The authors report the identification of novel gene targets of miR-204 and show that this microRNA may play an important role in the regulation of multiple functions in human trabecular meshwork cells, including apoptosis, accumulation of damaged proteins, endoplasmic reticulum stress response, and expression of inflammatory mediators.
To investigate the biological functions of miR-204 in human trabecular meshwork (HTM) cells.
Changes in gene expression induced by miR-204 in HTM cells were evaluated by gene array analysis using arrays and confirmed by quantitative-PCR (Q-PCR). Direct targeting of miR-204 to 12 potential novel targets was confirmed using a luciferase system, and five of them were verified by Western blot analysis. Effects of miR-204 on apoptosis, cell viability, and accumulation of carbonylated proteins were evaluated in HTM cells treated with H2O2. Induction of endoplasmic reticulum (ER) stress markers by tunicamycin was analyzed by Q-PCR, and expression of IL-8 and IL-11 was analyzed by ELISA.
MiR-204 decreased the expression of multiple genes in HTM cells. Twelve genes (AP1S2, Bcl2l2, BIRC2, EDEM1, EZR, FZD1, M6PR, RAB22A, RAB40B, SERP1, TCF12, and TCF4) were validated as direct targets of miR-204. Downregulation of expressions at protein levels of Bcl2l2, BIRC2, EZR, M6PR, and SERP1 were confirmed by Western blot analysis. HTM cells transfected with miR-204 showed increased levels of apoptosis, decreased viability, increased accumulation of oxidized proteins after H2O2 treatment, decreased induction of ER stress response markers, and reduced expression of inflammatory mediators IL-8 and IL-11.
MiR-204 potentially plays an important role in the regulation of multiple functions in HTM cells including apoptosis, accumulation of damaged proteins, ER stress response, and expression of inflammatory mediators.