环状RNA circRTN4IP1在肝细胞癌中的生物信息学分析及其与预后的关系

Bioinformatics analysis of circRTN4IP1 in hepatocellular carcinoma and its relationship with clinical prognosis

目的探讨环状RNA circRTN4IP1在肝细胞癌(HCC)中的表达及其与预后的关系。方法使用R软件的Limma包分析GEO数据集GSE 97332、GSE 94508、GSE 78520,联合筛选差异表达的环状RNA。通过CSCD网站预测circRTN4IP1结合的microRNA(miRNA),使用TargetScan、miRDB、miRTarBase数据库联合预测miRNA的靶基因,并对其进行基因本体(GO)功能和京都基因与基因组百科全书(KEGG)通路富集分析。应用STRING在线工具分析靶基因的功能和蛋白质相互作用(PPI)网络,筛选关键基因。采用Kaplan-Meier Plotter数据库分析其与预后的关系。收集2016年8月至2018年6月于中山大学孙逸仙纪念医院行手术切除的60例HCC患者癌组织及配对癌旁组织,荧光定量RT-PCR检测组织circRTN4IP1表达水平,并分析其与HCC患者临床病理特征和预后的关系。两组circRTN4IP1 mRNA相对表达量比较采用t检验,率的比较采用χ2检验。生存分析采用Kaplan-Meier法和Log-rank检验。结果GEO数据集分析筛选HCC中显著上调的circRTN4IP1,预测出其结合的60个miRNA及581个靶基因。GO和KEGG分析显示其靶基因主要参与转录、翻译、蛋白泛素化、细胞周期等生物学进程,以及p38MAPK、PI3K/AKT、FoxO及TGF-β/Smad等信号通路。共筛选出8个关键基因,其中UBE2D1、H2AFX、UBE2V1、LRRC41、POLR2D高表达的HCC患者总生存时间明显低于低表达患者(HR=1.61,1.93,1.72,1.88,1.51;P<0.05)。荧光定量RT-PCR结果显示,HCC癌组织中circRTN4IP1 mRNA平均相对表达量为0.759±0.020,明显高于癌旁组织的0.625±0.024(t=8.385,P<0.05)。circRTN4IP1在HCC组织中的表达与AFP水平有关(χ^(2)=4.267,P<0.05)。circRTN4IP1高表达组患者的无病生存明显差于低表达组(χ^(2)=5.055,P<0.05)。结论circRTN4IP1在HCC中高表达,可能通过上调靶基因UBE2D1、H2AFX、UBE2V1、LRRC41、POLR2D参与调控HCC的发生、发展,是潜在的预后标志物及治疗靶点。

Objective To explore the expression of circular RNA circRTN4IP1 in hepatocellular carcinoma(HCC)and its relationship with clinical prognosis.Methods GEO datasets of GSE 97332,GSE 94508 and GSE 78520 were analyzed using Limma package of R software,and the differentially-expressed circular RNAs were screened.The combining microRNAs(miRNAs)of circRTN4IP1 were predicted on CSCD website.The target genes of miRNA were jointly predicted through TargetScan,miRDB and miRTarBase databases and then subjected to gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.The function of target genes and protein-protein interaction(PPI)network were analyzed using STRING online tool,and the key genes were selected.The relationship between the genes and prognosis was analyzed by Kaplan-Meier Plotter database.The cancer tissues and paired adjacent tissues were collected from 60 HCC patients who underwent surgical resection in Sun Yat-sen Memorial Hospital of Sun Yat-sen University from August 2016 to June 2018.The expression level of circRTN4IP1 in the tissues was detected by fluorescence quantitative RT-PCR,and its relationship with the clinicopathological features and prognosis of HCC patients was analyzed.The relative expression levels of circRTN4IP1 mRNA were compared between two groups by t test,and the rate was statistically compared by Chi-square test.Survival analysis was performed with Kaplan-Meier method and Log-rank test.Results Significantly up-regulated circRTN4IP1 in HCC was screened out by GEO datasets and its 60 combining miRNAs and 581 target genes were found.GO and KEGG analysis showed that the target genes were mainly involved in the transcription,translation,protein ubiquitination,cell cycle and other biological processes,as well as the p38MAPK,PI3K/AKT,FoxO and TGF-β/Smad signaling pathways.8 key genes were selected.The overall survival of HCC patients with high expression of UBE2D1,H2AFX,UBE2V1,LRRC41 and POLR2D was significantly shorter than that of HCC patients with low expression of these genes(HR=1.61,1.93,1.72,1.88,1.51;P<0.05).Fluorescence quantitative RT-PCR demonstrated that the average relative expression of circRTN4IP1 mRNA in HCC tissues was 0.759±0.020,significantly higher than 0.625±0.024 in adjacent tissues(t=8.385,P<0.05).The expression level of circRTN4IP1 was significantly correlated with the AFP level in HCC tissues(χ^(2)=4.267,P<0.05).The disease-free survival of patients with high circRTN4IP1 expression was significantly worse compared with their counterparts with low circRTN4IP1 expression(χ^(2)=5.055,P<0.05).Conclusions circRTN4IP1 is highly expressed in HCC patients,which participates in regulating the incidence and development of HCC probably by up-regulating the target genes of UBE2D1,H2AFX,UBE2V1,LRRC41 and POLR2D.circRTN4IP1 is a potential prognostic marker and therapeutic target.