miR-195-5p调控VEGFA对骨肉瘤MG-63细胞转移能力的影响

Effect of mir-195-5p on Metastasis of Osteosarcoma MG-63 Cells by Targeting VEGFA

目的研究miR-195-5p对骨肉瘤MG43细胞转移能力的影响。方法RT-PCR检测miR-195-5p和VEGFAmRNA表达水平,基因预测软件预测miR-195-5p靶向基因,荧光素酶报告实验分析靶向关系;利用Li-pofectamine.2000转染试剂盒将miR-195-5pmimic和pcDNA-VEGFA单独或联合转染骨肉瘤MG63;TRANSWELL检测细胞侵袭,划痕实验检测细胞迁移,Western印迹检测VEGFA、p-p38、MMP-2、MMP-9表达水平。结果与正常组织相比,骨肉瘤MG-63细胞中miR-195-5p表达降低、VEGFA表达升高;miR-195-5p与VEGFA在3'UTR区存在结合位点,miR-195-5p直接负作用于VEGFA;miR-195-5p过表达后,MG-63细胞侵袭数目减少、划痕变宽,伤口愈合率降低,VEGFA,p-p38、MMP-2和MMP-9的蛋白表达量下调(P<0.01)。结论miR-195-5p通过靶向抑制VEGFA来抑制骨肉瘤MG-63细胞迁转移能力。

Objective To investigate the effect and mechanism of mir-195-5p on the metastasis of osteosarcoma MG-63 cells by targeting VEGFA. Methods The expression levels of miR-195-5p and VEGFA mRNA were detected by RT-PCR. The miR-195-5p targeting gene was predicted by gene prediction software, and the targeting relationship was analyzed by luciferase reporter assay. miR-195-5p mimic and pcDNA-VEGFA were separately or jointly transfected into osteosarcoma MG63 cells by using Li-pofectamineTM2000 transfection kit. Cell invasion was detected by Transwell, cell migration by scratch test, and expression levels of VEGFA, p-p38 , MMP-2 and MMP- 9 by Western blotting. Results The expression of miR-195-5p was decreased and the expression of VEGFA was increased in osteosarcoma MG-63 cells as compared with those in normal tissues. miR195-5p and VEGFA had a binding site in the 3'UTR region, and miR-195-5p directly and negatively acted on VEGFA. After miR-195-5p overexpression, the number of invading osteosarcoma MG-63 cells was significantly reduced, the scratch wound was significantly wider, the wound healing rate was significantly decreased, and the protein expression levels of VEGFA, p-p38, MMP-2 and MMP-9 were significantly down-regulated (P < 0. 01).Conclusion miR-195-5p inhibits the metastasis of osteosarcoma MG-63 cells by targeting inhibition of VEGFA.