Research On Effects And Mechanism Of MiR-134 In Human Osteosarcoma Proliferation And Angiogenesis

Objective:To investigate the effects and the mechanism of miR-134 function in human osteosarcoma angiogenesis and proliferation and establish some theoretical basis for osteosarcoma clinical diagnosis and therapeutic targets for further research.Methods:(1)To detect the differential expression of miR-134 in three osteosarcoma cell lines(MG-63,U2-OS and Saos-2)and osteosarcoma tissues compared to primary osteoblasts and non-tumor bone tissues by using qRT-PCR.(2)To verify the biological function of miR-134 in virto,Saos-2 and MG-63 cells were transduced with miR-134 mimic and control and detect the function of miR-134 affected cell proliferation,apoptosis,and angiogenesis.(3)To investigate the function of miR-134 to osteosarcoma angiogenesis and proliferation in vivo,qRT-PCR analysis of the relative levels of miR-134 and VEGFA/VEGFR1 in the implanted tumors were performed.(4)The levels of VEGFA and VEGFR1 expression in human osteosarcoma and non-tumor bone tissues were determined qRT-PCR and Western blot.To verify the function of miR-134 to VEGFA/VEGFR1,ELISA were performed on the transfected cells.(5)The potential motifs for miR-134 binding in the VEGFA and VEGFR1 were predicted by bioinformatics and confirmed by using Western blot.Results:(1)The relative levels of miR-134 in MG-63,U2-OS and Saos-2 cells were similar and significantly lower than that in osteoblasts(P<0.05).Similarly,the relative levels of miR-134 in OS tissues were significantly lower than that in the non-tumor bone tissues(P < 0.0001).(2)The positive fluorescence cells transfected with miR-134 mimic or control scramble miRNA accounted for > 90%.The qRT-PCR indicated that transfection with miR-134 increased the relative levels of miR-134 expression by near 600 folds(P <0.0001).miR-134 over-expression may inhibit the proliferation of Saos-2 and MG-63 cells(P <0.001)and significantly increased the percentages of apoptotic Saos-2 cells(P<0.05)and suppressed the secretion of angiogenic stimulators(P <0.001).(3)miR-134 over-expression significantly inhibited the growth of implanted tumors and tumor blood flow(angiogenesis)in individual tumors(P <0.001).(4)We detected significantly higher levels of VEGFA and VEGFR1 expression in human osteosarcoma tumors than the non-tumor bone tissues(P <0.001).Transfection with miR-134 mimic significantly reduced the VEGF and VEGFR1 expression in both SaoS-2 and MG-63 cells in a time-dependent and dose-dependent manner(P <0.001).(5)Dual luciferase assays indicated that co-transfection with the luciferase reporter containing the 3’UTR of VEGFA/VEGFR1,together with mir-134 mimic,but not the control miRNA,significantly reduced the levels of luciferase activity in Saos-2 cells.miR-134 over-expression significantly reduced the relative levels of AKT expression and phosphorylation and the relative levels of PCNA expression in Saos-2 cells by using Western-blot.Conclusions:(1)The expression of miR-134 in osteosarcoma was closely correlated with the capacity of progression and angiogenesis.(2)miR-134 may act as a tumor suppressor in inhibiting the progression and facilitating apotosis in osteosarcoma in vitro.(3)miR-134 may be a tumor suppressor in inhibiting the progression and angiogenesis in osteosarcoma in vivo.(4)VEGFA/VEGFR1 signaling is crucial for the progression and angiogenesis of osteosarcoma.(5)miR-134 can simultaneously bind to the receptor and its ligand to inhibit the VEGFA/VEGFR1 signaling.