MicroRNA-494 is Highly Expressed in Human Cornea Epithelium Cells(HCECs) and inhibits Nerve Growth Factor(NGF)-Induced Cell Proliferation through Targeting Cyclin D1

Abstract

 

Purpose

 

To reveal novel miRNA regulators implicated in NGF-induced HCECs proliferation.

 

Methods

 

The differential miRNA expression profiles in HCECs with or without 25ng/ml NGF treatment were identified by microarray analysis and confirmed by RT-PCR.The lentiviral vector contained object miRNA were transfected into HCECs.The expression level of NGF-related miRNA and its target gene were examined by RT-PCR,western blot.Furthermore, the cell cycle distribution was performed by flow cytometry.

 

Results

 

Five miRNAs were up-regulated and three miRNAs down-regulated in NGF treated HCECs when compared with non-treated cells.The most obvious change was the apparent decrease of miR-494(P<0.05).Transfection of the lentivirus pLV-THM-miR-494(figure 1) greatly recovered miRNA-494 expression(P<0.05,figure 1).Cyclin D1 was predicted as a novel target of miRNA-494 through bioinformatics analysis.Overexpression of the miR-494 suppressed cyclin D1 protein and mRNA expression and increased cell distribution in G1 period(P<0.05,figure 2),which showed obviously suppressed proliferation ability.However,NGF treatment largely reversed the effects induced by miR-494, suggesting that the regulative effect of miR-494 on HECEs growth was indeed mediated by NGF(P<0.05,figure1 and figure 2)

 

Conclusions

 

MiRNA-494 and its downstream target Cyclin D1 could be a crucial axis for NGF in regulation the proliferation of HCECs.Specific modulation of miR-494 in human cornea epithelium cells may represent an attractive approach for the treatment of cornea epithelium defects diseases.

 

 

Fig.1 Stable lentivirus pLV-THM-miR-494-HCEC lines. Lentivirus pLV-THM-miR-494-HCEC examined by phase contrast microscopy(A) and flurescent microscopy(B). HCECs were transfected with lentivirus-negative or lentivirus-miR-494 and then treated with or without NGF; NGF decreased miRNA-494 expression and miRNA-494 expression was detected by RT-PCR(C).(*p < 0.05, *** p < 0.001).

 

 

Fig.2 MiRNA-494 regulated cyclin D1 expression and the increase of miRNA-494 induced G1 arrest. HCECs were transfected with lentivirus-negative or lentivirus-miR-494 and then treated with or without NGF; Cyclin D1 expression was detected by RT-PCR (A) and western blot(B). Cell cycle was detected by flow cytometry. The percentage of cells in different cell cycle phases was calculated(C) .(*p<0.05,** p<0.01).

 

Keywords: 482 cornea: epithelium • 533 gene/expression • 449 cell survival