MicroRNA-34a suppresses aggressiveness of hepatocellular carcinoma by modulating E2F1, E2F3, and Caspase-3

Rui Han,1,2 Xinyi Chen,1 Ya Li,1,2 Shunjia Zhang,2 Ruibai Li,1 Lingeng Lu3,41Department of Oncology and Hematology, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700 People’s Republic of China; 2Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520, USA; 3Department of Chronic Disease Epidemiology, Yale School of Public Health, School of Medicine, Yale University, New Haven, CT, 06520-8034, USA; 4Center for Biomedical Data Science, Yale Cancer Center, Yale University, New Haven, CT, USABackground: Accumulating evidence suggests an antineoplastic role of MicroRNA-34a (miR-34a) in human cancer. However, its precise biological functions stay largely elusive.Purpose: Our study was aimed to investigate the impact of miR-34a on hepatocellular carcinoma (HCC) and its underlying apoptosis related mechanisms in vitro, as well as the association of miR-34a,E2F1andE2F3expression with patient survival of HCC using publicly accessed datasets.Methods: The HBV-expressing Hep3B and SNU-449 cell lines with or without enforced expression of miR-34a were in vitro cultured for cell proliferation, colony formation, wound healing, cell invasion, and 3D spheroid formation. Quantitative reverse transcription PCR (RT-qPCR) was performed forE2F1, E2F3expression. Caspase-3 (CASP3) activity was determined using a CaspACETM,Assay System. Kaplan–Meier survival curves were used to analyze the associations of miR-34a,E2F1andE2F3expression and overall survival in HCC. Meta-analysis was performed to examine the differential expression ofE2F1andE2F3between primary HCC vs normal tissues.Results:The results in vitro showed that enforced miR-34a expression significantly inhibited cell proliferation, migration, and invasion of both Hep3B and SNU-449. RT-qPCR results demonstrated that miR-34a could significantly suppressE2F1andE2F3expression, particularly in SNU-449. CASP3 activity in both Hep3B and SNU-449 increased in miR-34a treatment group. OverexpressedE2F1andE2F3were observed in primary HCC vs normal tissues. Survival analyses showed that HCC patients with either high miR-34a, or lowE2F1, or lowE2F3expression had better survival than their opposite counterparts, respectively.Conclusion: Our study suggested thatmiR-34a can modulate the expression ofE2F1, E2F3, and CASP3 activity, thereby repressing tumor aggressiveness and expediting apoptosis in liver cancer cellsKeywords: hepatocellular carcinoma, microRNA-34a, E2Fs, Caspase-3, prognosis