HCV核心蛋白调控miR-486-5p/AKR1B10促进肝癌细胞生长的实验研究

Experimental study of HCV core protein regulates miR-486-5p/AKR1B10 to promote growth of hepatocellular carcinoma cells

目的:探讨丙型肝炎病毒核心蛋白(HCVc)对肝癌HepG2细胞增殖凋亡和miR-486-5p以及醛酮还原家族1B10(AKR1B10)蛋白表达的影响。方法:以重组腺病毒Ad-GFP-HCVc感染HepG2细胞,采用RT-PCR和Western blot检测HepG2细胞HCVc表达,MTT法和流式细胞术分别检测HepG2细胞增殖和凋亡情况,qRT-PCR检测HepG2细胞中miR-486-5p表达,Western blot检测AKR1B10蛋白表达;以miR-486-5p抑制剂转染HepG2细胞,qRT-PCR和Western blot分别检测HepG2细胞中miR-486-5p和AKR1B10蛋白表达;采用双荧光素酶报告基因实验检测miR-486-5p和AKR1B10的靶向关系。结果:Ad-GFP-HCVc感染后,HepG2细胞中HCVc表达升高;与阴性对照组相比,HCVc组细胞增殖活力和AKR1B10蛋白表达明显升高,而细胞凋亡率和miR-486-5p表达明显降低(P<0.05);与anti-miR-NC组相比,转染miR-486-5p抑制剂的HepG2细胞miR-486-5p表达明显降低,而AKR1B10蛋白表达明显升高(P<0.05);双荧光素酶报告基因实验证实miR-486-5p可与AKR1B10靶向结合。结论:HCVc可能通过下调miR-486-5p表达引起其靶基因AKR1B10表达升高,促进HepG2细胞异常生长。

Objective:To investigate effects of hepatitis C virus core protein(HCVc)on proliferation and apoptosis of HepG2 cells,expressions of miR-486-5p and aldehyde reductase family 1 member B10(AKR1B10)protein.Methods:After HepG2 cells were infected with recombinant adenovirus Ad-GFP-HCVc,expression of HCVc in HepG2 cells was detected by RT-PCR and Western blot,proliferation and apoptosis of HepG2 cells were detected by MTT and flow cytometry,expression of miR-486-5p in HepG2 cells was detected by qRT-PCR,expression of AKR1B10 protein was detected by Western blot.After transfection of HepG2 cells with miR-486-5p inhibitor,protein expressions of miR-486-5p and AKR1B10 in HepG2 cells were detected by qRT-PCR and Western blot.Double luciferase reporter gene assay was used to detect targeting relationship between miR-486-5p and AKR1B10.Results:After HepG2 cells were infected with Ad-GFP-HCVc,HCVc expression in HepG2 cells was significantly increased.Compared with negative control group,proliferation activity and AKR1B10 protein expression of HepG2 cells in HCVc group were significantly increased,while apoptosis rate and miR-486-5p expression were significantly decreased(P<0.05).Compared with anti-miR-NC group,expression of miR-486-5p of HepG2 cells transfected with miR-486-5p inhibitor was significantly decreased,while expression of AKR1B10 protein was significantly increased(P<0.05);double luciferase reporter gene assay confirmed that miR-486-5p could targeting combine with AKR1B10.Conclusion:HCVc may increase target gene AKR1B10 and promote abnormal growth of HepG2 cells by downregulating of miR-486-5p.