Title: Long non‐coding <scp>RNA MALAT</scp> 1 regulates retinal neurodegeneration through <scp>CREB</scp> signaling
Abstract: Research Article10 March 2016Open Access Source DataTransparent process Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling Jin Yao Jin Yao Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Xiao-Qun Wang Xiao-Qun Wang Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Yu-Jie Li Yu-Jie Li Eye Hospital, Nanjing Medical University, Nanjing, China Search for more papers by this author Kun Shan Kun Shan Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Hong Yang Hong Yang Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Yang-Ning-Zhi Wang Yang-Ning-Zhi Wang Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Mu-Di Yao Mu-Di Yao Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Chang Liu Chang Liu Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Xiu-Miao Li Xiu-Miao Li Eye Hospital, Nanjing Medical University, Nanjing, China Search for more papers by this author Yi Shen Yi Shen Eye Hospital, Nanjing Medical University, Nanjing, China Search for more papers by this author Jing-Yu Liu Jing-Yu Liu Eye Hospital, Nanjing Medical University, Nanjing, China Search for more papers by this author Hong Cheng Hong Cheng Department of Neurology, Jiangsu Province Hospital, Nanjing, China Search for more papers by this author Jun Yuan Jun Yuan Department of Neurology, Jiangsu Chinese Medicine Hospital, Nanjing, China Search for more papers by this author Yang-Yang Zhang Yang-Yang Zhang Department of Cardiac Surgery, The first School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Qin Jiang Corresponding Author Qin Jiang Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Biao Yan Corresponding Author Biao Yan Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Jin Yao Jin Yao Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Xiao-Qun Wang Xiao-Qun Wang Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Yu-Jie Li Yu-Jie Li Eye Hospital, Nanjing Medical University, Nanjing, China Search for more papers by this author Kun Shan Kun Shan Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Hong Yang Hong Yang Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Yang-Ning-Zhi Wang Yang-Ning-Zhi Wang Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Mu-Di Yao Mu-Di Yao Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Chang Liu Chang Liu Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Xiu-Miao Li Xiu-Miao Li Eye Hospital, Nanjing Medical University, Nanjing, China Search for more papers by this author Yi Shen Yi Shen Eye Hospital, Nanjing Medical University, Nanjing, China Search for more papers by this author Jing-Yu Liu Jing-Yu Liu Eye Hospital, Nanjing Medical University, Nanjing, China Search for more papers by this author Hong Cheng Hong Cheng Department of Neurology, Jiangsu Province Hospital, Nanjing, China Search for more papers by this author Jun Yuan Jun Yuan Department of Neurology, Jiangsu Chinese Medicine Hospital, Nanjing, China Search for more papers by this author Yang-Yang Zhang Yang-Yang Zhang Department of Cardiac Surgery, The first School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Qin Jiang Corresponding Author Qin Jiang Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Biao Yan Corresponding Author Biao Yan Eye Hospital, Nanjing Medical University, Nanjing, China The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Author Information Jin Yao1,2,‡, Xiao-Qun Wang1,2,‡, Yu-Jie Li1,‡, Kun Shan1,2, Hong Yang1,2, Yang-Ning-Zhi Wang1,2, Mu-Di Yao1,2, Chang Liu1,2, Xiu-Miao Li1, Yi Shen1, Jing-Yu Liu1, Hong Cheng3, Jun Yuan4, Yang-Yang Zhang5, Qin Jiang 1,2 and Biao Yan 1,2 1Eye Hospital, Nanjing Medical University, Nanjing, China 2The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China 3Department of Neurology, Jiangsu Province Hospital, Nanjing, China 4Department of Neurology, Jiangsu Chinese Medicine Hospital, Nanjing, China 