Objectives: The high morbidity and mortality of colorectal cancer (CRC) have posed great threats to human health. Circular RNA (circRNA) and microRNA (miRNA), acting as competing endogenous RNAs (ceRNAs), have been found to play vital roles in carcinogenesis. This paper aims to construct a circRNA/miRNA/mRNA regulatory network so as to explore the molecular mechanism of CRC.
Methods: The sequencing data of circRNA from CRC were obtained from Gene Expression Omnibus (GEO). The differential circRNA was screened and its structure was identified by Cancer-specific CircRNA Database (CSCD); the sequencing data of miRNA and messenger RNA (mRNAs) were downloaded from The Cancer Genome Atlas (TCGA) database and the differentially expressed genes were screened; the corresponding miRNA of differential circRNAs were predicted by CircInteractome database; DIANA, Miranda, PicTar, and TargetScan databases were used to predict the target genes of different miRNAs; the target genes from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched by R language; String database combined with Cytoscape 3.7.2 software was used to construct protein-protein interaction (PPI) network and hub genes were screened; the expressions of mRNAs in the Top10 hub genes were verified in CRC. The network diagrams of circRNAs/miRNAs/mRNAs and circRNAs/miRNAs/Top10 hub mRNAs were constructed by Cytoscape3.7.2. Real-time PCR was used to examine the expression levels of hsa_circRNA_0065173, hsa-mir-450b, hsa-mir-582, adenylate cyclase 5 (ADCY5), muscarinic acetylcholine receptor M2 (CHRM2), cannabinoid receptor 1 (CNR1), and lysophosphatidic acid receptor 1 (LPAR1) in the CRC tissues and the adjacent normal tissues.
Results: A total of 14 differential circRNAs were identified, and 8 were found in CSCD; 34 miRNAs targeted by circRNAs were obtained. The PPI network was constructed, and the Top10 hub genes were identified, which were CHRM2, melanin concentrating hormone receptor 2 (MCHR2), G-protein gamma 3 subunit (GNG3), neuropeptide Y receptor Y1 (NPY1R), CNR1, LPAR1, ADCY5, adenylate cyclase 2 (ADCY2), gamma 7 (GNG7) and chemokine 12 (CXCL12), respectively. The expressions of Top 10 hub genes were also verified, and the results showed that the Top 10 hub genes were down-regulated in CRC; the constructed network diagram showed that hsa_circRNA_0065173 may regulate ADCY5, CHRM2, and Hsa-mir-450b by modulating hsa-mir-450b and hsa-mir-582. CNR1 and LPAR1 genes might serve as potentially relevant targets for the treatment of CRC. Real-time PCR results showed that the expression levels of hsa_circRNA_0065173, ADCY5, CHRM2, CNR1 and LPAR1 in the CRC tissues were significantly reduced compared with the adjacent normal tissues (all P<0.05); the expression levels of hsa-mir-450b and hsa-miR-582 were significantly increased (both P<0.05).
Conclusions: In this study, a potential circRNAs/miRNAs/mRNAs network is successfully constructed, which provides a new insight for CRC development mechanism through ceRNA mediated by circRNAs.
目的: 结直肠癌(colorectal cancer，CRC)的高发病率和高病死率对人类健康构成了巨大威胁。环状RNA(circRNAs)和微RNA(microRNAs，miRNAs)作为相互竞争的内源性RNA(endogenous RNAs，ceRNAs)，已被发现在致癌过程中发挥了重要作用。本文通过构建CRC中的circRNA/miRNA/mRNA调控网络，旨在探讨CRC发生过程中的具体分子机制。方法: 从基因表达综合(Gene Expression Omnibus，GEO)数据库获得CRC的circRNA测序数据，筛选差异circRNA并用癌症特异性circRNA数据库(Cancer-specific CircRNA Database，CSCD)鉴定其结构；采用癌症基因组图谱(The Cancer Genome Atlas，TCGA)数据库下载miRNAs和mRNAs测序数据并筛选差异基因；应用CircInteractome数据库预测差异circRNA对应的miRNA；采用DIANA、miRanda、PicTar和TargetScan数据库预测差异miRNA对应靶基因；应用R语言进行靶基因的基因本体(Gene Ontology，GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)的富集分析；采用蛋白质相互作用分析数据库String结合网络可视化分析软件Cytoscape3.7.2构建蛋白质-蛋白质相互作用(protein-protein interaction，PPI)网络图和筛选核心基因；验证前10位核心基因(Top10 hub基因)的mRNA表达量；采用Cytoscape3.7.2构建circRNAs/miRNAs/mRNAs和circRNAs/miRNAs/Top10 hub mRNAs网络图；采用real-time PCR检测hsa_circRNA_0065173、hsa-mir-450b、hsa-mir-582、腺苷酸环化酶5(adenylate cyclase 5，ADCY5)、毒蕈碱型乙酰胆碱受体M2(muscarinic acetylcholine receptor M2，CHRM2)、大麻素I型受体(cannabinoid receptor 1，CNR1)和溶血磷脂酸受体1(lysophosphatidic acid receptor 1，LPAR1)在CRC组织和癌旁组织中的表达水平。结果: 共筛选出14个差异circRNAs，在CSCD上可查询到8个；获得circRNAs靶向miRNAs 34个；mRNA在进行PPI和Top10 hub基因筛选时，得到Top10 hub基因分别为CHRM2、黑色素浓集激素受体2(melanin concentrating hormone receptor 2，MCHR2)、G蛋白γ3亚单位(G-protein gamma 3 subunit，GNG3)、神经肽Y受体Y1(neuropeptide Y receptor Y1，NPY1R)、CNR1、LPAR1、ADCY5、腺苷酸环化酶2(adenylate cyclase 2，ADCY2)、G蛋白偶联受体γ7抗体G(gamma 7，GNG7)、趋化因子12(chemokine 12，CXCL12)；Top10 hub基因的表达量验证显示：Top10 hub基因在CRC中均下调；构建的网络图表明hsa_circRNA_0065173可能通过调控hsa-mir-450b、hsa-mir-582而调控ADCY5、CHRM2、CNR1、LPAR1基因，这些基因可能可作为治疗CRC的潜在相关靶点。Real-time PCR结果显示：与癌旁正常组织比较，CRC组织中hsa_circRNA_0065173、ADCY5、CHRM2、CNR1和LPAR1的表达水平均显著降低(均P<0.05)；hsa-mir-450b、hsa-mir-582的表达水平均显著升高(均P<0.05)。结论: 本研究构建了潜在的circRNAs/miRNAs/mRNAs网络，它可为circRNA通过ceRNA机制介导CRC的发生、发展提供新的见解。.
Keywords: Top10 hub gene; circRNAs/miRNAs/mRNAs; colorectal cancer.