Jianming Qiu

Alterations in microRNA expression in stress-induced cellular senescence.

We investigated miRNA expression changes associated with stress-induced premature senescence (SIPS) in primary cultures of human diploid fibroblast (HDF) and human trabecular meshwork (HTM) cells. Twenty-five miRNAs were identified by miRNA microarray analysis and their changes in expression were validated by TaqMan real-time RT-PCR in three independent cell lines of HTM and HDF. SIPS in both HTM and HDF cell types was associated with significant down-regulation of four members of the miR-15 family and five miRNAs of the miR-106b family located in the oncogenic clusters miR-17-92, miR-106a-363, and miR-106b-25. SIPS was also associated with up-regulation of two miRNAs (182 and 183) from the miR-183-96-182 cluster. Transfection with miR-106a agomir inhibited the up-regulation of p21(CDKN1A) associated with SIPS while transfection with miR-106a antagomir led to increased p21(CDKN1A) expression in senescent cells. In addition, we identified retinoic acid receptor gamma (RARG) as a target of miR-182 and showed that this protein was down-regulated during SIPS in HDF and HTM cells. These results suggest that changes in miRNA expression might contribute to phenotypic alterations of senescent cells by modulating the expression of key regulatory proteins such as p21(CDKN1A) as well as by targeting genes that are down-regulated in senescent cells such as RARG.

Targeting of Integrin β1 and Kinesin 2α by MicroRNA 183

MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H2O2. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells.

Role of miR-204 in the Regulation of Apoptosis, Endoplasmic Reticulum Stress Response, and Inflammation in Human Trabecular Meshwork Cells

PURPOSE To investigate the biological functions of miR-204 in human trabecular meshwork (HTM) cells. METHODS 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 H(2)O(2). 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. RESULTS 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 H(2)O(2) treatment, decreased induction of ER stress response markers, and reduced expression of inflammatory mediators IL-8 and IL-11. CONCLUSIONS 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.

Targeting of Integrin beta 1 and Kinesin 2 alpha by miR-183

MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H2O2. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells.

Resveratrol prevents the expression of glaucoma markers induced by chronic oxidative stress in trabecular meshwork cells

Elevated intraocular pressure (IOP) constitutes the best characterized risk for primary open-angle glaucoma (POAG). Elevated IOP is believed to result from an increase in aqueous humor outflow resistance at the level of the trabecular meshwork (TM)/Schlemms canal. Malfunction of the TM in POAG is associated with the expression of markers for inflammation, cellular senescence, oxidative damage, and decreased cellularity. Current POAG treatments rely on lowering IOP, but there is no therapeutic approach available to delay the loss of function of the TM in POAG patients. We evaluated the effects of chronic administration of the dietary supplement resveratrol on the expression of markers for inflammation, oxidative damage, and cellular senescence in primary TM cells subjected to chronic oxidative stress (40% O2). Resveratrol treatment effectively prevented increased production of intracellular reactive oxygen species (iROS) and inflammatory markers (IL1alpha, IL6, IL8, and ELAM-1), and reduced expression of the senescence markers sa-beta-gal, lipofuscin, and accumulation of carbonylated proteins. Furthermore, resveratrol exerted antiapoptotic effects that were not associated with a decrease in cell proliferation. These results suggest that resveratrol could potentially have a role in preventing the TM tissue abnormalities observed in POAG.

Cross-talk between miR-29 and Transforming Growth Factor-Betas in Trabecular Meshwork Cells

