Iinhibitory effects of miR-200a on proliferation and migrating ability of conjunctival fibroblasts and its mechanism

Background

Scarring of surgical area, the most important factor, leads to the failure of glaucoma filtering surgeries.Therefore, more and more attentions are paid to the causes and process of scar formation.

Objective

This study was to compare the differences of proliferation and migrating abilities of fibroblasts between filtering bleb scar tissue and normal Tenon capsular tissue, and to investigate the inhibitory effects of miRNA-200a (miR-200a) on biological behavior of conjunctival fibroblasts.

Methods

Normal Tenon capsular tissue and filtering bleb scar tissue were collected during the strabismus surgery and glaucoma filtering surgery, respectively for the primarily culture of fibroblasts.The proliferation (absorbency, A) of the cells was assayed by cell counting kit-8 (CCK8) method; the relative migrating distance of the cells was measured by cell scratch test; and the relative expressions of transforming growth factor-β₁ (TGF-β₁)mRNA and miR-200a mRNA in the cells were detected by real-time fluorescence quantitative PCR.TGF-β₁ mimic of 0, 1, 2 and 5 ng/ml was added in the medium of human normal Tenon capsular-derived fibroblasts (HTFs), and 0.00, 0.25, 0.50, 1.00 μg/ml TGF-β₁ inhibitor was added in the medium of human scarring-derived fibroblasts (HSFs) for 24 hours, respectively, and CCK8 was used to evaluate the proliferation of the cells.The relative migrating distance as well as the relative expressions of miR-200a mRNA were analyzed in the 2 ng/ml TGF-β₁ mimic-or 1.00 μg/ml TGF-β₁ inhibitor-treated cells.

Results

The primary conjunctival presented the spindle and star-like in shape with large body and oval nuclei.The cells showed the positive response for keratin and vimentin antibodies.The A values were 1.476±0.110 in the HSFs and 0.958±0.074 in the HTFs, with a significant difference between them (t=24.900, P=0.016). The relative expressions of TGF-β₁ mRNA were significantly higher in the HSFs than those in the HTFs, and the relative expressions of miR-200a were evidently lower in the HSFs than those in the HTFs, showing significant differences between them (t=6.358, P=0.024; t=7.394, P=0.018). Compared with the 2 ng/ml TGF-β₁ mimic-treated HTFs, the relative migrating distance increased, while the expression level of miR-200a mRNA was significantly reduced in the 2 ng/ml TGF-β₁ mimic-treated HSFs (all at P<0.05); Compared with the 1.00 μg/ml TGF-β₁ inhibitor-treated HTFs, the relative migrating distance decreased, but the expression level of miR-200a mRNA was significantly elevated in the 1.00 μg/ml TGF-β₁ inhibitor-treated HSFs (all at P<0.05).

Conclusions

The proliferation and migrating abilities are stronger in the HSFs than those in the HTFs, which probably is regulated by the expression of miR-200a in the cells.The miR-200a plays a negative feedback for the effect of TGF-β₁ promoting proliferation and migration of fibroblasts.

MicroRNAs; Fibroblasts; Signal transduction; Transforming growth factor beta1; Cell movement; Cell proliferation; Message RNA; Glaucoma