Inhibitory effect of miR-204 on corneal epithelial cell proliferation in vitro

Abstract

Background As the main cellular constituent of corneal epithelium, corneal epithelial cells play critical roles in regulating and controlling the migration, proliferation and differentiation of cells during the repair of damage. MicroRNA (miRNA) is endogenously expressed small non-coding RNAs, which participates in a variety of biological processes. Previous studies demonstrated that miR-204 is highly expressed in normal corneal epithelium, but its function is still unclear. Objective This study was to investigate the function and mechanism of miR-204 in corneal epithelial cell proliferation. Methods Corneal epithelial tissue was collected during the corneal refractive surgery from the patients with refractive error under the informed consent, and human corneal epithelial cells(HCECs)were cultured and passaged. The relative expressing levels of miR-204 mRNA in the normal corneal epithelium and HCECs were detected by real time quantitative PCR. Cultured HCECs were evenly divided into three groups. The liposome with miR-204 mimic was transfected into the cells of the miR-204 mimic group, and blank liposome was transfected in the cells of the positive control group, and regularly cultured cells served as the normal control group. Cell proliferation capability was evaluated by colony-forming assay, and the percentage of the cells in different cell cycles was analyzed by flow cytometry. Western blot assay was employed to detect the expression levels of p-RB, E2F1, p27, CyclinA, CDC2, p-CDC2 and CDK2 proteins in the cells. Results The relative expression levels of miR-204 mRNA were 1.077±0.268 in the normal corneal epithelium and 0.041±0.018 in the HCECs, showing a significant difference between them (t=7.700, P<0.001). The cloning cell number was evidently decreased in the miR-204 mimic group in comparison with the positive control group and normal control group. The percentage of cells in the G1 phase was 47.75% in the miR-204 mimic group, which was significantly higher than 37.23% in the positive control group and 40.72% in the normal control group. The expression levels of E2F1 and p27 proteins in the cells were elevated (t=14.87, 25.11; both at P<0.01) and those of CDK2 and p-CDC2 proteins were decreased (t=5.39, 10.65; both at P<0.01) in the miR-204 mimic group in comparison with the positive control group. Conclusions The overexpression of miR-204 in the normal corneas probably is associated with non-proliferation status of corneal epithelial cells. Transfection of miR-204 into corneal epithelial cells can inhibit the proliferation of corneal epithelial cells probably by up-regulating the expression of E2F1 and p27 and suppressing the expression of CDK2/CyclinA and p-CDC2/CyclinA, which lead to cell arrest in G1 phase. Key words: [Key words] Epithelial cells, corneal; MicroRNAs; Proliferation; Cyclins; Gene expression; Humans; miR-204