miRNA-373-3p靶向调控CD44表达对肾母细胞瘤细胞增殖的影响  

Effects of miRNA-373-3p on the proliferation of nephroblastoma cells through targeted regulation of CD44 expression

作　　者：王艳君[1] 孔艳霞 全雪丽 原艳丽[1] 段勇涛 王风 王洁[1] Wang Yanjun;Kong Yanxia;Quan Xueli;Yuan Yanli;Duan Yongtao;Wang Feng;Wang Jie(Department of Surgical Intensive Care Unit,Children's Hospital Affiliated to Zhengzhou University,Children's Hospital of Henan Province,Zhengzhou 450000,China;Department of Hematological Oncology,Children's Hospital Affiliated to Zhengzhou University,Children's Hospital of Henan Province,Zhengzhou 450000,China)

机构地区：[1]郑州大学附属儿童医院河南省儿童医院外科重症监护室,郑州450000 [2]郑州大学附属儿童医院河南省儿童医院小儿血液肿瘤科,郑州450000

出　　处：《肿瘤研究与临床》2022年第2期86-91,共6页Cancer Research and Clinic

基　　金：河南省医学科技攻关计划(联合共建项目) (LHGJ20190976)。

摘　　要：目的:探讨miRNA-373-3p(miR-373-3p)靶向调控CD44表达对肾母细胞瘤G401细胞增殖的影响。方法:利用生物信息学方法,基于miRDB、miRanda、PITA以及DIANA-microT生物信息学数据库预测miR-373-3p可能的靶基因。取G401细胞,分别转染miR-373-3p模拟物、模拟物阴性对照、miR-373-3p抑制剂、抑制剂阴性对照,采用CCK-8法检测细胞增殖能力;采用克隆形成实验检测克隆形成的数目;采用实时荧光定量聚合酶链反应(qRT-PCR)检测CD44 mRNA的相对表达量;蛋白质印迹法检测CD44蛋白的表达水平。利用HEK-293T细胞进行双荧光素酶报告基因实验验证miR-373-3p靶基因。结果:生物信息学分析结果提示CD44是miR-373-3p的靶基因。自转染24 h起,miR-373-3p模拟物组G401细胞增殖能力较模拟物阴性对照组均下降(均 P<0.05);自转染48 h起,miR-373-3p抑制剂组细胞增殖能力较抑制剂阴性对照组均增强(均 P <0.05)。miR-373-3p模拟物组克隆形成数少于模拟物阴性对照组[(55.3±2.5)个比(90.7±2.9)个],差异有统计学意义( t=14.57, P<0.01);miR-373-3p抑制剂组克隆形成数多于抑制剂阴性对照组[(115.0±2.7)个比(92.0±2.4)个],差异有统计学意义( t=8.86, P<0.01)。双荧光素酶报告基因实验显示,CD44是miR-373-3p的直接靶基因。miR-373-3p模拟物组、模拟物阴性对照组G401细胞中CD44 mRNA的相对表达量分别为0.62±0.03、1.00±0.01,差异有统计学意义( t=11.28, P<0.01);miR-373-3p抑制剂组、抑制剂阴性对照组CD44 mRNA的相对表达量分别为1.31±0.02、1.00±0.00,差异有统计学意义( t=12.65, P<0.01)。miR-373-3p模拟物组CD44蛋白表达降低,而miR-373-3p抑制剂组CD44蛋白表达升高。 结论:在肾母细胞瘤中,miR-373-3p可以通过靶向调控CD44的表达抑制肿瘤细胞的增殖。Objective To explore the effects of miRNA-373-3p(miR-373-3p)on the proliferation of nephroblastoma G401 cells through targeted regulation of CD44 expression.Methods Bioinformatic method was used to predict the possible targeted genes of miR-373-3p based on bioinformatic databases including miRDB,miRanda,PITA and DIANA-microT.G401 cells were taken and transfected with miR-373-3p mimic,mimic negative control,miR-373-3p inhibitor or inhibitor negative control,respectively.Cell proliferation ability was detected by using CCK-8 assay.The number of clones was detected by using clone formation assay.The relative expression level of CD44 mRNA was detected by using real-time fluorescent quantitative polymerase chain reaction(qRT-PCR),and the expression level of CD44 protein was detected by using Western blotting.The dual luciferase gene reporter assay was carried out in HEK-293T cells to vertify the target gene of miR-373-3p.Results Bioinformatic analysis indicated that CD44 was a targeted gene of miR-373-3p.After 24 h transfection,the proliferation activity of G401 cells in miR-373-3p mimic group was decreased compared with that in mimic negative control group(all P<0.05).After 48 h transfection,the proliferation activity of tumor cells in miR-373-3p inhibitor group was increased compared with that inhibitor negative control group(all P<0.05).The formed number of clones in miR-373-3p mimic group was reduced compared with that in the mimic negative control group(55.3±2.5 vs.90.7±2.9),and the difference was statistically significant(t=14.57,P<0.01).The formed number of clones in miR-373-3p inhibitor group was more than that in inhibitor negative control group(115.0±2.7 vs.92.0±2.4),and the difference was statistically significant(t=8.86,P<0.01).The dual-luciferase gene reporter assay showed that CD44 was a direct targeted gene of miR-373-3p.The relative expression levels of CD44 mRNA in miR-373-3P mimic and mimic negative control group were 0.62±0.03 and 1.00±0.01,respectively,and the difference was statistically sig

关 键 词：WILMS瘤 微RNAS 抗原 CD44 细胞增殖 

分 类 号：R737.11[医药卫生—肿瘤]