二甲双胍通过miR-194-5p/RBM6通路抑制肝细胞癌细胞的恶性细胞表型的机制研究

目的 观察二甲双胍对肝细胞癌（hepatocellular carcinoma，HCC）细胞HepG2增殖、凋亡的影响，并探究其潜在机制与miR-194-5p/RNA结合基序蛋白6（RNA binding motif protein 6，RBM6）通路之间的关系。 方法 随机将100例接受手术的HCC患者分为对照组、试验组，每组50例。对照组患者给予安慰剂口服，试验组给予二甲双胍口服。脂质体法将antagomiRNA、antagomiR-194-5p、pcDNA 3.1、pcDNA 3.1-RBM6、二甲双胍+si-NC、二甲双胍+si-RBM6、antagomiR-194-5p+si-NC、antagomiR-194-5p+si-RBM6转染至HepG2细胞。荧光定量聚合酶链式反应实验检测血清、组织、细胞中miR-194-5p、RBM6的表达；噻唑蓝法、5-溴-2-脱氧尿嘧啶染色检测细胞增殖；流式细胞术检测细胞凋亡；双荧光素酶报告基因实验检测细胞荧光活性；蛋白免疫印迹实验检测细胞RBM6蛋白。 结果 试验组患者的2年无瘤生存期显著延长，患者治疗后血清miR-194-5p表达明显降低（P＜0.05）。体外细胞研究显示，二甲双胍处理后的HepG2细胞中miR-194-5p表达降低，细胞的增殖能力显著降低，凋亡能力显著升高（P＜0.05）。癌组织、HepG2细胞中miR-194-5p表达显著高于癌旁组织或正常肝细胞，抑制miR-194-5p后，削弱了HepG2细胞的增殖能力，增强了凋亡能力（P＜0.05）。miR-194-5p靶向负调控RBM6的表达。miR-194-5p、RBM6在肝癌组织中呈明显的负相关性（r=0.672，P＜0.05）。过表达RBM6具有与抑制miR-194-5p相似的功能。敲减RBM6明显的抑制二甲双胍、抑制miR-194-5p对HepG2细胞的增殖、凋亡调控。 结论 二甲双胍抑制肝细胞癌细胞增殖，促进凋亡，提高患者的DFS，其潜在的机制与miR-194-5p/RBM6通路有关。

Mechanism of metformin inhibiting malignant cell phenotype of hepatocellular carcinoma cells through miR-194-5p/RBM6 pathway

Objective To observe the effect of metformin on the proliferation and apoptosis of hepatocellular carcinoma (HCC) cells HepG2, and to explore the relationship between its potential mechanism and miR-194-5p/RNA binding motif protein 6 (RBM6) pathway.Methods A total of 100 patients with HCC undergoing surgery were randomly divided into control group and experimental group, with 50 cases in each group. The control group was given placebo orally and the experimental group was given metformin orally. Antigomirna, antigomiR-194-5p, pcDNA 3.1, pcDNA 3.1-RBM6, metformin+si-NC, metformin+si-RBM6, antigomiR-194-5p+si-NC and antigomiR-194-5p+si-RBM6 were transfected into HepG2 cells by liposome method. The expressions of miR-194-5p and RBM6 in serum, tissues and cells were detected by fluorescence quantitative polymerase chain reaction (FQ-PCR).MTT assay and 5-bromo-2-deoxyuracil staining were used to detect cell proliferation, and apoptosis was detected by flow cytometry. Double luciferase reporter gene assay was used to detect the fluorescence activity of cells, and RBM6 protein was detected by Western blot.Results The two-year disease-free survival (DFS) of the experimental group was significantly prolonged, and the expression of serum miR-194-5p decreased significantly after treatment (P＜0.05). In vitro cell study showed that the expression of miR-194-5p decreased, the proliferation ability decreased and the apoptosis ability increased significantly in HepG2 cells treated with metformin (P＜0.05). The expression of miR-194-5p in cancer tissues and HepG2 cells was significantly higher than that in adjacent tissues or normal hepatocytes. Inhibition of miR-194-5p weakened the proliferation and enhanced the apoptosis of HepG2 cells (P＜0.05). miR-194-5p negatively regulated the expression of RBM6. There was a significant negative correlation between miR-194-5p and RBM6 in hepatocellular carcinoma tissues (r=0.672, P＜0.05). Overexpression of RBM6 had a similar function with inhibition of miR-194-5p. Knockdown of RBM6 significantly inhibited metformin and the regulation of miR-194-5p on proliferation and apoptosis of HepG2 cells.Conclusion Metformin inhibits the proliferation of hepatocellular carcinoma cells, promotes apoptosis and improves DFS of patients. Its potential mechanism may be related to miR-194-5p/RBM6 pathway.