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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 regulatory effect of microRNA-25 (miR-25) on the expression of Klf4 gene in human hepatocellular carcinoma cells, The impact of ability. Methods: Bioinformatics tools were used to predict the possible target genes of miR-25. After miR-25 mimics and inhibitors were transfected into human hepatocellular carcinoma cells Sk-hep-1 and Bel-7402 respectively, real-time fluorescent quantitative PCR, Western blot and dual luciferase reporter assay were used to verify the effect of miR-25 on Klf4 gene Meanwhile, the Klf4 overexpression plasmid was co-transfected into the above cell lines, and then the effects of the changes of miR-25 and Klf4 expression on the proliferation and migration of hepatoma cells were detected by CCK-8 assay and Transwell cell migration assay. Results: Bioinformatics analysis showed that miR-25 binds to two potential binding sites in the 3 ’untranslated region of Klf4 gene. The expression of Klf4 gene could be regulated by miR-25. The expression of Klf4 was down-regulated after miR-25 mimics transfection (P <0.05), while the expression of Klf4 was up-regulated after miR-25 inhibitor transfection (P <0.05). miR-25 mimics could promote the proliferation and migration of hepatocellular carcinoma Sk-hep-1 and Bel-7402 cells (all P <0.05), miR-25 suppressor could inhibit the expression of Sk-hep-1 and Bel- 7402 cells (P <0.05). However, the effect of miR-25 mimics on proliferation and migration of hepatoma cells was significantly weaker (P <0.05) after transfection of Klf4 overexpression plasmid into hepatoma cells The inhibitory effect of -25 inhibitor on the proliferation and migration of hepatocellular carcinoma cells was significantly enhanced (P <0.05). Conclusion: miR-25 can promote the proliferation and migration of hepatocellular carcinoma cells, and its mechanism may be related to the regulation of Klf4 expression.