肝细胞癌中miR-574-5p的表达与作用及其与预后的关系

背景与目的 研究表明，miR-574-5p与多种肿瘤预后密切相关，但尚未见miR-574-5p与肝细胞癌（HCC）的关系报道。因此，本研究初步探讨miR-574-5p在HCC中表达及其与患者预后的关系。方法 用qRT-PCR检测130例HCC与癌旁组织及HCC细胞系（HepG2、MHCC-97H）与正常肝细胞系（L-02）中miR-574-5p的表达，用X-tile软件在患者生存数据中确定miR-574-5p表达量的最佳截断值后，分析miR-574-5p表达水平与患者临床病理因素于术后生存率的关系。分析HCC患者预后的影响因素。用TargetScan预测miR-574-5p的靶基因，并用双荧光素酶报告基因实验验证与Western blot实验确证。结果 miR-574-5p表达水平在HCC癌组织中明显高于癌旁组织，在HCC细胞系中明显高于正常肝细胞系（均P<0.05）。miR-574-5p表达与TNM分期（P=0.002）和分化程度（P=0.000）明显有关。miR-574-5p高表达组的总生存率与无瘤生存率均明显低于miR-574-5p低表达组（均P<0.01）。单因素与多因素分析结果显示，miR-574-5p高表达（HR=4.101，95% CI=1.348~8.968，P=0.03）、III~IV期（HR=5.403，95% CI=1.266~13.860，P=0.02）及中低分化（HR=3.655，95% CI=2.165~6.984，P=0.00）是HCC患者总生存率的独立危险因素，miR-574-5p高表达（HR=7.168，95% CI=1.144~18.260，P=0.01）与III~IV期（HR=7.436，95% CI=1.123~20.916，P=0.00）是HCC患者无瘤生存率的独立危险因素。TargetScan软件发现FOG2存在和miR-574-5p直接结合位点，双萤光素酶报告基因实验表明FOG2是miR-574-5p的直接靶基因。Western blot结果显示，FOG2蛋白在HCC组织中的表达量低于癌旁组织；Pearson相关分析表明，HCC组织中miR-574-5p表达水平与FOG2蛋白表达水平呈负相关（r=-0.499，P<0.05）。结论 miR-574-5p在HCC中普遍升高，miR-574-5p高表达可作为HCC患者预后不良的分子标志物，miR-574-5p可能通过下调其靶基因FOG2发挥促癌作用。

Expression and action of miR-574-5p in hepatocellular carcinoma and its association with prognosis

Background and Aims Previous studies demonstrated that miR-574-5p is closely associated with the prognosis of a variety of tumors. However, the relationship between miR-574-5p and hepatocellular carcinoma (HCC) has not been reported yet. Therefore, this study was designed to investigate the expression of miR-574-5p in HCC and its relationship with prognosis of the patients.Methods The expressions of miR-574-5p in 130 paired specimens of HCC and adjacent tissue as well as in HCC cell lines (HepG2 and MHCC-97H) and normal hepatic cell line (L-02) were detected by qRT-PCR method. After the optimal cut-off value of miR-574-5p expression level was determined from the survival data of the patients using X-tile software, the relations of miR-574-5p expression with the clinicopathologic factors and postoperative survival rates of the patients were analyzed. The influencing factors for the prognosis of HCC patients were determined. The target genes of miR-574-5p were predicted by TargetScan, and then were verified by dual-luciferase reporter assay and confirmed by Western blot analysis.Results The expression levels of miR-574-5p were significantly increased in HCC tissue than that in adjacent tissue, and increased in HCC cell lines than that in normal hepatic cell line (all P<0.05). The expression of miR-574-5p was significantly associated with TNM stage (P=0.002) and degree of differentiation (P=0.000). Both overall survival rate and tumor-free survival rate were significantly lower in patients with high miR-574-5p expression than those in patients with low miR-574-5p expression (both P<0.01). The results of univariate and multivariate analysis showed that high miR-574-5p expression (HR=4.101, 95% CI=1.348-8.968, P=0.03), stage III-IV (HR=5.403, 95% CI=1.266-13.860, P=0.02) and moderate/poor differentiation (HR=3.655, 95% CI=2.165-6.984, P=0.00) were independent risk factors for overall survival of HCC patients, and high miR-574-5p expression (HR=7.168, 95% CI=1.144-18.260, P=0.01) and stage III-IV (HR=7.436, 95% CI=1.123-20.916, P=0.00) were independent risk factors for disease-free survival of HCC patients. Targetscan software analysis showed that there was a direct binding site between FOG2 and miR-574-5p, and dual luciferase reporter gene assay showed that FOG2 was the target gene of miR-574-5p. Western blot analysis showed that the protein expression level of FOG2 in HCC tissue was lower than that in adjacent tissue, and Pearson correlation analysis showed that the miR-574-5p expression was negatively correlated with the protein expression of FOG2 in HCC tissue (r=-0.499, P<0.05).Conclusion The expression of miR-574-5p is generally increased in HCC. High miR-574-5p expression can be considered as a molecular marker for poor prognosis of HCC patients. MiR-574-5p may probably exert a tumor-promoting action through down-regulating its target gene FOG2.