Fig 1.
Comparison of expression levels of miRNAs in the PVD and MH samples.
(A, B) Clustering analyses of the expression patterns of microRNAs (miRNAs) in the vitreous of PVD (n = 3) and control MH (n = 3) samples. Red indicates high expression and blue indicates low expression. (C) Volcano plot of real-time polymerase chain reaction (PCR)-based miRNA array data from the vitreous humor of PVD and control MH samples. Profiling for 377 miRNAs was performed on the vitreous humor of PVD (n = 3) and control MH (n = 3) samples. The y-axis depicts the negative log10 of p-values of the t-tests (the horizontal line corresponds to a p-value of 0.05; a higher value indicates greater significance), and the x-axis depicts the difference in expression between the two experimental groups as log2 fold changes. (D, E) Expression levels of miRNAs detected by real-time PCR-based miRNA arrays that were significantly different (p < 0.05) between PVD (n = 3) as compared with control MH (n = 3) samples. The average of ΔCt values of control MH samples (white bars) and proliferative vitreoretinal disease (PVD) samples (black bars) were calculated with standard deviations. To reverse the inverse relationship between ΔCt values and miRNA expression levels, ΔCt values were transferred into negative ΔCt values. The differences of ΔCt values between PVD and control MH samples are shown by relative expression levels of downregulated miRNAs (D) and upregulated miRNAs (E) in PVD. CTR; control, PVD; proliferative vitreoretinal disease; MH, macular hole. ** p < 0.01; *p < 0.05.
Fig 2.
Comparisons of expression levels of miR-21, miR-204, and let-7e in the vitreous humor of MH, RRD, PVR, and PDR samples.
(A, B, and C) Results of quantitative PCR (qPCR) analyses of miR-21, miR-204, and let-7e levels from the vitreous humor of MH (n = 7), RRD (n = 5), PVR (n = 5), and PDR (n = 10) samples. Fold changes in expression levels of miR-21(A), miR-204 (B), and let-7e (C) in RRD, PVR, and PDR samples were determined relative to the control MH sample. CTR, control; RRD, rhegmatogenous retinal detachment; MH, macular hole; PVR, proliferative vitreoretinopathy; PDR, proliferative diabetic retinopathy. **p < 0.01; *p < 0.05.
Fig 3.
Expression of miR-21 is induced by signaling and high glucose conditions in RPECs.
(A, B, and C) Results of quantitative (q) PCR analyses of miR-21, miR-204, and let-7e levels in TGF-β-stimulated ARPE-19 cells. Fold changes in expression levels of miR-21 (A), miR-204 (B), and let-7e (C) in TGF-β-stimulated ARPE-19 cells were determined relative to the unstimulated control cells. (D) Results of qPCR analyses of TGF-β-induced expression of miR-21 in ARPE-19 cells under low or high glucose conditions. Fold changes in expression levels of miR-21 in TGF-β-stimulated ARPE-19 cells under low or high glucose culture conditions were determined relative to the unstimulated control cells. RPECs, retinal pigment epithelial cells; TGF-β, transforming growth factor-β; LG = low glucose; HG = high glucose. **p < 0.01; *p < 0.05.
Fig 4.
Effect of miR-21 expression on cell proliferation of ARPE-19 cells.
(A, B) Results of quantitative (q)PCR analyses of miR-21 in miR-21 mimic- or inhibitor-transfected ARPE-19 cells. Fold changes in expression levels of miR-21 in miR-21 mimic-transfected ARPE-19 cells were determined relative to the negative control mimic-transfected cells (A). Expression level of miR-21 in miR-21 inhibitor-transfected ARPE-19 cells in the presence or absence of TGF-β2 was calculated by comparing with negative control inhibitor-transfected cells (B). (C) Effect of miR-21 mimic or miR-21 inhibitor transfection on GFP reporter gene expression of CMV-EGFP or CMV-EGFP-3′ UTR miR-21-target reporter plasmid in ARPE19 cells. ARPE-19 cells were co-transfected with CMV-EGFP or CMV-EGFP-3′ UTR miR-21-target reporter plasmid, together with miR-21 mimics or miR-21 inhibitor, as indicated. After 24 h, GFP expression was analyzed under the fluorescence microscope. All fluorescence images were taken at the same exposure time (1/15s). (D, E) The migration ability of miR-21 mimic and miR-21 inhibitor-treated ARPE-19 cells was examined using the wound healing assay. (D) Representative images of wound sealing of miR-21 mimic and miR-21 inhibitor-transfected cells cultured with or without TGF-β at 0 and 12 h. (E) Quantification of the wound healing assay. The figure shows the average percentage of wound closure ± SD. The transfection of the miR-21 mimic significantly increased cell migration of ARPE-19 cells without TGF-β stimulation, and transfection of miR-21 inhibitor inhibited TGF-β-induced enhanced migration. (F, G) Cell viability was measured with CCK-8 kits following the treatment of miR-21 mimic and miR-21 inhibitor for 12, 24, 48, 72, and 96 h (F). The miR-21 mimic treatment resulted in increased cell proliferation, and miR-21 inhibitor treatment resulted in decreased cell proliferation at 72 h (G). NC, negative control. ***p < 0.001; **p < 0.01; *p < 0.05.
Fig 5.
Effect of miR-21 expression on epithelial-mesenchymal transition (EMT)-related gene expression during TGF-β2-induced EMT of ARPE-19 cells.
(A, B, and C) Results of qPCR analyses of fibronectin, αSMA, and N-cadherin mRNA expression in miR-21 mimic- or inhibitor-transfected ARPE-19 cells in the presence or absence of TGF-β2. Fold changes in expression levels of fibronectin (A), αSMA (B), and N-cadherin (C) mRNAs in miR-21 mimic-transfected ARPE-19 cells were determined relative to the untransfected control. NC, negative control; αSMA, alpha smooth muscle actin; EMT, epithelial-mesenchymal transition. ***p < 0.001; **p < 0.01; *p < 0.05; n.s; not significant.