基于二代全转录组测序技术探究人胃癌组织中非编码RNA的差异表达及功能注释分析  

Differential Expression and Functional Annotation Analysis of Non-coding RNA in Human Gastric Cancer Tissuess Based on Second-generation Wholetranscriptome Sequencing

作　　者：朱萌 张宁[2] 马经伟[3] 黄宁波[1] ZHU Meng;ZHANG Ning;MA Jingwei;HUANG Ningbo(Basic Medical College,Ningxia Medical University,Yinchuan,750004;Department of Pathology,General Hospital of Ningxia Medical University,Yinchuan,750004;The Second Department of Surgical Oncology,General Hospital of Ningxia Medical University,Yinchuan,750004)

机构地区：[1]宁夏医科大学基础医学院,银川750004 [2]宁夏医科大学总医院病理科,银川750004 [3]宁夏医科大学总医院肿瘤外二科,银川750004

出　　处：《基因组学与应用生物学》2023年第11期1247-1260,共14页Genomics and Applied Biology

基　　金：国家自然科学基金项目(82260591,81802416);宁夏自然科学基金项目(2022AAC03134,2023AAC05033);宁夏医科大学校级基金项目(XT2019007)共同资助。

摘　　要：本研究通过全转录组测序技术分析人胃癌组织和癌旁组织非编码RNA(non-coding RNA, ncRNA)的差异表达及功能注释,揭示ncRNA参与胃癌发生发展的潜在机制。收集手术切除人胃癌和癌旁新鲜标本各5例,建库进行RNA测序(RNA sequencing, RNA-seq),分别检测小RNA测序(microRNA sequencing, miRNA-seq)、环状RNA测序(circular RNA sequencing, circRNA-seq)、长链RNA测序(long-coding RNA sequencing, lncRNA-seq)表达谱并筛选差异表达基因(differentially expressed genes, DEGs),对DEGs进行GO和KEGG功能注释分析;采用全转录组联合分析并利用Cytoscape构建circRNA/lncRNA-miRNA-mRNA竞争性内源RNA(competing endogenous RNA, ceRNA)网络;采用RT-qPCR验证ceRNA关键miRNA表达。本研究在人胃癌组织中共检测出差异表达miRNA 157个,上调miRNA 98个、下调miRNA 59个,富集条目主要有转录调控、凋亡、酶活性等;检测出差异表达circRNA 365个,上调circRNA 180个、下调circRNA 185个,富集条目主要有酶活性、氨基酸代谢、氧化磷酸化等;检测出差异表达lncRNA 303个,上调lncRNA 123个、下调lncRNA 180个,富集条目主要有蛋白代谢过程的负调控、 ATP结合、 TNF信号通路、凋亡等。经miRNA靶向关系联合分析发现hsa-miR-196a/b、 hsa-miR-204是ceRNA网络关键miRNA。利用RT-qPCR验证hsa-miR-196a-5p、 hsa-miR-204-5p表达水平,其结果与测序结果一致。本研究揭示胃癌中的差异表达ncRNA可能通过凋亡、代谢、 TNF通路等参与胃癌的发病机制,hsa-miR-196a/b和hsa-miR-204可能是其中的关键miRNA节点。This study aims to reveal the potential mechanism of non-coding RNA(ncRNA)involved in the occurrence and develop-ment of gastric cancer by analyzing their differential expression and functional annotation in human gastric cancer and adjacent tissues through whole-transcriptome sequencing technology.Five human gastric cancer and adjacent fresh tissues were collected.The library was established for RNA sequencing(RNA-seq).The expression profiles of microRNA sequencing(miRNA-seq),circular RNA sequencing(circRNA-seq)and long-coding RNA sequencing(lncRNA-seq)were detected,and the diferentially expressed genes(DEGs)were screened,as well as GO and KEGG function were annotated.Whole-transcriptome combined analysis was performed and circRNA/lncRNA-miRNA-mRNA competing endogenous RNA(ceRNA)network was constructed by Cytoscape.RT-qPCR was used to verification of key miRNA in ceRNA.This study detected 157 differentially expressed miRNA in gastric cancer tissues,of which 98 were up-regulated and 59 were down-regulated.Functional annotation analysis showed that the enriched entries were mainly transcriptional regulation,apoptosis and enzyme activity.There were 365 differentially expressed circRNA,180 up-regulated and 185 downregulated.The main enrichment items were enzyme activity,amino acid metabolism and oxidative phosphorylation.303 IncRNA were differentially expressed,123 were up-regulated and 180 were down-regulated.The enrichment function items included negative regulation of protein metabolic process,ATP binding,TNF signaling pathway and apoptosis.Through miRNA targeting relationship analysis,hsa-miR-196a/b and hsa-miR-204 were found to be key miRNA of ceRNA network.RT-qPCR verified that the expression levels of hsamiR-196a-5p and hsa-miR-204-5p were consistent with the sequencing results.This study reveals that the differential expression of ncRNA in gastric cancer may participate in cancer pathogenesis through apoptosis,metabolism,TNF-related pathways,etc.hsa-miR-196a/b and hsa-miR-204 may be key miRNA nodes.

关 键 词：胃癌 全转录组测序 非编码RNA 竞争性内源RNA(ceRNA)网络 

分 类 号：R735.2[医药卫生—肿瘤]