Abstract
Purpose: :
We have previously reported that miR-29b inhibits the synthesis and deposition of multiple components of the extracellular matrix in human trabecular meshwork (HTM) cells. Here, we evaluated the role of miR-29b as a regulator of the responses induced by cyclic mechanical stress (CMS) in HTM cells and investigate the potential regulation by miR-29b of integrin alpha 5 (ITGA5), which has been identified as the main mechanoreceptor protein in several cell types.
Methods: :
For CMS, 2 HTM primary cell lines were incubated in triplicates on flexible bottom plates and subjected to 20% stretching at 1 cycle per second during 3 hours. Effects of miR29b and siITGA5 on the responses to CMS were evaluated in cells transfected with miR29b mimic or siITGA5 compared to controls transfected with scrambled or siControl. Induction of BMP2, HSP70, IL8 and MMP3 genes by CMS was evaluated by quantitative-PCR. Targeting of miR-29b to the 3’UTR of ITGA5 was analyzed using the Psicheck luciferase system, and the effects of miR-29b on ITGA5 protein expression were evaluated by western blot.
Results: :
Transfection of HTM cells with miR-29 mimic resulted in a significant decrease in the induction of BMP2, HSP70, IL8 and MMP3 by CMS. Luciferase assays confirmed a direct interaction between the 3’UTR of ITGA5 and miR-29b. Furthermore, miR-29b inhibited the expression of ITGA5 protein. Inhibition of ITGA5 with a specific siRNA resulted in a decrease in the induction of BMP2 and HSP70 by CMS.
Conclusions: :
Our results indicated that miR-29b can inhibit some of the responses induced by CMS in HTM cells and that part of these effects may be mediated by direct targeting of ITG5A. Therefore, miR-29b may regulate not only the production of ECM components, but some of the interactions between the actin cytoskeleton and the ECM that mediate mechanotransduction in HTM cells.
Keywords: trabecular meshwork • stress response • extracellular matrix