MicroRNA-483-3p Inhibits Extracellular Matrix Production by Targeting Smad4 in Human Trabecular Meshwork Cells

Abstract

PURPOSE. This study investigated the effects of microRNA-483-3p (miR-483-3p) on extracellular matrix (ECM) production, and clarified the regulatory mechanism of microRNA-483-3p in human trabecular meshwork cells (HTMCs) under oxidative stress. METHODS.The expression levels of ECM (fibronectin, laminin, collagen I) in HTMCs under oxidative stress were measured by Western blot. Changes of miR-483-3p expression in HTMCs were evaluated by quantitative polymerase chain reaction (qPCR). After using lentivirus stably expressing pri-miR-483, the effects of miR-483-3p on the ECM were assessed by qPCR and Western blot. Smad4, the potential target of miR-483-3p according to mRNA target-predicting algorithms, was confirmed by luciferase assay and Western blot. Furthermore, the effects of Smad4 knockdown on ECM expression were investigated by qPCR and Western blot.RESULTS. The mRNA and protein levels of ECM (fibronectin, laminin, collagen I) were upregulated in HTMCs induced by oxidative stress. The expression level of miR-483-3p decreased in HTMCs under oxidative stress, and the ectopic expression of miR-483-3p decreased the levels of ECM. In addition, miR-483-3p targeted Smad4 through two binding sites, resulting in a decrease of Smad4 expression. Furthermore, knockdown of Smad4 reduced the levels of ECM in HTMCs.CONCLUSIONS. MicroRNA-483-3p has an inhibitory effect on ECM production in HTMCs through downregulating Smad4, which indicates that miR-483-3p may serve as a potential therapeutic target in glaucoma.

HES1 promotes extracellular matrix protein expression and inhibits proliferation and migration in human trabecular meshwork cells under oxidative stress

“…As shown by western blots (Figure 2A), the relative protein levels of these ECM genes in the H 2 O 2 -treated cells were 1.81 ∼ 3.03 fold higher than the normal controls (Figure 2B, H 2 O 2 vs normal, p < 0.01 for Fibronectin, Laminin, and α-SMA; p < 0.05 for Collagen I). These results were consistent with previous studies [23, 24]. The results of immunofluorescence revealed that the fluorescence intensities of the ECM proteins in the cytoplasm under oxidative stress were substantially greater than those under normal condition (Figure 2C-2J).…”

“…The up-regulation of the ECM proteins in the HTMCs upon an acute exposure to oxidative stress (Figure 2A and 2B) was consistent with the studies conducted by us [23] and others [24, 31]. The increased production and cytoplasmic accumulation of the ECM proteins (Figure 2C-2J) will likely result in an increased deposition of key ECM components, such as fibronectin, collagen I, and laminin, in TM.…”

“…69 μM. Whereas in the experimental studies, H 2 O 2 at the concentrations ranging from 100 to 800 μM has been applied to the HTMCs to induce oxidative stress, serving as an in vitro pathological model for glaucoma research [23, 29, 30]. Therefore, in this study, H 2 O 2 at the concentrations of 100, 200, 300, 400, 600, 800, and 1000 μM was transiently applied to the HTMC cultures, and cell viability was examined (Figure 1).…”

“…Therefore, we hypothesize that HES1 may promote profibrotic ECM protein expression in TM cells under oxidative stress, contributing to the increased AH outflow resistance and elevated IOP that ultimately lead to the POAG. To test this hypothesis, we subjected human TM cells (HTMCs), a primary cell culture commonly used in glaucoma research [20–23], to oxidative stress induced by a sublethal dose of H 2 O 2 . We found the up-regulated expression of ECM and HES1 genes, as well as the compromised cell functions, such as migration and proliferation, in H 2 O 2 -stimulated HTMCs.…”

Relationship between the Pathogenesis of Glaucoma and miRNA

“…In addition, the miRNA-29 family might regulate the effect of TGF-β on the extracellular matrix [32] . Our research team showed that miRNA-483-3p had an inhibitory effect on extracellular matrix production in the human trabecular meshwork cells through downregulating Smad4 [33] , which targeted TGF-β/bone morphogenetic protein [34] and laminin [35,36] . As a consequence, we may draw the conclusion that miRNA serves as a potential therapeutic target in influencing the development of glaucoma by regulating TGF-β, which affects the extracellular matrix.…”