调控食管胃结合部腺癌增殖、侵袭能力的关键环状RNA分子的筛选与初步鉴定

Screening and preliminary identification of key circRNAs that regulate the proliferation and invasion of adenocarcinoma of the esophagogastric junction

目的:利用RNA高通量测序和生物信息学分析筛选出调控食管胃结合部腺癌(adenocarcinoma of esophagogastric junction,AEG)增殖、侵袭能力的关键环状RNA分子,并对其作用方式进行初步探究。方法:提取22对食管胃结合部腺癌组织及癌旁正常黏膜组织的RNA。采用RNA高通量测序(测序深度200×),利用生物信息分析云平台进行差异基因筛选,同时对差异表达的基因构建共表达网络和circRNAs-miRNAs-mRNAs间的内源性RNA竞争机制(ceRNA)网络;利用RNA图谱共表达网络构建和内源性竞争RNA负相关分析,筛选出在AEG发生过程中可能发挥关键作用的circRNAs。并预测可与该circRNA存在ceRNA关系的miRNAs和mRNAs;通过qRT-PCR等方法对该circRNA在胃癌组织、癌旁组织中的表达进行验证。结果:通过RNA高通量测序,共预测得到46553个circRNAs,对测序数据进行严格质控,得到26个差异表达的circRNAs(fold change≥2或fold change≤0.5,false discovery rate<0.05),其中10个circRNAs表达上调,16个circRNAs表达下调。对组织样本进行m RNA和miRNA测序分析,构建circRNAs-miRNAs-mRNAs间的ceRNA网络、RNA图谱共表达网络和内源性竞争RNA负相关分析。结果表明,hascirc0007766在AEG肿瘤组织中特异性高表达,可能在AEG发生发展过程中起关键作用;同时发现,miR4492与circRNA0007766和MET mRNA均有结合位点。结论:hascirc0007766可能通过miR4492-circRNA0007766-MET m RNA的ceRNA网调控食管胃结合部腺癌增殖、侵袭,是AEG发生发展的关键circRNA。

Objective:To screen out the key circRNAs that regulate the proliferation and invasion of adenocarcinoma of the esophagogastric junction(AEG)by high-throughput RNA sequencing and bioinformatics analysis,and to preliminarily explore their modes of action.Methods:RNAs were extracted from 22 pairs of adenocarcinoma of the esophagogastric junction and adjacent normal mucosa tissues.Differentially expressed genes were screened out by high-throughput RNA sequencing(sequencing depth 200×)and bioinformatics cloud platform.Meanwhile,a co-expression network and an endogenous RNA competition mechanism(ceRNA)network among circRNAs-miRNAs-mRNAs were constructed for differentially expressed genes.The construction of co-expression network of RNA maps and the negative correlation analysis of endogenous competitive RNAs were used to screen for circRNAs that may play a key role in the development of AEG,and to predict the expression of miRNAs and m RNAs which have ceRNA relationship with the circRNA.qRT-PCR and other methods were used to verify the expression of circRNA in gastric cancer tissues and adjacent tissues.Results:A total of 46553 circRNAs were predicted by high-throughput RNA sequencing,and 26 differentially expressed circRNAs were obtained after strict quality control of sequencing data(fold change≥2 or fold change≤0.5,false discovery rate<0.05).Among them,10 circRNAs were up-regulated and 16 circRNAs were down-regulated.Tissue samples were analyzed by mRNA and miRNA sequencing,and the ceRNA network among circRNAs-miRNAs-mRNAs,co-expression network of RNA maps,and endogenous competitive RNA negative correlation analysis were constructed.The results showed that hascirc0007766 was highly specifically expressed in AEG tumors,which may play a key role in the development and progression of AEG.Meanwhile,it was also found that miR4492 had binding sites with both circRNA0007766 and MET mRNA.Conclusion:The hascirc0007766 may regulate the proliferation and invasion of adenocarcinoma of the esophagogastric junction through the ceRNA network of mir4492-and circRNA0007766-MET mRNA,which may be the key circRNA for the development and progression of AEG.