5Department of Cardiac Surgery, The first School of Clinical Medicine, Nanjing Medical University, Nanjing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 25 86677699; E-mail: [email protected] *Corresponding author. Tel: +86 25 86677677; Fax: +86 25 86677677; E-mail: [email protected] EMBO Mol Med (2016)8:346-362https://doi.org/10.15252/emmm.201505725 Correction(s) for this article Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling07 December 2022 Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling01 September 2016 This article has the following note(s): Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling07 February 2022 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non-coding RNAs (lncRNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up-regulated in the retinas, Müller cells, and primary retinal ganglion cells (RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Müller cell activation, and RGC survival in vivo and in vitro. MALAT1-CREB binding maintains CREB phosphorylation by inhibiting PP2A-mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling. Synopsis Long non-coding RNA MALAT1 knockdown decreases retinal reactive gliosis, Müller cell activation, and RGC survival via interactions with CREB signaling. MALAT1 expression is up-regulated in retinas, Müller cells, and primary retinal ganglion cells (RGCs) under stress. MALAT1 knockdown decreases reactive gliosis, Müller cell activation, and RGC survival in vivo and in vitro. MALAT1 interacts with the CREB signaling pathway to regulate Müller cell and RGC function. MALAT1 dysregulation is implicated in several human neurological diseases. Introduction The eye is known as an extension of the brain. It displays many similarities to the brain in terms of anatomy, functionality, stress response, and immunology (London et al, 2013). Several neurodegenerative changes in the brain have similar manifestations in the eye (Kerrison et al, 1994; Berisha et al, 2007; Baker et al, 2008). Thus, understanding the mechanism of neurodegeneration in eye would provide new insights into the mechanism of neurodegeneration in the brain and provide novel therapeutic targets for central nervous system (CNS) diseases. Long non-coding RNAs (lncRNAs) are non-coding transcripts > 200 nucleotides. They regulate gene expression at transcriptional, epigenetic, or translational levels, thereby altering cellular responses to various stresses (Esteller, 2011; Wapinski & Chang, 2011). Aberrant lncRNA expression is implicated in several human diseases, such as tumorigenesis, neurological diseases, and cardiovascular diseases (Qureshi et al, 2010). Nervous system development, homeostasis, stress response, and plasticity are regulated by complicated gene network (Qureshi & Mehler, 2012). Given the importance of lncRNAs in gene expression regulation, we speculated that aberrant lncRNA expression might be involved in the pathogenesis of neurological diseases. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA, is up-regulated in many solid tumors and associated with cancer metastasis and recurrence (Gutschner et al, 2013). The role of MALAT1 in nervous system has been gradually recognized. In neurons, MALAT1 regulates gene expression involved in nuclear and synapse function and synaptogenesis (Bernard et al, 2010). MALAT1 is significantly up-regulated in the cerebellum, hippocampus, and brain stem of human alcoholics (Kryger et al, 2012). We reveal that MALAT1 is involved in diabetes-induced retinal microvascular dysfunction (Liu et al, 2014). Blood vessels and nerves are two important channels in vivo and usually share common signaling regulators of differentiation, growth, and navigation (Zacchigna et al, 2007). We speculated that MALAT1 was a potential regulator of retinal neurodegeneration. In this study, we determined the expression pattern of MALAT1 and defined its role in retinal neurodegeneration. Results In vivo and in vitro