of miR-221 in MG63 and U-20S cells were increased significantily.Therefore MG63 and U-20S cells were used for further experiments.Part 2:Effects of miR-221 inhibitor on the biological behavior of osteosarcoma cellsObjective:To study the effects of miR-221 inhibitor on cell cycle,proliferation,apoptosis,migration and invasion of osteosarcoma cells,and provide evidences for further mechanism research.Methods:1.The expression of miR-221 in MG63 and U-20S cells transfected with miR-221 inhibitor or NC inhibitor were detected by qRT-PCR.2.MTT assays were used to examine cell proliferation at 0,24,48 and 72 hours in MG63 and U-20S cells after transfected with miR-221 inhibitor or NC inhibitor.3.The cell cycle and apotosis of MG63 and U-20S cells transfected with miR-221 inhibitor or NC inhibitor were analyzed by flow cytometry assays.4.The migration and invasion of MG63 and U-20S cells transfected with miR-221 inhibitor or NC inhibitor were detected by transwell assaysResults:1.The miR-221 inhibitor significantly downregulated the expression of miR-221 in MG63 and U-20S cells(P<0.01).The significant decrease of miR-221 expression demonstrated the success of cell transfection.2.The proliferation of MG63 and U-20S cells were obviously inhibited at 24(P<0.01),48(P<0.01)and 72(P<0.01)hours after transfected with miR-221 inhibitor compared with those transfected with NC inhibitor.3.The percentage of G0-G1 phase cells in MG63 and U-20S cells transfected with miR-221 inhibitor were significantly increased compared with those transfected with NC inhibitor(P<0.01).The apoptosis rate in MG63 and U-20S cells transfected with miR-221 inhibitor were significantly increased compared with those transfected with NC inhibitor(P<0.01).4.The number of migrated and invasive cells in MG63 and U-20S cells transfected with miR-221 inhibitor were significantly reduced compared with those transfected with NC inhibitor(P<0.01).Conclusion:Downregulation of miR-221 expression can significantly inhibit the cell proliferation,increase cell apoptosis rate,promote cell cycle arrest,and decrease the ability of cell migration and invasion in MG63 and U-20S cells.Part 3:Mechanisms of miR-221 in osteosarcoma cellsObjective:1.To search for the target gene of miR-221.2.To study the effects on the biological behavior of osteosarcoma cells by altering the expression of target gene of miR-221.3.To reveal the mechanisms of miR-221 affecting the biological behavior of osteosarcoma cells.Methods:1.Targetscan analysis were used to identify the target gene of miR-221.2.pGL3-CDNK1B-3'-UTR(wild type or mutant plasmid)together with pRL-SV40 renilla plasmid were cotransfected with miR-NC or miR-221 mimics into MG63 and U-20S cells,and activities of luciferase were detected by the dual-luciferase reporter system.3.The expression of CDKN1B/p27 in MG63 and U-20S cells transfected with miR-NC or miR-221 mimics were detected by qRT-PCR and Western blot.4.The effects of CDKN1B knockdown on U-20S cell cycle,proliferation,apoptosis,migration and invasion of cells were investigated.5.The protein expressions of p27,cyclin E,cyclin D1,Bcl-2,caspase-3,Snail,and Twistl were measured by Western blot analysis.Results:1.According to the Targetscan analysis,CDKN1B/p27 was a binding target of miR-221.2.The relative luciferase activities of osteosarcoma cells transfected with miR-221 mimics were significantly lower than which transfected with miR-NC(P<0.01).The luciferase activity assays showed that overexpression of miR-221 inhibited transcription of the CDKN1B/p27 through binding the 3'-UTR site of the latter.3.The expression of CDKN1B mRNA were significantly downregulated in MG63 and U-20S cells after transfected with miR-221 mimics(P<0.01).The expression of p27 were significantly downregulated in MG63 and U-20S cells after transfected with miR-221 mimics(P<0.05).4.Transfection of U-20S cells with miR-221 inhibitor and siCDKNIB significantly increased cell proliferation(P<0.01),inhibited G0/G1 arrest(P<0.01),decreased apoptosis rate(P<0.01),and increased the cell number of migration and invasion(P<0.01).5,miR-221 inhibitor markedly increased the levels of Bax and caspase-3 and decreased cyclin E,cyclin D1,Bcl-2,Snail,and Twistl protein in U-20S cells(P<0.01).However,downregulation of CDKN1B in U-20S cells significantly reversed these protein expressions induced by miR-221 inhibitor(P<0.01).Conclusion:miR-221 regulates osteosarcoma cell cycle,proliferation,apoptosis,migration and invasion by targeting CDKN1B/p27.Our study demonstrated the functional existence of miR-221/CDKN1B/p27 in osteosarcoma cells.The effects of miR-221 downregulation in osteosarcoma cells depend specifically on CDKN1B increase.