MicroRNA-193a通过靶向WT1抑制肝细胞癌发生的机制研究

Mechanism of MicroRNA-193a Suppressing the Tumorigenesis of Hepatocellular Carcinoma Through Targeting WT1

目的:通过检测miR-193a在肝细胞癌(Hepatocellular carcinoma,HCC)组织中以及其对肝癌细胞的影响探讨其在HCC中的机制。方法:肝癌组织和相邻非癌的正常组织取自武汉市中医医院的75名肝癌患者,采用定量实时聚合酶链反应(qRT-PCR)法比较miR-193a和Wilms'肿瘤1基因(WT1)在肝癌组织和相邻非癌的正常组织的表达水平。探讨肝癌组织中miR-193a的表达与肿瘤分期、肿瘤是否转移、分化程度的相关性。检测肝癌细胞系MHCC97H,HCCLM3,Huh7和人正常肝细胞系LO2中miR-193a表达。此外,过表达和抑制miR-193a后通过细胞计数CCK-8法对HCC细胞增殖进行检测。通过异种移植物肿瘤形成试验观察抑制miR-193a是否加速HCC的肿瘤生长。通过双重荧光素酶测定法证实WT1是否为miR-193a的靶点基因。结果:相对于非癌组织,肝癌组织中miR-193a相对于表达显著降低(P<0.01),WT1基因表达显著升高,同时miR-193a的表达在正常肝细胞系中的表达显著低于肝癌细胞系(P<0.05~P<0.01)。肝癌患者TNM分期(P=0.004)以及淋巴结转移程度(P=0.013)在miR-193a低表达组患者相对于高于miR-193a高表达组更为严重。生存分析表明低表达miR-193a的肝癌患者的预后较差。此外,发现过表达miR-193a抑制HCC细胞增殖,而抑制miR-193a表达促进了HCC增殖。在体外miR-193a过表达抑制了HCC的肿瘤生长。荧光素酶试验证实WT1是miR-193a的靶点基因。结论:miR-193a可通过靶向WT1抑制HCC发生。

Objective:To elucidate the function of miR-193a in HCC.Methods:The expression levels of miR-193a and Wilms'tumor-1(WT1)were detected by quantitative real Time-polymerase chain Reaction(qRT-PCR)assay.In addition,miR-193a expression was also investigated in MHCC97H,HCCLM3,Huh7 and LO2 cell lines.Moreover,the HCC proliferation was examined through cell counting Kit-8(CCK-8)assay after miR-193a overexpression or inhibition.Tumor growth of HCC was observed via xenograft tumor formation assay.The relationship between miR-193a and WT1 was verified by the dual-Luciferase assay.Results:Downregulation of miR-193a whereas upregulation of WT1 were identified in HCC,which predicted a worse prognosis in HCC patients.Meanwhile,miR-193a was significantly decreased in LO2 cell lines compared with MHCC97H,HCCLM3,Huh7 cell lines.Low miR-193a expression HCC patients showed severer TMN stage and presence of lymph node metastasis.Furthermore,it was found that miR-193a mimic suppressed cell proliferation by inhibiting WT1.miR-193a also inhibited tumor growth of HCC.Conclusion:miR-193a suppressed tumorigenesis of HCC by targeting WT1.