The Effects Of MicroRNA-145on The Biological Behaviour Of Osteosarcoma Cells And Correlated Mechanisms

Part1The expression of microRNA-145and vascular endothelial growth factor (VEGF) in osteosarcoma tissuesObjective:To investigate the expression of microRNA-145(miRNA-145) and vascular endothelial growth factor (VEGF) in osteosarcoma tissues.Methods:Twenty-eight osteosarcoma tissue samples diagnosed by pathological examination and28normorl bone tissue samples from the same patients were investigated. The expression of miRNA-145were detected in osteosarcoma tissues, the edge of osteosarcoma tissues and normal tissues by quantitative real-time RT-PCR analysis.The expression of VEGF were detected in osteosarcoma tissues, the edge of osteosarcoma tissues and normal tissues by quantitative real-time RT-PCR analysis and immunohistochemistry.Results:Under-expression of miRNA-145was observed in25of28osteosarcoma tissues in comparison with the corresponding normal tissues. MiRNA-145expression in the edge of osteosarcoma tissues was more than that in tumor tissues but less than that in the normal tissues. Over-expression of VEGF was observed in28osteosarcoma tissues in comparison with the corresponding normal tissues. VEGF expression in the edge of osteosarcoma tissues was less than that in tumor tissues but more than that in the normal tissues. The differences were all statistically significant(P<0.05).Conclusions:MiRNA-145is under-expressed in osteosarcoma tissues and VEGF is over-expressed in osteosarcoma tissues. MiRNA-145and VEGF are participated in the occurrence and development of osteosarcoma. MiRNA-145and VEGF may have correlated control relationships. Part2The effects of microRNA-145on the biological behaviour of osteosarcoma cells in vitroObjective:To investigate the effects of microRNA-145(miRNA-145) on the proliferation, apoptosis, adhesiveness, invasiveness and angiogenesis of osteosarcoma cells (MG63) in vitro.Methods:MG63cells were randomly allocated into3groups:blank group(non-transfected group), negative control transfected group and miRNA-145transfected group. The cells in miRNA-145transfected and negative control transfected groups were transfected with the miRNA-145mimics and mimics negative control respectively, by using Lipofectamine2000according to the manufacturer’s instruction. After transfection, transfection efficiency was observed.MiRNA-145expression level was detected by quantitative real-time RT-PCR. VEGF expression level was detected by Western Blot. Cell proliferation was detected by MTT proliferation assay. Cell cycle and apoptosis of MG63cells in each group were detected by flow cytometry. The adhesiveness and invasiveness of cells were detected by cell adhesion assay and transwell invasion assay respectively. Angiogenesis were detected by endothelial cell tube formation assay.Results:MiRNA-145expression in the miRNA-145transfected group was significantly up-regulated compared to the blank group and negative control group(P<0.05). VEGF expression in the miRNA-145transfected group was significantly down-regulated compared to blank group and negative control group(P<0.05). Meanwhile, cell proliferation, cycle and apoptosis, adhesiveness, invasiveness and angiogenesis of osteosarcoma cells were all significantly inhibited in the miRNA-145transfected group(P<0.05).Conclusions:MiRNA-145can dramatically inhibit proliferation, apoptosis adhesiveness, invasiveness and angiogenesis of osteosarcoma cells. MiRNA-145can promote apoptosis of osteosarcoma cells.MiRNA-145is a tumor suppressor gene for osteosarcoma. Part3The study of regulatory relationship of miRNA-145on VEGF expression in osteosarcomaObjective:To investigate the effect of miRNA-145on VEGF expression in osteosarcoma.Methods:The luciferase-UTR reporter constructs were generated by introducing the VEGF-3’UTR containing the wild-type and mutant into pMiR-Report vector. MG63cells were cotransfected with the pMiR-report vector containing the3’-UTR variants and microRNA-145mimics or mimics negative control. Experiments were divided into three groups:blank group(plasmid transfected group), negative control and plasmid cotransfected group, plasmid and miRNA-145cotransfected group.Luciferase activity was measured48h after transfection using the Dual-Glo luciferase assay system according to manufacturer’s instructions.Results:After co-transfection with pMiR-report vectors and miRNA-145or negative control, the cells over-expressing miRNA-145were observed with a significant decline in luciferase activity compared to other control groups(P<0.05).Conclusions:MiRNA-145regulate VEGF expression at the translational level by interacting with the3’UTR of VEGF in osteosarcoma. VEGF is a direct target gene of microRNA-145. Part4The effects of microRNA-145on the biological behaviour of osteosarcoma cells in vivoObjective:To investigate the effects of microRNA-145(miRNA-145) on the growth and angiogenesis of osteosarcoma in vivo.Methods:Sixteen nude mice(Balb/c) were randomly divided into2groups, experimental group and control group,8mice in each group. The animal model of experimental group was constructed by subcutaneous inoculation of osteosarcoma cells transfected with miRNA-145into nude mice and control group by subcutaneous inoculation of osteosarcoma cells.The osteosarcoma-bearing nude mice were feeded for4weeks. After4weeks, tumor formation rate, size and weight of tumor were observed in each group. VIII factor expression and VEGF expression of tumor were detected by immunohistochemistry and microvessel density were calculated.Results:The tumor formation rate in control group is87.5%and experimental group is37.5%.The size and weight of tumor in experimental group is significantly less than control group(P<0.05). The VEGF expression and MVD of tumor in experimental group is significantly lower than those in control group(P<0.05).Conclusions:MiRNA-145can inhibit the osteosarcoma formation rate of nude mice, and inhibit the growth and angiopoiesis of tumor in vivo.