微RNA-25通过调控Klf4表达促进肝癌细胞的增殖和迁移

MicroRNA-25 promotes growth and migration of liver cancer cells by regulating Klf4 gene expression

目的 :探讨肝癌细胞中微RNA-25(microRNA-25,miR-25)对核转录因子Krüppel样因子(4Krüppel-like factor 4,Klf4)基因表达的靶向调控作用,以及对肝癌细胞增殖和迁移能力的影响。方法 :通过生物信息学工具预测miR-25可能调控的靶基因。将miR-25模拟物和抑制子分别转染人肝癌细胞Sk-hep-1和Bel-7402后,采用实时荧光定量PCR、蛋白质印迹法和双荧光素酶报告基因实验验证miR-25对Klf4基因的调控作用;同时,将Klf4过表达质粒共转染至以上细胞株,然后采用CCK-8法和Transwell细胞迁移实验分别检测miR-25和Klf4表达变化对肝癌细胞增殖和迁移的影响。结果 :生物信息学分析表明,miR-25可与Klf4基因3’端非翻译区的2个潜在结合位点相结合。miR-25可以调控Klf4基因的表达,其中miR-25模拟物转染后Klf4表达明显下调(P<0.05),而miR-25抑制子转染后Klf4表达明显上调(P<0.05)。miR-25模拟物转染能促进肝癌Sk-hep-1和Bel-7402细胞的增殖和迁移能力(P值均<0.05),miR-25抑制子可抑制Sk-hep-1和Bel-7402细胞的增殖和迁移能力(P值均<0.05);而肝癌细胞转染Klf4过表达质粒后,miR-25模拟物对肝癌细胞增殖和迁移能力的促进作用明显减弱(P值均<0.05),miR-25抑制子对肝癌细胞增殖和迁移能力的抑制作用则明显增强(P值均<0.05)。结论 :miR-25可促进肝癌细胞的增殖和迁移,其作用机制可能与靶向调控抑癌基因Klf4的表达有关。

Objective： To investigate the regulating effects of microRNA-25 （miR-25） on the expression of nuclear transcription factor Kruppel- like factor 4 （Klf4） gene, and the proliferation and migration abilities of hepatocellular carcinoma cells.Methods： The bioinformatics tool was used to predict the target gene of miR-25. The mimic and inhibitor of miR-25 were transfected into human hepatocellular carcinoma cell lines Sk-hep-1 and Bel-7402, respectively. Then the regulation effect of miR-25 on Klf4 gene expression was validated by real-time fluorescent quantitative PCR, Western blotting and dual luciferase reporter experiment. After Sk-hep-1 and Bel-7402 cells were transfected with Klf4 overexpression plasmids combined with miR-25-mimic and -inhibitor, the effects of miR-25 and Klf4 expression on the proliferation and migration of hepatocellular carcinoma cells were detected by CCK-8 method and Transwell migration assay, respectively. Results： Bioinformatics analysis showed that miR-25 could combine with two potential binding sites in 3＇-terminal non-translation region of Klf4 gene. MiR-25 could regulate the expression of Klf4 gene. The expression of Klf4 was down-regulated in Sk-hep-1 cells transfected with miR-25-mimic （P 〈 0.05）, while which was up-regulated in Bel-7402 cells transfected with miR-25-inhibitor （P 〈 0.05）. Moreover, the proliferation and migration of Sk-hep-1 cells were significantly promoted after transfection with miR-25-mimic （both P 〈 0.05）, while those of Bel-7402 cells were significantly inhibited after transfection with miR- 25-inhibitor （both P 〈 0.05）. In addition, after co-transfection with Klf4 overexpression plasmids, the effects of miR-25-mimic on the proliferation and migration of hepatocellular carcinoma Sk-hep-1 cells were significantly weakened （both P 〈 0.05）, but the effects of miR-25-inhibitor were enhanced in Bel-7402 cells （both P 〈 0.05）. Condusion： MiR-25 can promote the proliferation and migration of hepatocellular carcinoma cells, and its mechanism may be related to regulating the expression of tumor suppressor gene Klf4.