microRNA-199b-5p在尤文肉瘤细胞系中的功能研究  

FUNCTION OF MicroRNA-199b-5p IN EWING'S SARCOMA CELL LINES

作　　者：李卫华[1] 王晓[1] 张永乐[1] 郭建阔[1] 潘华刚[1] 

机构地区：[1]河南大学淮河医院骨科,河南开封475000

出　　处：《中国修复重建外科杂志》2015年第5期595-599,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(51375142)~~

摘　　要：目的探讨microRNA-199b-5p(mi R-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mi R-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mi R-199b-5p m RNA相对表达量,并与MSCs比较;细胞计数试剂盒8法检测mi R-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mi R-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mi R-199b-5p m RNA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mi R-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 mi R-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。Objective To investigate the function and possible mechanism of microRNA-199b-5p（mi R-199b-5p） in Ewing’s sarcoma cell lines so as to provide theoretical basis for biological treatment in the future.Methods A673 cells and TC252 cells were adopted as Ewing’s sarcoma cell lines in vitro.mi R-199b-5p oligonucleotide fragments（mimic） and scramble control（mimic control） were used to transfect TC252 cells and A673 cells,respectively.The expression of mi R-199b-5p was measured in 2 cell lines by real-time quantitative-PCR and compared with that in mesenchymal stem cells（MSCs）.The cell proliferation was examined by cell counting kit 8.The cell cycle and apoptosis were detected by flow cytometry.The possible targets of mi R-199b-5p were determined by luciferase assays.The protein expressions of possible targets were measured by Western blot.Results The expression of mi R-199b-5p in control group was significantly down-regulated in A673 cells and TC252 cells when compared with that in MSCs（P〈0.05）;and the expression of mi R-199b-5p in experimental group was significantly up-regulated when compared with that in control group（P〈0.05）.G1 phase cells increased obviously and S phase cells decreased significantly compared with corresponding cells in control group（P〈0.05）;but no significant difference was found in G2/M phase cells between 2 groups（P〉0.05）.The apoptosis rate increased significantly in experimental group when compared with that in control group（P〈0.05）.The possible targets of mi R-199b-5p were cyclin-L1（CCNL1） and c-Kit by luciferase assays.Western blot results showed that the CCNL1 and c-Kit protein expression levels in experimental group were significantly lower than those in control group（P〈0.05）.Conclusion mi R-199b-5p can suppress the cell proliferation,block the cell cycle,and promote the cell apoptosis,so mi R-199b-5p inhibits Ewing’s sarcoma cell lines by targeting CCNL1 and c-Kit.

关 键 词：尤文肉瘤 microRNA-199b-5p 细胞增殖 细胞周期调节蛋白1 C-KIT 

分 类 号：R738.1[医药卫生—肿瘤]