miRNA-199a-5p靶向CDCA7L对胶质瘤细胞迁移及侵袭的影响  

Effects of miRNA-199a-5p targeting CDCA7L on migration and invasion of glioma cells

作　　者：兰伟途 武峰 何建昌 兰文达 王万宏 Lan Weitu;Wu Feng;He Jianchang;Lan Wenda;Wiang Wanhong(Department of Neurosurgery,Cangzhou People*s Hospital,Cangzhou 061000,China)

机构地区：[1]河北省沧州市人民医院神经外科,沧州061000

出　　处：《中华细胞与干细胞杂志（电子版）》2021年第5期272-278,共7页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：河北省2020年度医学科学研究课题计划(20200172)。

摘　　要：目的探讨miR-199a-5p靶向细胞分裂周期相关7样蛋白(CDCA7L)对胶质瘤细胞迁移及侵袭的影响。方法采用实时荧光定量PCR(RT-qPCR)检测人正常星形胶质细胞HA1800与人胶质瘤细胞株U251中miR-199a-5p表达量;使用Lipofectamine 2000转染试剂盒对U251细胞进行转染,并分别命名为空白组(正常培养的U251细胞)、miR-NC组(转染miR-NC)、miR-199a-5p mimic组(转染miR-199a-5p mimic)、miR-199a-5p mimic+pcDNA组(共转染miR-199a-5p mimic和pcDNA)、miR-199a-5p mimic+pcDNA-CDCA7L组(共转染miR-199a-5p mimic和pcDNA-CDCA7L),Transwell实验检测U251细胞迁移、侵袭能力,Western blot检测CDCA7L蛋白表达情况;利用TargetScan软件预测miR-199a-5p和CDCA7L结合位点;双荧光素酶报告基因检测验证miR-199a-5p与CDCA7L靶向关系;HA1800、U251两组细胞间的比较采用两样本t检验;多组间比较采用单因素方差分析,两组间比较采用SNK-q检验。结果与HA1800细胞比较,U251细胞中miR-199a-5p表达水平降低(1.09±0.26比0.32±0.14),CDCA7L蛋白表达量升高(0.21±0.02比0.85±0.13),差异具有统计学意义(P均<0.001)。miR-199a-5p mimic组细胞迁移数、侵袭数及CDCA7L蛋白表达量均低于空白组、miR-NC组(50.12±3.36比94.57±7.14、93.86±6.11;42.53±3.34比71.49±4.35、73.76±4.21;0.25±0.07比0.83±0.12、0.89±0.09),差异具有统计学意义(P均<0.001)。CDCA7L为miR-199a-5p靶基因。过表达CDCA7L可逆转miR-199a-5p对U251细胞迁移及侵袭的抑制作用(P<0.05)。结论在胶质瘤细胞中miR-199a-5p呈低表达,上调miR-199a-5p可能通过靶向抑制CDCA7L蛋白表达,进而抑制胶质瘤细胞的迁移和侵袭。Objective To investigate the effects of miR-199a-5p targeting cell division cycle-associated 7-like protein (CDCA7L) on the migration and invasion of glioma cells.Methods Real-time fluorescent quantitative PCR (RT-qPCR)was used to detect the expression of miR-199a-5p in human normal astrocyte HA1800 and human glioma cell line U251,U251 cells were transfected with Lipofectamine 2000 transfection kit and named blank group (normally cultured U251 cells),miR-NC group (transfected with miR-NC),miR-199a-5p mimic group (transfected with miR-199a-5p mimic),miR-199a-5p mimic+pcDNA group (co-transfection of miR-199a-5p mimic and pcDNA) and miR-199a-5p mimic+pcDNA-CDCA7L group (co-transfection of miR-199a-5p mimic and pcDNA-CDCA7L).Transwell experiment was used to detect the migration and invasion abilities of U251 cells.Western blot was used to detect the CDCA7L protein expression.TargetScan software was used to predict the binding sites of miR-199a-5p and CDCA7L,and dual luciferase reporter gene was used to verify the targeting relationship between miR-199a-5p and CDCA7L.The comparison between the HA1800 and U251 groups was performed by two-sample t test,the comparison among multiple groups was performed by one-way analysis of variance,and the comparison between the two groups was performed by SNK-q test.Results Compared with HA1800 cells,the expression level of miR-199a-5p in U251 cells was decreased (1.09±0.26 vs 0.32±0.14),while the expression of CDCA7L protein in was increased (0.21±0.02 vs 0.85±0.13),and the difference was statistically significant (P < 0.001).The number of cell migration,invasion and CDCA7L protein expression in miR-199a-5p mimic group were significantly lower than those in blank group and miR-NC group (50.12±3.36 vs 94.57±7.14,93.86±6.11) (42.53±3.34 vs 71.49±4.35,73.76±4.21) (0.25±0.07 vs 0.83±0.12,0.89±0.09),and the difference was statistically significant (P < 0.001).CDCA7L was a target gene of miR-199a-5p.Overexpression of CDCA7L could reverse the inhibitory effects of up-regulatin

关 键 词：胶质瘤 miR-199a-5p 细胞周期蛋白质类 细胞迁移 细胞侵袭 

分 类 号：R739.41[医药卫生—肿瘤]