Role of miR-29c-3p in regulation of extracellular matrix synthesis

Clark, Abbot PhD
Lopez, Navita
Tovar-Vidales, Tara PhD
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Purpose Glaucoma is a group of optic neuropathies characterized by cupping of the optic nerve head (ONH) and degeneration of retinal ganglion cell (RGC) axons that lead to loss of visual function. Primary open angle glaucoma (POAG) is the most common form of glaucoma, with a global prevalence of 65.5 million, approximately 74% of glaucoma cases. The initial site of damage in POAG is within the lamina cribrosa (LC) region of the ONH. There is upregulation of the pro-fibrotic cytokine, TGFβ2, and marked disparity in the distribution and organisation of extracellular matrix (ECM) proteins. TGFβ2 induced downregulation of miR-29 has been shown, in part, to drive ECM protein synthesis in trabecular meshwork cells. Our purpose was to determine the effect of TGFβ2 on miRNA expression, in cells that populate the LC. We hypothesise that increased TGFβ2 signalling downregulates the expression of anti-fibrotic miRNAs, stimulating a fibrotic response and remodelling of the glaucomatous LC. Methods Primary human LC cells were grown to 100% confluency, treated with TGFβ2 (5ng/ml) or control for 24hours and differences in expression of miRNAs were analysed by PCR arrays. LC cells were transfected with miR-29c-3p (10nM) mimic, inhibitor or non-targeting controls and analysed by Q-PCR to confirm overexpression or knockdown of miR-29c-3p. mRNA targets of miR-29c-3p were determined through protein expression analysis by immunocytochemistry. The effects of miR-29c-3p and TGFβ2 on collagen type (COL) I and IV protein expression were evaluated in cells transfected with miR-29c-3p mimic, inhibitor or control and treated with TGFβ2 expression. Results TGFβ2 treatment downregulated the expression of miR-29c-3p in LC cells (n=4, pa-smooth muscle actin,COL (collagen) I and IV. Transfection of miR-29c-3p mimic or inhibitor showed upregulation and downregulation of miR-29c-3p respectively, confirming transfection efficiency. miR-29c-3p was found to be a key regulator of COL I and IV synthesis. Overexpression of miR-29c-3p decreased TGFβ2 induced COL I and IV expression in LC cells. Inhibition of miR-29c-3p exacerbated the effects of TGFβ2 on COL I and IV expression. Conclusion This suggests that elevated TGFβ2 signalling may stimulate a pro-fibrotic response through downregulation of miR-29c-3p. Although miR-29c-3p may be protective by decreasing the effects of TGFβ2 induced ECM protein synthesis, we will need to further elucidate the role of TGFβ2 and miR-29c-3p in maintaining the balance of ECM synthesis.