MicroRNA-1 regulates fibronectin expression in human trabecular meshwork cells under oxidative stress

Abstract

Objective: To investigate the expression of microRNA-1 (miR-1) and its regulatory function on fibronectin (FN) in human trabecular meshwork cells (HTMC) under oxidative stress. Methods: Experimental study. After HTMC were treated with 0, 60, 100, 200, 400 μmol/L hydrogen peroxide (H(2)O(2)) for 6 h, respectively, the cells were placed in culture medium for 24 h. The expression of miR-1 and FN mRNA in these cells were detected by real-time quantitative PCR. According to bioinformatics analysis, the target gene of miR-1 is predicted to be FN; pcDNA3/pri-miR-1 vectors, pcDNA3/enhanced green fluorescent protein (EGFP)-FN-3'UTR vectors and pcDNA3/EGFP-FN-3'UTRmut vectors were constructed. pcDNA3/pri-miR-1 were co-transfected with pcDNA3/EGFP-FN-3'UTR or pcDNA3/EGFP-FN-3'UTRmut respectively into HTMC. pDsRed2-N1 was taken as internal reference. After 48 h transfection, the absorbance of EGFP and red fluorescent protein (REP) was detected with fluorescence spectrophotometer to explore the effect of miR-1 on FN expression. HTMC was stimulated with 200 μmol/L H(2)O(2) for 24 h after overexpression plasmid of miR-1 was transfected into it, and then FN mRNA and protein levels were detected via real time PCR, Western blotting and immunofluorescence. Data were analyzed via one-way analysis of variance or t test. Results: With the increase of H(2)O(2) concentration, miR-1 decreased (F=390.80, P<0.01) while FN increased (F=13.16, P<0.01). The level of miR-1 in HTMC stimulated by 200 μmol/L and 400 μmol/L H(2)O(2) decreased to 0.608±0.014 (t=21.67, P<0.01) and 0.409±0.020 (t=29.91, P<0.01), respectively, compared with untreated control cells (1.000); whereas, the mRNA levels of FN increased to 1.630±0.233 (t=4.47, P=0.011) and 1.903±0.246 (t=6.15, P=0.003), respectively, compared with untreated control cells(1.000). Through bioinformatics analysis, miR-1 might have candidate binding site in FN mRNA 3'-UTR. Meanwhile, these cells co-transfected with pcDNA3/pri-miR-1 and pcDNA3/EGFP-FN-3'UTRmut (0.562±0.018) had higher EGFP expression than cells co-transfected with pcDNA3/pri-miR-1 and pcDNA3/EGFP-FN-3'UTR (0.329±0.015) (t=17.39, P<0.01). Compared with the control (1.000), after overexpressing miR-1 the mRNA expression and the protein level of FN decreased to 0.294±0.081 (t=11.01, P<0.01) and 0.584±0.022 (t=5.57, P<0.01), respectively. Conclusions: MiR-1 decreases while FN increased in HTMC under oxidative stress. MiR-1 inhibits FN expression through targeting FN 3'-UTR. (Chin J Ophthalmol, 2019, 55: 355-360).