PURPOSE To investigate the interactions between microRNA-29 (miR-29), a negative regulator of extracellular matrix (ECM), and transforming growth factors (TGF)β-1 and TGFβ-2. METHODS Changes in expression of the miR-29 family were analyzed by quantitative-PCR (Q-PCR) after treatment with TGFβ1 and TGFβ2 (1 ng/mL). TGFβ1 and TGFβ2 were evaluated at gene expression and protein levels by Q-PCR and ELISA, respectively, in human trabecular meshwork (HTM) cells transfected with miR-29b or scramble control. TGFβ1 promoter activity was analyzed using an adenovirus with the reporter SEAP. The effects of miR-29b and TGFβ2 on ECM gene expression were evaluated in cells transfected with miR-29b or scramble control and treated with TGFβ2, and the expression of ECM genes was analyzed by Q-PCR. RESULTS TGFβ2 but not TGFβ1, downregulated the three members of the miR-29 family. Overexpression of miR-29b antagonized the effects of TGFβ2 on the expression of several ECM components. MiR-29b decreased the expression of TGFβ1 at the promoter, transcript, and protein levels but had only a minor effect on the expression of active TGFβ2. The inhibition of TGFβ1 by miR-29b was partially recovered after co-transfection with a plasmid-expressing bone morphogenetic protein 1. CONCLUSIONS Results showed some level of crosstalk between TGFβs and miR-29. Specifically, the downregulation of miR-29 by TGFβ2 contributed to the induction of several ECM components by this cytokine in TM cells. This observation, together with the inhibitory effects of miR-29b on the expression of TGFβ1, suggests that the miR-29 family could play an important role in modulating TGFβs on the outflow pathway.

Modulation of Inflammatory Markers by miR-146a during Replicative Senescence in Trabecular Meshwork Cells

PURPOSE To investigate the alterations in microRNA (miRNA) expression during replicative senescence (RS) in human trabecular meshwork (HTM) cells. METHODS Two HTM cell lines were serially passaged until they reached RS. Changes in expression of 30 miRNAs were assessed by real-time quantitative (q)-PCR. The effects of miR-146a on gene expression were analyzed with gene arrays and the results confirmed by real-time q-PCR. Protein levels of IRAK1 and PAI-1 were analyzed by Western blot and those of IL6 and IL8 by ELISA. Senescence-associated markers were monitored by flow cytometry and cell proliferation by BrdU incorporation. RESULTS RS of HTM cells was associated with significant changes in expression of 18 miRNAs, including the upregulation of miR-146a. miR-146a downregulated multiple genes associated with inflammation, including IRAK1, IL6, IL8, and PAI-1, inhibited senescence-associated beta-galactosidase (SA-beta-gal) activity and production of intracellular reactive species (iROS), and increased cell proliferation. Overexpression of either IRAK1 or PAI-1 inhibited the effects of miR-146a on cell proliferation and iROS production in senescent cells. CONCLUSIONS RS in HTM cells was associated with changes in miRNA expression that could influence the senescent phenotype. Upregulation of the anti-inflammatory miR-146a may serve to restrain excessive production of inflammatory mediators in senescent cells and limit their deleterious effects on the surrounding tissue. Among the different proteins repressed by miR-146a, the inhibition of PAI-1 may act to minimize the effects of senescence on the generation of iROS and growth arrest and prevent alterations of the extracellular proteolytic activity of the TM.

Extracellular Release of ATP Mediated by Cyclic Mechanical Stress Leads to Mobilization of AA in Trabecular Meshwork Cells

PURPOSE To investigate the mechanisms that mediate the release of ATP induced by cyclic mechanical stress (CMS) and the role of extracellular ATP in the mobilization of arachidonic acid (AA) and prostaglandin secretion. METHODS Porcine trabecular meshwork (pTM) cells were subjected to CMS. Extracellular ATP was detected with a luciferin-luciferase assay in the presence or absence of transport inhibitors and a lipid raft disrupter. ATP vesicles were visualized with quinacrine. The release of AA (AA 1-14C) was measured with and without ATP, ATP inhibitors, and phospholipase-A and -C inhibitors. Prostaglandin E2 (PGE2) and viability were measured with ELISA and a lactate dehydrogenase assay, respectively. RESULTS CMS induced ATP release that was inhibited by the vesicle inhibitors N-ethylmaleimide (NEM) and monensin. Lipid raft disruption significantly increased the extracellular ATP induced by CMS. CMS induced AA release (1-4-fold increase) and its metabolic product PGE2 (3.9-fold increase). The AA mobilization induced by CMS could be mimicked by the addition of extracellular ATP and was partially inhibited by a P2 antagonist, by an ATP inhibitor, and by inhibitors of phospholipase-A2 and -C. Addition of PGE2 (10 microM) to the media exerted cytoprotective effects against long-term CMS. CONCLUSIONS Extracellular release of ATP induced by CMS in TM cells is mediated by exocytosis of ATP-enriched vesicles into lipid rafts. The resulting activation of purinergic receptors leads to mobilization of AA from the plasma membrane. The subsequent release of PGE could exert protective effects by preventing TM cell loss that may result from chronic exposure to CMS.

