上调的miR-452-5p抑制RORα的表达并促进肝癌细胞的增殖和迁移

Up-regulated miR-452-5p suppressed the expression of RORαand promoted the proliferation and migration of HCC cells

为了探究肝细胞癌中miR-452-5p对患者预后生存的影响及其对肝癌细胞增殖、迁移的作用。采用肿瘤基因组图谱(TCGA)数据库中肝细胞肝癌数据集对miR-452-5p进行差异表达分析和Kaplan-Meier生存分析。采用TargetscanHuman和miRDB靶基因数据库对miR-452-5p靶基因进行预测;采用基因差异表达分析和加权基因共表达网络分析(WGCNA)的方法对数据集GSE14520进行分析计算。用Lipofectmine-2000将miR-452-5p模拟物、模拟物阴性对照和miR-452-5p抑制剂、抑制剂阴性对照分别转染到Huh7细胞中。分别采用RT-qPCR和Westernblot实验,检测4组细胞中RORα在mRNA和蛋白水平上的表达情况。采用CCK-8、Transwell实验,检测4组细胞的增殖活力以及迁移能力。双荧光素酶报告基因实验验证Huh7细胞中miR-452-5p与RORα的调控关系。经TCGA数据分析,miR-452-5p在肝癌组织中高表达且显著影响患者预后总生存期。通过miRNA靶基因预测、基因差异表达分析、WGCNA分析,筛选出关键基因RORα和LAMC1。通过Kaplan-Meier生存分析得知RORα的异常表达显著影响肝癌患者的预后总生存期。肝癌细胞中miR-452-5p的过表达,会降低RORα的mRNA和蛋白表达,同时增强肝癌细胞的增殖能力和迁移能力。双荧光素酶报告基因实验结果证实了miR-452-5p靶向RORα的3′UTR区。在肝细胞癌患者中高表达的miR-452-5p靶向抑制了RORα的表达,促进了肝癌细胞增殖和迁移,进而加剧了肝癌的进展和预后不良。

To study the prognosis-related regulation mechanism of miR-452-5p and its influence on the proliferation and migration of hepatocellular carcinoma(HCC)cells,liver hepatocellular carcinoma(LIHC)dataset in The Cancer Genome Atlas(TCGA)was used to validate the differential expression of miR-452-5p and perform the Kaplan-Meier analysis of overall survival(OS).Target genes of miR-452-5p from TargetscanHuman and miRDB databases were predictived;and differentially expressed genes(DEGs)and weighted gene co-expression network analysis(WCGNA)were completed with GSE14520.Lipofectmine-2000 was used to transfect miR-452-5p mimics,mimics negative control,miR-452-5p inhibitor and inhibitor negative control into Huh7 cells,respectively.The mRNA and protein expression level of RORαin 4 groups were determined by RT-qPCR and Western blot.CCK-8 assay and Transwell assay were conducted to testify the capabilities of proliferation and migration.The regulation between miR-452-5p and RORαwas confirmed by the dual luciferase reporter assay.After analysis in the TCGA-LIHC dataset,miR-452-5p had higher expression in HCC tissue than that in normal tissue,which was also associated with a shorter OS.RORαand LAMC1 were discriminated by intersecting of DEGs,WGCNA module genes,and predictive target genes.Survival analysis exhibited that dysregulation of RORαwas significantly related to the OS.Overexpression of miR-452-5p in HCC cells suppressed the expression of RORαboth in mRNA and protein,and also enhanced the viability and migration of HCC cells.The results of the dual luciferase reporter assay showed that miR-452-5p targeted 3′UTR of RORα.Up-regulated miR-452-5p inhibited the expression of RORα,facilitated the proliferation and migration of HCC cells,and promoted the progression and poor prognosis of HCC.