MiR-30靶向BECLIN1在胃癌中的作用及其机制研究

Study on the effect of MiR-30 targeting BECLIN1 in the diagnosis of gastric carcinoma and its mechanism

目的基于二代测序技术分析胃癌组织中miRNA-30的表达差异,采用生物信息学方法分析其靶基因及相关功能注释和信号通路富集,以期揭示miRNA-30在胃癌中的诊断价值及临床意义。方法收集2015年9月—2016年12月本院住院胃癌患者60例和同期对照健康人群50例标本,用高通量测序技术检测其中3例胃癌及癌旁组织中差异表达的miRNA,利用R 3.4.0软件绘制热图,并用荧光定量PCR(q PCR)验证miRNA-30和beclin1的差异表达。应用miRBase数据库预测miRNA-30的靶基因。利用cytoscape 3.5.1及其插件Clue GO进行GO(gene ontology)富集,利用DAVID数据库进行KEGG(Kyoto Encyclopedia of Genes and Genomes)信号通路注释。利用miRNet数据库绘制miRNA-30与mRNA表达调控网络。结果二代测序结果显示,胃癌组织中miRNA-30表现为下调。qRT-PCR验证支持这一结果(P<0.001)。GO功能注释主要富集于MAPKK活性、干细胞发育、STAT蛋白酪氨酸磷酸化的调控、蛋白质磷酸化氨基酸结合、酪氨酸磷酸化肽、调节脂质激酶活性、磷蛋白聚合、核转录mRNA聚A尾缩短等生物学过程和分子功能(P<0.01);KEGG信号通路富集主要涉及TP53基因调节细胞死亡基因的转录、TOR信号通路、Wnt信号通路、Hippo信号通路中(P<0.01)。调控网络表明miRNA-30参与包括beclin1在内的多种靶基因的直接调控。结论 miRNA-30在胃癌组织中表达水平下调,作为胃癌的抑癌因子,miRNA-30的靶基因、功能注释和信号通路注释富集于与消化肿瘤相关的生物信息中。

Objective Analyze the difference expression of miRNA-30 in gastric cancer tissues using the second generation sequencing technology,and use bioinformatics methods were used to analyze the target genes beclin1 and related annotation and signal pathway concentrate,aimed to reveal the diagnostic value and clinical significance in gastric cancer.Methods From September 2015 to December 2016,50 healthy volunteers and 60 cases of gastric cancer were collected.Differentially expressing miRNAs in tumor tissue and paracancerous tissue of 3 cases in cancer group were detected via high-throughput sequencing.The expression of miRNA-30 and Beclin1 were verified by fluorescence quantitative RT-PCR（q PCR）.The target gene of miRNA-30 was predicted using the miRBase database.Cytoscape 3.5.1 and its plug-in unit Clue GO were used to classify the gene ontology（GO） function.KEGG（Kyoto Encyclopedia of Genes and Genomes） pathway was enriched by DAVID database.MiRNA-30 and its genes interaction network diagram were drawn using the miRNet database.Results Sequencing showed that miRNA-30 was down-regulated in carcinoma group（P 0.001）.So was the qRT-PCR result.GO functional annotation mainly enriched in biological process and molecular function,for instance,stem cell development,MAPKK activity,STAT protein tyrosine phosphorylation regulation,protein phosphorylation,tyrosine phosphorylation of amino acid binding peptide,regulating lipid kinase activity,polymerization,poly nuclear phosphoprotein mRNA A tail shortened（P 0.01）.KEGG pathway analysis involved TP53 genes regulating cell death gene transcription,TOR signaling pathway,Wnt signaling pathway,Hippo signaling（P〈0.01）.The interaction network showed that miRNA-30 is involved in the direct regulation of multiple target genes including beclin1.Conclusions miRNA-30 was down-regulated in patients with gastric carcinoma.MiRNA-30 may be a tumor inhibiting factor in gastric carcinoma.The target gene,functional and signaling pathways annotation of miRNA-30 was largely enriched in the biological message associated with tumor digestion.