MiR-136通过下调靶基因ITGAV表达抑制骨肉瘤细胞增殖及迁移的分子机制

Molecular mechanism of miR-136 inhibiting proliferation and migration of osteosarcoma cells by down-regulating the target gene integrin subunit alpha V

目的:分析miR-136对骨肉瘤(osteosarcoma,OS)细胞生物学行为的调控机制研究,揭示miR-136在OS中的作用及其机制。方法:检测OS组织和癌旁正常组织中miR-136和整合素αV亚基(integrin subunit alpha V,ITGAV)的表达;采用TargetScan和双荧光素酶报告基因实验、反转录PCR和蛋白质印迹法证实ITGAV是否为miR-136的靶基因;采用CCK-8法、流式细胞法和细胞划痕试验检测miR-136和ITGAV对MG63细胞增殖、周期和迁移的影响。构建OS小鼠模型验证miR-136以及ITGAV对体内肿瘤生长的影响。结果:在OS癌组织中,miR-136明显降低,而ITGAV的表达则明显升高。miR-136能特异性结合ITGAV的3'-UTR并负调控ITGAV。细胞实验结果显示:ITGAV组可以提高OS细胞MG63和U2OS增殖、S期细胞比例以及迁移能力,而miR-136组可抑制OS细胞MG63和U2OS增殖、S期细胞比例以及迁移能力,而miR-136+ITGAV组可以逆转miR-136组对MG63和U2OS细胞增殖、S期细胞比例以及迁移能力的抑制效果。OS小鼠模型显示:miR-136在裸鼠体内具有抑制OS生长的作用,ITGAV在miR-136 mimic组中的表达较对照组明显降低,而在miR-136 inhibitor组中出现相反的效果。结论:MiR-136的异常表达可能与OS有关,miR-136通过靶向调控ITGAV的表达,从而抑制OS细胞的生物学行为以及体内肿瘤生长,提示miR-136可能具有抑制OS的作用。

Objective:To analyze the regulatory mechanism of miR-136 on the biological behavior of osteosarcoma cells,and reveal the role and mechanism of miR-136 in osteosarcoma.Methods:The expressions of miR-136 and ITGAV in OS tissues and adjacent normal tissues were detected.TargetScan,Dual-Luciferase Reporter Assay,reverse transcription polymerase chain reaction(RT-PCR)and Western blotting were used to confirm whether ITGAV was the target gene of miR-136.The effects of miR-136 and ITGAV on the proliferation,cycle,and migration of MG63 cells were determined by using CCK-8,flow cytometry and Scratch-wound assay.The mouse model of osteosarcoma was established,and the effect of miR-136 on ITGAV expression was detected by using RT-PCR and Western blotting.Results:We found that the expression of miR-136 in OS tissues was significantly decreased,while the expression of ITGAV was increased.MiR-136 could specifically bind to the 3'-UTR of ITGAV and negatively regulate ITGAV.Adopting cell experiments,cell proliferation,S-phase cell ratio,and migration ability of MG63 and U2OS osteosarcoma cell were increased in ITGAV group,while such ability was inhibited in miR-136 group,and the miR-136+ITGAV group could reverse the inhibitory effects of miR-136 group on MG63 and U2OS cell proliferation,S-phase cell ratio,and migration ability.In nude mouse model,miR-136 could inhibit osteosarcoma development,and ITGAV expression was decreased in the miR-136 mimic group as comparing with the control group,but an opposite result occurred in the miR-136 inhibitor group.Conclusion:The abnormal expression of miR-136 may be associated with osteosarcoma.MiR-136 inhibits the biological behavior of osteosarcoma cells and in vivo tumor growth by targeting the expression of ITGAV,suggesting that miR-136 may pose tumor-suppressive effects.