Downregulation of microrna-122 promotes proliferation, migration, and invasion of human hepatocellular carcinoma cells by activating epithelial–mesenchymal transition
Human HCC cell lines HepG2 and huh7 were obtained from the Department of Oncology, The Affiliated Jiangyin Hospital, School of Medicine, Southeast University, Jiangyin, People’s Republic of China. Cell suspension (1 mL) was cultured in Roswell Park Memorial Institute-1640 medium (HyClone, Logan, UT, USA) supplemented with heat-inactivated 10% fetal bovine serum (FBS), 1,000 U/mL penicillin, and 100 mg/mL streptomycin and maintained in a 5% CO2 humidified incubator at 37°C. Cells were digested with pancreatin after they reached 80%–90% confluence and passaged using standard methods.15 All the cell lines were provided from participants in our hospital after they signed the informed consent. The study was approved by the ethics committee of Affiliated Jiangyin Hospital, School of Medicine, Southeast University.miRNA target gene software predicted that Wnt1 may be the target gene of miR-122 (Figure 1A). To confirm that Wnt1 is a direct target gene of miR-122, luciferase reporter vector reconstructed plasmids, pWnt1-Wt and pWnt1-Mut, that were inserted with Wnt1 mRNA3′-untranslated region. Luciferase activity test results showed that in HepG2 and huh7 cells with the cotransfected miR-122 mimics and recombinant plasmids pWnt1-Wt or pWnt1-Mut, miR-122 mimics had no significant effect on the intensity of luciferase activity in the mutant pWnt1-Mut group, but decreased its intensity by approximately 47% in the wild-type pWnt1-Wt group of HepG2 cells and by approximately 69% in huh7 cells, with statistically significant differences (both P<0.001) (Figure 1B); however, miR-NC had no obvious effects on the luciferase activity intensity of pWnt1-Mut and pWnt1-Wt groups in the HepG2 and huh7 cells.We acknowledge our instructors for their valuable advice. We also thank the reviewers for their helpful comments on this paper.DisclosureThe authors report no conflicts of interest in this work.