miR-200c通过AKT2对骨肉瘤细胞增殖的影响

Effects of miR-200c on proliferation of osteosarcoma cells by targeting AKT2

[目的]探讨过表达mi R-200c通过对AKT2的相关调控对骨肉瘤细胞增殖能力的影响,为骨肉瘤的靶向治疗提供新的依据。[方法](1)检测mi R-200c在正常成骨细胞和4种骨肉瘤细胞株中的表达差异;(2)将mi R-200c转染至骨肉瘤细胞株MG-63中,构建稳定过表达mi R-200c骨肉瘤细胞株;(3)预测mi R-200c与AKT2基因的相关结合位点,构建相应的荧光素酶基因表达载体,连同过表达mi R-200c质粒共转染至骨肉瘤细胞株MG-63中;(4)检测两组骨肉瘤细胞株MG-63荧光素酶活性;(5)检测两组骨肉瘤细胞株MG-63中AKT2表达水平;(6)观察两组骨肉瘤细胞株MG-63的增殖能力。[结果](1)与正常成骨细胞(NHOst)相比,mi R-200c在4种骨肉瘤细胞株(HOS、U2OS、Saos-2、MG-63)中表达量明显降低(P<0.05);(2)与mi R-NC组相比,过表达mi R-200c组骨肉瘤细胞株MG-63中AKT2 3′-UTR序列结构野生型(WT)的荧光素酶活性明显降低(P<0.05),而突变型(MUT)的荧光素酶活性无明显差异(P>0.05);(3)与mi R-NC组相比,过表达mi R-200c的骨肉瘤细胞株MG-63中AKT2表达明显降低(P<0.05);(4)与mi R-NC组相比,过表达mi R-200c骨肉瘤细胞株MG-63增殖能力明显下降(P<0.05)。[结论]mi R-200c在骨肉瘤细胞中低表达,而过表达mi R-200c通过靶向调控AKT2表达抑制骨肉瘤细胞的增殖。因此mi R-200c可能对未来骨肉瘤的预防及治疗提供新的策略。

[Objective] To explore the effects of overexpressed mi R-200 c on the proliferation of osteosarcoma cells through regulating AKT2,and provide new evidences for the targeting treatment of osteosarcoma.[Methods] The expression difference of mi R-200 c in normal osteoblasts and osteosarcoma cell lines was detected.In addition,the mi R-200 c into osteosarcoma cell MG-63 was transfected,then stable overexpressed mi R-200 c osteosarcoma cell line was set up.Furthermore,the related binding sites of mi R-200 c and AKT2 gene were predicted,after that,corresponding luciferase gene expression vector,and co-transfect the overexpressed mi R-200 c plasmid into osteosarcoma cell MG-63 were created.The activity of luciferase in two groups of osteosarcoma cell MG-63 and the expression of AKT2 in two groups of osteosarcoma cell MG-63 were detected.Moreover,the proliferation ability of two osteosarcoma cell MG-63 was observed.[Results] Compared with normal osteoblasts（NHOst）,mi R-200 c expression in osteosarcoma cell lines（HOS,U2 OS,Saos-2,MG-63） decreased significantly（P0.05）.Compared with group mi R-NC,the luciferase activity of AKT2 3＇-UTR sequence structure（WT） in mi R-200 c overexpression group was significantly decreased（P0.05）,while the activity of luciferase in mutant type（MUT） kept unchanged（P〈0.05）.Compared with the mi R-NC group,the expression of AKT2 in osteosarcoma cells expressing mi R-200 c was significantly decreased（P〉0.05）.Compared with the mi R-NC group,the proliferation ability of overexpressed mi R-200 c osteosarcoma cells decreased significantly（P〈0.05）.[Conclusion] Mi R-200 c has low expression in osteosarcoma cells,and overexpression of mi R-200 c can inhibit osteosarcoma cell proliferation by targeting AKT2.Therefore,mi R-200 c may provide a new strategy for the prevention and treatment of osteosarcoma in the future.