肝癌环状RNA-100338/微小RNA-141-3p轴与磷酸肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路的关系

Relationship between circRNA-100338/microRNA-141-3p axis and phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin signaling pathway in hepatocellular carcinoma

目的探讨肝癌(HCC)环状RNA(circRNA)竞争性内源RNA(ceRNA)分子机制及其关联信号通路。方法共收集10对HCC及癌旁组织,应用circRNA芯片检测其中4对HCC及癌旁组织,对circRNA芯片数据进行聚类分析,结合生物信息分析的结果筛选出目标circRNA并用实时定量反转录聚合酶链反应(RT-qPCR)验证;基于公开的同类型测序数据库,采用Cytoscape软件构建微小RNA(miRNA,miR)-Target-Network、miRNA-Gene-Act-Network,分析与目标circRNA关联密切的信号通路;通过免疫组织化学方法验证信号通路的关键蛋白。结果肝癌及癌旁组织RNA提取及质检合格,环状RNA芯片检测肝癌及癌旁组织circRNA,图像清晰可见、扫描数据可靠;通过聚类分析及RT-qPCR验证结果获得circRNA_100338为候选靶标,吸附miR-141-3p,HCC中circRNA_100338水平(0.012±0.007)高于癌旁circRNA_100338水平(0.005±0.003)(P<0.05),HCC中miR-141-3p水平(0.885±0.354)低于癌旁miR-141-3p水平(4.066±1.628)(P<0.01);基于我们的芯片数据,联合同类型测序数据库(GSE25599、GSE77276)分析结果显示,miR-141-3p与RHEB的mRNA表达水平的负相关性值为-0.602,circRNA_100338与RHEB显著正相关(Correlation test,P<0.05),推断circRNA_100338/miR-141-3p调控PI3K/AKT/mTOR信号通路;免疫组化结果显示该信号通路关键蛋白RHEB在circRNA_100338高表达(circRNA_100338/GAPDH > 0.05)的HCC组织中表达增强(评分≥ 3),与对照组比较差异有统计学意义(P<0.05)。结论肝癌circRNA_100338/miR-141-3p轴参与调控PI3K/Akt/mTOR信号通路,RHEB蛋白表达强度与circRNA_100338水平明显相关。

Objective To explore the molecular mechanism of competing endogenous RNA (ceRNA) and related signaling pathway for circular RNA (circRNA) of hepatocellular carcinoma (HCC). Methods Ten pairs of HCC and adjacent tissues were collected, and four pairs of specimens were detected by circular RNA chip. Cluster analysis, bioinformatics analysis and real-time quantitive reverse transcriptase-polymerase chain reaction (RT-qPCR) were applied for the target circRNAs. Base on the similar type of open sequencing database, we constructed the microRNA (miRNA, miR)-Target-Network and miRNA-Gene-Act-Network by using Cytoscape software and analyzed signal pathways that was closely related to the target circRNAs. The key protein in this signaling pathway was validated by immunohistochemical (IHC) analysis. Results Tissue RNA extraction and quality inspection were qualified. Circular RNA chip was used to detect circRNAs in HCC and adjacent tissues. The images were clear, and the scanning data were reliable. Cluster analysis and RT-qPCR validation results showed that circRNA_100338 was a candidate target to adsorb microRNA-141-3p, the level of circRNA_100338 in HCC (0.012±0.007) was higher than that in para-cancer (0.005±0.003)(P<0.05), and the level of microRNA-141-3p in HCC (0.885±0.354) was lower than that in para-cancer (4.066±1.628)(P<0.01). Based on our chip data analysis, combined with the similar sequencing databases (GSE25599, GSE77276), the results showed that the value of negative correlation between the miR-141-3p and RHEB mRNA expression was -0.602, circRNA_100338 was significantly positive correlated with RHEB (P<0.05), which inferred that circRNA_100338/miR-141-3p regulated PI3K/AKT/mTOR signaling pathway. Results of IHC showed that expression of RHEB, the key protein of the signaling pathway, was enhanced in HCC tissues (scores ≥ 3) with high circRNA_100338 expression (circRNA_100338/GAPDH > 0.015) when compared with control group (P<0.05). Conclusion The circRNA_100338/miR-141-3p axis is involved in the regulation of PI3K/Akt/mTOR signaling pathway in HCC, and the expression intensity of RHEB protein is correlated with the level of circRNA_100338.