Research On Effects Of MiR-15a And MiR-16-1on Apoptosis And Proliferation Of Human Osteosarcoma Cell Line SOSP-9607

BackgroundOsteosarcoma, the most common primary malignant bone tumor in childhood andadolescence, causes many deaths due to its rapid proliferation and drug resistance.Recent studies have shown that microRNAs play a key regulatory role duringtumorigenesis, proliferation, metastasis, etc. The aim of the study is to determinethe role of miR-15a and miR-16-1in controlling SOSP-9607cell lineproliferation, cell cycle and apoptosis.ObjectiveThe purpose of this research is to find the possible changes of SOSP-9607inapoptosis, proliferation and cell cycle which caused by upregulate (ordownregulate) miR-15a and miR-16-1by microRNA mimics (or inhibitors).Methods （1） miR-15a and miR-16-1mimics (inhibitors) were transfected intoSOSP-9607by using LipofectamineT M2000, which may deregulate theexpression of miR-15a and miR-16-1. The transfection rate was measuredafter4-6hours.（2） To find the deregulation of apoptosis in SOSP-9607,the cells werecollected and checked through flow cytometer48hours after transfection.Meanwhile, TUNEL assay was also used to examine the apoptotic cells.（3） To check the cell cycle changes of SOSP-9607,cells was collected andused for flow cytometer48hours after transfection.（4） MTT was performed in24,48,72, and96h, respectively. Then draw thecurve of cell proliferation and examine the proliferation efficiency.Result（1） The SOSP-9607cells transfected with miR-15a and miR-16-1mimicsobtain higher apoptosis ratio compared with the inhibitor and control groups.There was no observed difference between the inhibitor and control groups.TUNEL assay which was used to confirm DNA breakage and chromatincondensation showed that more apoptotic cells are significantly found inmiR-15a and miR-16-1groups, and no difference is found among the levelsof apoptosis in the inhibitor, negative, and blank control groups.（2） Cell cycle analysis showed that more cells are in the G0/G1phase in themicroRNAs treated cells than in the controls. In contrast, cells transfectedwith miR-15a and miR-16-1inhibitors had no significant changes in cellcycle.（3） MTT assay indicated that the proliferation curves of cells transfectedwith miR-15a and miR-16-1are slower compared with that of the othergroups. However, no differences are found compared with the inhibitor,negative control and blank groups, respectively Conclusion（1） Upregulation of miR-15a and miR-16-1could induce apoptosis ofSOSP-9607. There were no significant changes among cells transfected withmicroRNA inhibitors, which indicate in SOSP-9607cell line, miR-15a andmiR-16-1was low expressed or inhibited.（2） miR-15a and miR-16-1could arrest cell cycle and let them stay atG0/G1phase.（3） Upregulation of miR-15a and miR-16-1may inhibit the proliferation ofSOSP-9607cells. While downregulation of miR-15a and miR-16-1bymicroRNA ibhibitors had no effect on cell proliferation. This result couldalso demonstrate the hypothesis that miR-15a and miR-16-1was lowexpressed or inhibited in nomal SOSP-9607cells.