The effect of microRNA-101 on biological behaviord of human prostate cancer cell line PC-3 by targeting enhancer of zeste homolog 2

Abstract

Objective To investigate the regulatory effect of microRNA (miR)-101 on the biological features of human prostate cancer cell line PC-3 by targeting enhancer of zeste homolog 2 (EZH2).Methods The expression of miR-101 and EZH2 in prostate cancer cell lines (PC-3,and LNCaP) and normal prostate epithelial RWPE-1 cell line was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting respectively.Dual luciferase reporter gene assay was employed to determine if miR-101 could target EZH2 through binding to 3' untranslated region (3 '-UTR) of EZH2 mRNA.PC-3 cells were transfected with synthetic miR-101 mimic.Methyl thiazol tetrazolium (MTT) assays were used to measure the proliferation of the PC-3 cells.Flow cytometry was used to analyze the cell cycle distribution.Annexin V/propidium iodide (PI) double staining was used to measure cell apoptosis.The wound scrape assays were used to measure the cell motility.Transwell invasion assays were used to analyze the invasiveness in vitro.Results The expression levels of miR-101 in PC-3 and LNCaP cell lines were lower than in the RWPE-1 cell line,while the expression levels of EZH2 mRNA and protein were higher.There was a negative correlation between the expression of miR-101 and EZH2 (P ＜ 0.05).Dual luciferase reporter gene assay confirmed EZH2 was one of regulatory genes by miR-101.After miR-101 mimic was transfected into prostate cancer PC-3 cells,the expression of miR-101 was up-regulated (P ＜ 0.05),and that of EZH2 mRNA and protein was down-regulated significantly (P ＜ 0.05).The growth rate in miR-101 group was lower significantly than that in two control groups at 48,72 and 96 h after transfeetion (P ＜ 0.05).Flow eytometry analysis showed there was G0/G1 phase arrest in miR-101 group compared to two control groups (P ＜ 0.05).Cell apoptosis rate in transfected experimental group with miR-101 mimics (miR-101 group),blank control group and negative control group at 48 h after transfection was (9.84 ± 0.83) ％,(2.53 ± 0.36) ％ and (2.71 ± 0.42) ％ respectively.The apoptosis rate in miR-101 group was higher than two control groups (P ＜ 0.01).Scarification test demonstrated that the migration of cells in miR-101 group was more slowly than two control groups (P ＜ 0.01).Transwell test showed that the number of PC-3 cells penetrating the Matrigel gel in miR-101 group was significantly less than two control groups (P ＜ 0.01).Conclusion EZH2 may be one of target genes of miR-101 that plays a negative regulatory role.The upregulation of miR-101 can lead to the lower expression of EZH2 in PC3 cells,suggesting miR-101 exerts significantly inhibitory effect on prostate cancer. Key words: Prostate cancer; MicroRNA-101 ; Enhancer of zeste homolog 2