Regulation of Trabecular Meshwork Cell Contraction and Intraocular Pressure by miR-200c

Lowering intraocular pressure (IOP) delays or prevents the loss of vision in primary open-angle glaucoma (POAG) patients with high IOP and in those with normal tension glaucoma showing progression. Abundant evidence demonstrates that inhibition of contractile machinery of the trabecular meshwork cells is an effective method to lower IOP. However, the mechanisms involved in the regulation of trabecular contraction are not well understood. Although microRNAs have been shown to play important roles in the regulation of multiple cellular functions, little is known about their potential involvement in the regulation of IOP. Here, we showed that miR-200c is a direct postranscriptional inhibitor of genes relevant to the physiologic regulation of TM cell contraction including the validated targets Zinc finger E-box binding homeobox 1 and 2 (ZEB1 and ZEB2), and formin homology 2 domain containing 1 (FHOD1), as well as three novel targets: lysophosphatidic acid receptor 1 (LPAR1/EDG2), endothelin A receptor (ETAR), and RhoA kinase (RHOA). Consistently, transfection of TM cells with miR-200c resulted in strong inhibition of contraction in collagen populated gels as well as decreased cell traction forces exerted by individual TM cells. Finally, delivery of miR-200c to the anterior chamber of living rat eyes resulted in a significant decrease in IOP, while inhibition of miR-200c using an adenoviral vector expressing a molecular sponge led to a significant increase in IOP. These results demonstrate for the first time the ability of a miRNA to regulate trabecular contraction and modulate IOP in vivo, making miR-200c a worthy candidate for exploring ways to alter trabecular contractility with therapeutic purposes in glaucoma.

Protective Effects of Resveratrol in Experimental Retinal Detachment

Background Oxidative stress is one of the major factors that trigger photoreceptor apoptosis. To investigate whether resveratrol, a potent antioxidant and small molecule activator of the FoxO pathway, would be neuroprotective against photoreceptor cell death in a rodent model of retinal detachment. Methods Retinal detachment was created in adult Brown Norway rats by subretinal injection of sodium hyaluronate. The animals were treated daily with vehicle or resveratrol (20mg/kg) intraperitoneal injection. Photoreceptor death was assessed by counting the number of apoptotic cells with TdT-dUTP terminal nick-end labeling (TUNEL) and measurement of the outer nuclear layer (ONL) thickness 3 days after RD. Changes in expression of FoxO1a, FoxO3a, and FoxO4 were analyzed by western blot. The activity of caspase 3, caspase 8, caspase 9, spectrin and their cleavage forms were studied. Results Three days after retinal detachment, caspase 3, caspase 8 and caspase 9 were significantly activated in the detached retina. Spectrin cleavage products at 120 and 145 kDa were also detected. Both caspase and calpain activation are involved in apoptotic photoreceptor cell death in detached retinas. Treatment with resveratrol increases FoxO1a, FoxO3a, and FoxO4 protein expression in detached retinas only. Resveratrol treatment decreases activation of intrinsic and extrinsic caspase apoptotic pathways triggered by RD. The number of TUNEL-positive cells decreases from 1301±51 cells/mm2 in control groups to 430±35 cells/mm2 in treatment groups (p<0.05). Resveratrol treatment also demonstrates 59% less ONL thickness loss compared to controls. Conclusions Resveratrol treatment up-regulates the FoxO family and blocks Caspase3, 8, and 9 activation. Resveratrol has the potential to be used as a novel therapeutic agent for preventing vision loss in diseases characterized by photoreceptor detachment.