人肝癌耐药细胞中差异表达微小RNA的筛选及生物学功能分析  

Screening of differentially expressed microRNAs in human hepatocellular carcinoma resistant cells and analysis of their biological functions

作　　者：耿海静 王海生 张建宇 李薇 邓秀玲 GENG Haijing;WANG Haisheng;ZHANG Jianyu;LI Wei;DENG Xiuling(Basic Medical College,Inner Mongolia Medical University,Hohhot 010000,China)

机构地区：[1]内蒙古医科大学基础医学院,呼和浩特010000

出　　处：《山东医药》2023年第2期11-15,共5页Shandong Medical Journal

基　　金：内蒙古医科大学科技百万工程项目(YKD2013KJBW001)。

摘　　要：目的通过生物信息学方法筛选人肝癌耐药细胞株Bel-7402/5-FU、Bel-7402/ADR与亲本细胞株Bel-7402差异表达的微小RNA(miRNA),分析差异表达miRNA的生物学功能。方法利用miRNA芯片分析亲本细胞和耐药细胞中miRNA的差异表达情况,筛选差异倍数较高的10个miRNA进行靶基因预测,对获取的靶基因进行GO富集分析、KEGG信号通路富集分析。采用实时定量PCR法验证差异表达显著的miRNA。结果与Bel-7402细胞比较,Bel-7402/5-FU细胞中存在58个差异表达miRNA,其中上调27个、下调31个;Bel-7402/ADR细胞中存在87个差异表达miRNA,其中上调17个、下调70个。对耐药细胞中差异倍数较高的10个miRNA进行靶基因预测,GO分析发现,这些靶基因主要涉及RNA聚合酶Ⅱ启动子的转录调控、生物合成过程正向调控和氮化合物代谢过程负向调控、酶结合、大分子复合物结合和核酸结合转录因子活性、高尔基体、催化复合物和转移酶复合物等过程;KEGG分析发现,靶基因富集通路涉及轴突引导、泛素介导的蛋白水解、PAR1通路、神经营养因子信号通路、MAPK信号通路、TGF-β信号通路。实时定量PCR检测结果显示,miR-31-5p、miR-4446-3p表达上调,miR-205-5p、miR-675-3p、miR-373-5p、miR-372-3p、miR-371a-3p、miR-675-5p、miR-10a-5p和miR-4532表达下调(P均<0.01),与基因芯片检测结果一致。结论肝癌耐药细胞中存在多种差异表达miRNA,这些miRNA可能通过靶基因介导PAR1通路、MAPK信号通路和TGF-β信号通路的异常参与肝癌耐药的发生发展。Objective To screen the differentially expressed microRNAs(miRNAs)between human hepatocellular carcinoma resistant cell lines Bel-7402/5-FU,Bel-7402/ADR and parental cell line Bel-7402 by bioinformatic methods and to analyze the biological functions of differentially expressed miRNAs.Methods The miRNA microarray was used to analyze the differential expression of miRNAs in parental cells and drug-resistant cells.Ten miRNAs with high differential fold were screened for target gene prediction,and GO enrichment analysis and KEGG signaling pathway enrichment analysis were performed on the obtained target genes.Real-time quantitative PCR was used to validate the miRNAs with significant differential expression.Results Compared with Bel-7402 cells,there were 58 differentially expressed miRNAs in Bel-7402/5-FU cells,of which,27 were up-regulated and 31 were down-regulated,and 87 differentially expressed miRNAs in Bel-7402/ADR cells,of which,17 were up-regulated and 70 were down-regulated.We conducted the target gene prediction on 10 miRNAs with high differential fold in drug-resistant cells.GO analysis revealed that these target genes were mainly involved in the transcriptional regulation of RNA polymerase Ⅱ promoter,positive regulation of biosynthetic process and negative regulation of nitrogen compound metabolic process,enzyme binding,macromolecular complex binding and nucleic acid binding transcription factor activity,Golgi apparatus,catalytic complex and transferase complex processes.KEGG analysis revealed that the target gene enrichment pathways involved axon guidance,ubiquitin-mediated protein hydrolysis,PAR1 pathway,neurotrophic factor signaling pathway,MAPK signaling pathway,and TGF-β signaling pathway.The results of real-time quantitative PCR showed that the expression of miR-31-5p and miR-4446-3p was up-regulated,and the expression of miR-205-5p,miR-675-3p,miR-373-5p,miR-372-3p,miR-371a-3p,miR-675-5p,miR-10a-5p and miR-4532 was down-regulated(both P<0.01),which were consistent with the results of gene microar

关 键 词：微小RNA 肿瘤耐药 基因芯片检测 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤]