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

摘要:

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 junc－tion，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46 553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mRNA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，has_circ_0007766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：has_circ_0007766可能通过miR4492-circRNA0007766-MET mRNA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。

Abstract:

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 mRNAs 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 has_circ_0007766 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 has_circ_0007766 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.