miR-499在肝癌发生和发展中的作用机制研究

Study on the mechanism of miR-499 in the genesis and development of liver cancer

目的以肝癌细胞系Huh7细胞为模型,研究miR-499在肝癌发生和发展中的作用机制。方法通过TCGA和GEO数据库分析miR-499在肝癌患者及健康人中的表达差异,并通过实时荧光定量聚合酶链反应(RT-qPCR)检测肝癌组织及正常癌旁组织中miR-499的表达差异。通过临床病例分析miR-499与肝癌患者临床指标的关系。采用CCK-8试验、集落形成试验检测miR-499对Huh7细胞活性及集落形成的影响。采用流式细胞术检测miR-499对Huh7细胞周期及相对凋亡率的影响。用TargetScan7.1、MicroRNA.org、MiRDB等数据库预测miR-499的下游靶基因。通过RT-qPCR及Western blot试验证明miR-499对RAB5C mRNA及蛋白水平的影响。结果TCGA和GEO数据库数据显示,miR-499在肝癌患者中呈低表达。RT-qPCR结果表明,miR-499在肝癌组织中的表达明显低于癌旁组织。临床数据整理表格显示,肝癌患者中miR-499高表达的患者普遍肿瘤体积较小、TNM分期较低、淋巴结转移能力较弱且分化良好。miR-499 mimics转染Huh7细胞后,通过CCK8试验和集落形成试验发现,miR-499抑制Huh7细胞增殖及集落形成能力。流式细胞术细胞周期与凋亡试验表明,miR-499抑制Huh7细胞的周期进程,并且促进Huh7细胞的凋亡能力。RT-qPCR及Western blot试验证明,RAB5C确为miR-499的靶基因。结论miR-499通过直接靶定并下调RAB5C的表达,从而抑制肝癌的发生和发展。

Objective To study the mechanism of miR-499 in the genesis and development of liver cancer by taking hepatocellular cell Huh7 as a model.Methods The gene expression differential display of miR-499 between hepatocellular cancer patients and healthy people were analyzed by TCGA and GEO database.Real-time PCR was used to determine the expression level of miR-499 in liver cancer tissues and the para-carcinoma tissues.Meanwhile the relationship between miR-499 and the clinical cases of liver cancer patients was displayed.CCK8 assay and colony formation assays were performed to investigate the influence of miR-499 on viability and colony formation of Huh7 cells.Flow cytometry was used to determine the influence of miR-499 on the cell cycle progression and apoptosis of Huh7 cells.The target genes of miR-499 were predicted by TargetScan7.1,MicroRNA.org and MiRDB databases.The influence of miR-499 on the expression levels of mRNA and protein of RAB5C was detected by Real-time PCR and Western blot.Results The expression level of miR-499 was low in hepatocellular cancers according to the TCGA and GEO database.Real-time PCR experiment revealed that the expression level of miR-499 was lower in the liver cancer tissues than that in para-carcinoma.Clinical data showed that the tumors of liver cancer patients with high expression of miR-499 had smaller volume,low TNM stage,lower lymphatic metastasis capacity and were well-differentiated.After tansfected with miR-499mimics,CCK8 assay and colony formation assay demonstrated that the viability and colony information of Huh7 cells were inhibited by miR-499.Flow cytometry experiment was further to prove that the cell cycle progression of Huh7 cells was inhibited and the apoptosis of Huh7 cells was promoted by miR-499 mimics.Real-time PCR and Western blot test revealed that RAB5C was one target gene of miR-499.Conclusion miR-499 inhibited the development of liver cancer by decreasing the expression of RAB5C.