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-  2015 

microRNA-199b-5p在尤文肉瘤细胞系中的功能研究

DOI: doi:10.7507/1002-1892.20150129

Keywords: 尤文肉瘤, microRNA-199b-5p, 细胞增殖, 细胞周期调节蛋白1, c-Kit

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Abstract:

目的探讨microRNA-199b-5p(miR-199b-5p)在尤文肉瘤细胞系中的功能,分析其作用机制,以期为临床生物学治疗提供理论依据。 方法取人尤文肉瘤细胞系A673、TC252,分别以miR-199b-5p寡核苷酸片段(mimic)及其阴性对照(mimic control)转染,作为实验组及对照组。实时荧光定量PCR检测miR-199b-5p mRNA相对表达量,并与MSCs比较;细胞计数试剂盒8法检测miR-199b-5p对细胞增殖的影响;流式细胞术法检测对细胞周期及凋亡的影响。双荧光报告基因实验初步检测miR-199b-5p可能作用的靶基因,Western blot验证靶基因。 结果实时荧光定量PCR检测示,对照组A673细胞及TC252细胞中的miR-199b-5p mRNA相对表达量与MSCs相比均显著下降(P<0.05),与实验组对应细胞相比亦显著下降(P<0.05)。与对照组对应细胞相比,实验组处于G1期细胞明显增多,S期细胞明显减少,比较差异均有统计学意义(P<0.05);而两组G2/M期细胞无明显差异(P>0.05)。实验组细胞凋亡率与对照组对应细胞相比显著增高(P<0.05)。双荧光报告基因实验检测miR-199b-5p可能作用的靶基因是细胞周期调节蛋白1(cyclin-L1,CCNL1)和c-Kit。Western blot检测显示,与对照组对应细胞相比,实验组CCNL1和c-Kit蛋白相对表达量明显下调,差异均有统计学意义(P<0.05)。 结论miR-199b-5p能抑制尤文肉瘤细胞增殖,阻滞细胞周期转换,促进细胞凋亡,其抑制作用是通过靶向CCNL1和c-Kit实现的

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