基于miRNA-mRNA网络筛查并验证胃癌诊断和预后评估相关的标志物及其潜在分子机制

Screening and validation of markers related to diagnosis and prognosis assessment of gastric cancer based on miRNA-mRNA network and their potential molecular mechanisms

目的:通过生物信息学方法探索并实验验证胃癌相关标志物miR-1-3p对胃癌细胞增殖的作用及其分子机制。方法:收集TCGA数据库中胃癌(n=375)及癌旁组织(n=45)的转录组数据,构建胃癌特异性mRNA-miRNA网络,筛选潜在的miRNA类标志物,利用TargetScan预测标志物的下游靶基因且分析它们的功能。选取人正常胃上皮细胞GES-1及胃癌细胞AGS、MKN45、NCI-N87,用q PCR法检测细胞中miR-1-3p和心肌蛋白(MYOCD)的表达,用lipofectamine 2000将miR-1-3p模拟物转染至胃癌细胞中,CCK-8法测定轨染后细胞的增殖能力,WB法测定MYOCD的表达量,双荧光素酶报告基因实验验证miR-1-3p与MYOCD之间的靶向结合关系。结果:通过数据库数据分析得到差异表达的259个miRNA和7 545个mRNA,构建胃癌特异性mRNAmiRNA调节网络,分析网络中脆弱结构后确定miR-1-3p为潜在的胃癌标志物,ROC曲线和Kaplan-Meier分析显示其对胃癌的诊断和预后评估有重要意义。细胞实验显示miR-1-3p在胃癌细胞中呈低表达(P<0.05),过表达miR-1-3p可抑制胃癌细胞AGS和MKN-45的增殖能力(P<0.05或P<0.01),且可抑制MYOCD的表达(P<0.01)。TargetScan数据库预测到MYOCD的3’UTR区域中有两个与miR-1-3p结合的位点,双荧光素酶报告基因实验证实miR-1-3p与MYOCD靶向结合且负调控MYOCD的表达(P<0.01)。结论:miR-1-3p可能是胃癌诊断和预后相关潜在的标志物,且miR-1-3p可能是通过靶向MYOCD来影响胃癌细胞的增殖。

Objective: To explore the role of miR-1-3p, a gastric cancer-related biomarker, on gastric cancer cell proliferation and its mechanism by bioinformatics approach. Methods: Transcriptomic data from gastric cancer(n=375) and paraneoplastic tissues(n=45) in the TCGA database were collected to construct a gastric cancer-specific mRNA-miRNA network, screen potential miRNA-like markers,predict the downstream target genes of the markers and analyze their functions using Target Scan. Human normal gastric epithelial cell and gastric cancer cell lines have been selected, and their miR-1-3p and myocardin(MYOCD expression was detected by q PCR. The miR-1-3p mimics were transfected into GES-1, AGS, and MKN45 cells using lipofectamine 2000. The proliferation ability of the cells was determined by CCK-8 assay, the expression of MYOCD was measured by WB assay, and the targeting relationship between miR-1-3p and MYOCD was verified by dual luciferase reporter assay. Results: Differentially expressed 259 miRNAs and 7 545 mRNAs were obtained by database data analysis to construct a regulatory network of gastric cancer-specific m RNA-miRNAs. Analysis of vulnerability structure in the network identified miR-1-3p as a potential gastric cancer marker, and in-depth analysis revealed its significance for the diagnosis and prognostic assessment of gastric cancer, while predicting MYOCD as its downstream target. Cellular assays showed that miR-1-3p was lowly expressed in gastric cancer cells(P<0.05);overexpression of miR-1-3p inhibited the proliferation ability of AGS and MKN-45 in gastric cancer cells(P<0.05 or P<0.01);and inhibited the expression of MYOCD(P<0.01);miR-1-3p was predicted to be associated with two binding sites in the 3’UTR region by the Target Scan database, and dual luciferase reporter assays showed that high expression of miR-1-3p at one of the wild-type predicted sites significantly inhibited MYOCD expression(P<0.01). Conclusion:miR-1-3p may be a potential diagnostic and prognostic-related marker for gastric cancer, and miR-1-3p may affect gastric cancer by targeting MYOCD.