[关键词]
[摘要]
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qPCR 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mRNA-miRNA 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。
[Key word]
[Abstract]
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 TargetScan. 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 qPCR. 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 mRNA-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 TargetScan 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.
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[基金项目]
四川省科技厅自然科学基金资助项目(No. 2022NSFSC0779);中央高校基本科研业务费基础培育项目(No. 2682021ZTPY016)