miR-370通过靶向FUS调控肝癌细胞增殖和迁移的作用机制

摘要/Abstract

摘要： 目的探讨miR-370通过靶向FUS调控肝癌细胞的增殖和迁移的作用机制。方法合成并构建miR-370的过表达载体pcDNA3/pri-miR-370,制备相应的反义寡聚核苷酸ASO-370。使用细菌转化法对miRNA进行扩增,并将其介导进入肝癌细胞系,以研究其对细胞功能的影响。将实验分为4组,pcDNA3 空载质粒组(pcDNA3 组)、pcDNA3/pri-miR-370质粒组(miR-370组)、乱序寡聚核苷酸组(ASO-ctrl 组)和miR-370 ASO组(ASO-370组)。CCK-8试验观察miRNA对肝癌细胞HepG2增殖能力的影响;Transwell试验观察miRNA对肝癌细胞系迁移能力和侵袭能力的影响;通过生物信息学筛选预测出miR-370的候选靶基因RNA结合蛋白FUS,实时荧光定量PCR及蛋白质印迹检测过表达miRNA的肝癌细胞系中靶基因FUS在mRNA和/或蛋白水平的表达。结果过表达miR-370后,HepG2肝癌细胞的增殖能力表现出明显的抑制效果,而抑制细胞中miR-370的表达则增强了细胞增殖能力;pcDNA3 组、miR-370组、ASO-ctrl 组、ASO-370组的迁移数相对值分别为(1.13±0.24)、(0.49±0.13)、(1.37±0.31)、(1.58±0.39),pcDNA3 组、miR-370组、ASO-ctrl 组、ASO-370组的FUS mRNA表达水平分别为1、(0.56±0.08)、1、(3.62±1.51),miR-370组FUS mRNA表达水平低于pcDNA3 组,而ASO--370组FUS mRNA表达水平高于ASO-ctrl 组;pcDNA3 组、miR-370组、ASO-ctrl 组、ASO-370组的FUS蛋白相对表达量分别为1、(0.59±0.12)、1、(1.38±0.29),miR-370组FUS蛋白表达水平低于pcDNA3 组,而ASO--370组蛋白表达水平高于ASO-ctrl 组,差异均有统计学意义(P<0.05)。结论 miR-370通过抑制RNA结合蛋白FUS的表达,降低肝癌细胞HepG2的增殖和迁移能力,表明miR-370可能作为抑制肝癌的潜在治疗靶点。

关键词: miR-370, 肝癌细胞, FUS蛋白, HepG2

Abstract: Objective To investigate the mechanism by which miR-370 targets FUS to regulate the proliferation and migration of hepatocellular carcinoma (HCC) cells. Methods A vector named pcDNA3/pri-miR-370 for overexpression miR-370 was synthesized and constructed, and the corresponding antisense oligonucleotide ASO-370 was prepared. MiRNA was amplified using bacterial transformation and subsequently transfected into HCC cell lines to investigate its effect on cellular functions. The experiment was divided into four groups: pcDNA3 empty vector group (pcDNA3 group), pcDNA3/pri-miR-370 plasmid group (miR-370 group), scrambled oligonucleotide group (ASO-ctrl group), and miR-370 ASO group (ASO-370 group). Cell proliferation ability was assessed using the CCK-8 assay; cell migration and invasion capabilities were evaluated using Transwell assays. Bioinformatics analysis was performed to predict the candidate target gene of miR-370, the RNA-binding protein FUS. The expression levels of FUS at both mRNA and protein levels in miR-370 overexpressing HCC cell lines were measured by real-time quantitative PCR and Western blotting. Results After overexpressing miR-370, the proliferative ability of HepG2 liver cancer cells were significantly inhibited, while inhibiting the expression of miR-370 in the cells enhanced their proliferative ability. The relative migration numbers of the pcDNA3 group, miR-370 group, ASO-ctrl group, and ASO-370 group were (1.13±0.24), (0.49±0.13), (1.37±0.31), and (1.58±0.39), respectively. The FUS mRNA expression levels in the pcDNA3 group, miR-370 group, ASO-ctrl group, and ASO-370 group were 1, (0.56±0.08), 1, and (3.62±1.51), respectively. The FUS mRNA expression level in the miR-370 group was lower than that in the pcDNA3 group, while the FUS mRNA expression level in the ASO-370 group was higher than that in the ASO-ctrl group. The relative expression levels of FUS protein in the pcDNA3 group, miR-370 group, ASO-ctrl group, and ASO-370 group were 1, (0.59±0.12), 1, and (1.38±0.29), respectively. The FUS protein expression level in the miR-370 group was lower than that in the pcDNA3 group, while the FUS protein expression level in the ASO-370 group was higher than that in the ASO-ctrl group. The differences were statistically significant (P<0.05). Conclusion miR-370 regulates the proliferation and migration of HCC line HepG2 by inhibiting the expression of the RNA-binding protein FUS, indicating that miR-370 may serve as a viable therapeutic target for suppressing HCC.

Key words: miR-370, Hepatocellular carcinoma cells, FUS protein, HepG2

王密斯, 马丽桃, 张丽娜, 王彬. miR-370通过靶向FUS调控肝癌细胞增殖和迁移的作用机制[J]. 肝脏, 2026, 31(1): 35-38.

WANG Mi-si, MA Li-tao, ZHANG Li-na, WANG Bin. The regulatory mechanism of miR-370 on the proliferation and migration of hepatocellular carcinoma cells via targeting RNA binding protein FUS[J]. Chinese Hepatolgy, 2026, 31(1): 35-38.

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