摘要
目的:探究Musashi RNA结合蛋白2(MSI2)如何通过Wnt/β-catenin信号通路发挥调控肝癌细胞增殖的作用。方法:通过转染短发夹RNA(shRNA)抑制MSI2表达,将细胞分为转染对照质粒(sh-Ctrl)组和sh-MSI2组。MSI2过表达实验中将细胞分为对照组(Vector组,转染空白质粒Vector)和过表达组(MSI2组,转染MSI2重组质粒)。CCK-8和平板克隆实验检测细胞增殖。蛋白质印迹法检测干预MSI2后β-catenin、转录因子7(TCF7)和淋巴增强因子1(LEF1)的表达。Rescue回复实验中将细胞分为MSI2+sh-Ctrl组(同时转染MSI2重组质粒和sh-Ctrl质粒)和MSI2+sh-β-catenin组(同时转染MSI2重组质粒和sh-β-catenin质粒)。在过表达MSI2的基础上敲低β-catenin进行肝癌细胞增殖能力检测。结果:sh-MSI2组HepG2和MHCC97H细胞的增殖率均低于sh-Ctrl组,差异具有统计学意义(P<0.05)。MSI2组的SMMC-7721和MHCC97L细胞增殖率均高于Vector组,差异具有统计学意义(P<0.05)。克隆形成实验结果显示,与sh-Ctrl组相比,sh-MSI2组的HepG2细胞克隆数量[(129.7±6.5)比(286.0±12.8)]和MHCC97H细胞克隆数量[(134.0±6.7)比(248.0±14.1)]均减少,差异具有统计学意义(P<0.05);与Vector组相比,MIS2组的SMMC-7721细胞克隆数量[(242.0±5.6)比(135.3±8.7)]和MHCC97L细胞克隆数量[(308.0±9.0)比(149.7±5.9)]均增加,差异具有统计学意义(P<0.05)。sh-MSI2组HepG2和MHCC97H细胞中β-catenin、TCF7和LEF1 mRNA和蛋白的表达均低于sh-Ctrl组,组间比较差异具有统计学意义(均P<0.05)。相反,与Vector组相比,MSI2组SMMC-7721和MHCC97L细胞的β-catenin、TCF7和LEF1 mRNA和蛋白表达水平均增加,差异均具有统计学意义(P<0.05)。与MSI2+sh-Ctrl组相比,MSI2+sh-β-catenin组细胞增殖能力下降,差异具有统计学意义(P<0.05)。平板克隆实验显示,MSI2+sh-β-catenin组细胞克隆形成数量少于MSI2+sh-Ctrl组[(138.3±7.0)比(246.3±8.0),P=0.028]。结论:MSI2通过Wnt/β-catenin信号通路促进肝癌细胞增殖,导致肿瘤进展。
Objective To investigate the mechanism that how Musashi RNA-binding protein 2(MSI2)regulates HCC growth.Methods Short hairpin RNA(shRNA)was transfected to inhibit MSI2 expression,and cells were divided into transfection control plasmid(sh-Ctrl)group and sh-MSI2 group.In the MSI2 overexpression experiment,cells were divided into control group(Vector group,transfected with blank plasmid Vector)and overexpression group(MSI2 group,transfected with MSI2 recombinant plasmid).Cell proliferation was detected by CCK-8 and plate cloning assays.Western blotting was used to detect the expressions ofβ-catenin,transcription factor 7(TCF7)and lymphoid enhancer factor 1(LEF1)after MSI2 intervention.In the Rescue recovery experiment,cells were divided into MSI2+sh-Ctrl group(transfected with MSI2 recombinant plasmid and sh-Ctrl plasmid at the same time)and MSI2+sh-β-catenin group(transfected with MSI2 recombinant plasmid and sh-β-catenin plasmid at the same time).On the basis of overexpression of MSI2,β-catenin was knocked down to detect the proliferation ability of HCC cells.Results The proliferation rates of HepG2 and MHCC97H cells in the sh-MSI2 group were lower than those in the sh-Ctrl group,and the difference was statistically significant(P<0.05).The proliferation rates of SMMC-7721 cells and MHCC97L cells in the MSI2 group were higher than those in the Vector group,and the difference was statistically significant(P<0.05).The results of the clone formation experiment showed that compared with the sh-Ctrl group,the number of HepG2 cell clones in the sh-MSI2 group[(129.7±6.5)vs.(286.0±12.8)]and the number of MHCC97H cell clones[(134.0±6.7)vs.(248.0±14.1)]were reduced,and the differences were statistically significant(P<0.05);compared with the Vector group,the number of SMMC-7721 cell clones[(242.0±5.6)vs.(135.3±8.7)]and MHCC97L cell clones[(308.0±9.0)vs.(149.7±5.9)]in the MIS2 group were increased,and the difference was statistically significant(P<0.05).The mRNA and protein expression ofβ-catenin,TCF7 and LEF1 in HepG2 and MHCC97H cells in the sh-MSI2 group were lower than those in the sh-Ctrl group,and the differences between groups were statistically significant(P<0.05).In contrast,compared with the Vector group,the mRNA and protein expression levels ofβ-catenin,TCF7 and LEF1 in SMMC-7721 and MHCC97L cells in the MSI2 group were all increased,and the differences were statistically significant(P<0.05).Compared with the MSI2+sh-Ctrl group,the cell proliferation ability of the MSI2+sh-β-catenin group was decreased,and the difference was statistically significant(P<0.05).Plate cloning experiments showed that the number of cell clones in the MSI2+sh-β-catenin group was less than that in the MSI2+sh-Ctrl group[(138.3±7.0)vs.(246.3±8.0),P=0.028].Conclusion MSI2 promotes HCC cell proliferation through Wnt/β-catenin signaling pathway,leading to tumor progression.
作者
李燕军
赵海潮
Li Yanjun;Zhao Haichao(Department of Hepatobiliary Surgery,Shanxi Bethune Hospital,Shanxi Academy of Medical Sciences,Tongji Shanxi Hospital,Taiyuan 030032,China;Department of Hepatobiliary Surgery,the Third Hospital of Shanxi Medical University,Taiyuan 030032,China)
出处
《中华肝胆外科杂志》
CAS
CSCD
北大核心
2022年第10期766-771,共6页
Chinese Journal of Hepatobiliary Surgery
基金
山西省应用基础研究计划项目(201901D111408)
山西省"136"兴医工程领军临床重点专科(普通外科)项目(2019XY002)。
关键词
癌
肝细胞
细胞增殖
WNT信号通路
Β连环蛋白
Carcinoma,hepatocellular
Cell proliferation
Wnt signaling pathway
Beta-catenin