expression pattern of lncRNA MALAT1 in different stress conditions Previous study reveals that MALAT1 expression levels are significantly up-regulated in the diabetic retinas of mice and rats (Liu et al, 2014). Here, we investigated the expression pattern of MALAT1 in the retinas of optic nerve transection (ONT) rat and mouse models. We found that MALAT1 expression levels were significantly up-regulated in the retinas of ONT rats and mice (Fig 1A). We also determined whether MALAT1 expression was altered in cultured retinal Müller cells and primary retinal ganglion cells (RGCs) under stress conditions. Hypoxia, high glucose, H2O2, and excitatory toxicity of glutamate treatment increased MALAT1 expression levels in both Müller cells and RGCs (Fig 1B and C). Collectively, these results show that MALAT1 expression is affected upon stress in vivo and in vitro. Figure 1. In vivo and in vitro expression pattern of lncRNA MALAT1 A. Quantitative reverse-transcription polymerase chain reactions (qRT–PCRs) were performed to detect MALAT1 levels in the mouse and rat retinas at 1, 2, and 4 weeks after optic nerve transection (ONT). Statistical differences were analyzed by Student's t-test (two-sided) from five independent experiments. Mice: *P = 0.0210 (1 week), *P = 0.0120 (2 weeks), *P = 0.0065 (4 weeks); rats: *P = 0.0125(1 week), *P = 0.0105 (2 weeks), *P = 0.0098 (4 weeks). B, C. Primary rat retinal ganglion cells (RGCs) and retinal Müller glial cells (rMC-1) were exposed to hypoxia (CoCl2, 200 μm), high glucose (HG, 30 mM), H2O2 (50 μm), and excitatory toxicity of glutamate (Glu, 3 mM) for the indicated time periods. qRT–PCRs were performed to detect MALAT1 levels. MALAT1 levels were shown as the relative change compared with the untreated group (0 h). Statistical differences were analyzed by Student's t-test (two-sided) from five independent experiments. RGCs: hypoxia: *P = 0.0246 (12 h), *P = 0.0187 (24 h), *P = 0.0114 (48 h); HG: *P = 0.0223 (12 h), *P = 0.0198 (24 h), *P = 0.0071 (48 h); H2O2: *P = 0.0201 (12 h), *P = 0.0138 (24 h), *P = 0.0076 (48 h); Glu: *P = 0.0208 (24 h), *P = 0.0134 (48 h); Müller cells: hypoxia: *P = 0.0306 (24 h), *P = 0.0125 (48 h); HG: *P = 0.0276 (12 h), *P = 0.0191 (24 h), *P = 0.0221 (48 h); H2O2: *P = 0.0183 (12 h), *P = 0.0087 (24 h), *P = 0.0065 (48 h); Glu: *P = 0.0299 (12 h), *P = 0.0194 (24 h), *P = 0.0230 (48 h). D. RNA fluorescence in situ hybridization (RNA-FISH) was performed to detect MALAT1 expression distribution. The samples were also hybridized with MALAT1 sense probe (negative control, NC) and U6 probe to verify RNA-FISH specificity. Quantification of RNA-FISH signal was performed in the GCL, INL, ONL, and RPE layer to determine MALAT1 expression difference between non-diabetes mellitus (Non-DM) and diabetes mellitus group (DM), or between ONT and non-ONT group (n = 5 independent experiments; analyzed by Mann–Whitney U-test). Rat MALAT1: *P = 0.0038 (GCL), *P = 0.0164 (INL), *P = 0.0199 (ONL), *P = 0.0066 (RPE); mice MALAT1: *P = 0.0059 (GCL), *P = 0.0177 (INL); *P = 0.0219 (ONL), *P = 0.0126 (RPE); human MALAT1: *P = 0.0032 (GCL), *P = 0.0188 (INL), *P = 0.0254 (ONL). GCL, ganglion cell layer; INL, inner nuclear layer; RPE, retinal pigment epithelium; ONL, outer nuclear layer; DM, diabetes mellitus; Wt, wild-type group; Exp, experimental group (ONT or DM group). Scale bar, 100 μm. E. RNA-FISH was performed to detect MALAT1 expression in different retinal cells. Tubulin was detected as the cytoplasmic marker to show cell boundary. Tubulin, green; Nuclei, blue; MALAT1, red. Scale bar, 50 μm. Data information: Data are represented as mean ± SEM. Download figure Download PowerPoint We then employed RNA fluorescence in situ hybridization (RNA-FISH) experiments to detect MALAT1 expression distribution in vivo. MALAT1 expression was detected in the retinal pigment epithelium (RPE) layer, outer nuclear layer, inner nuclear layer, and ganglion cell layer in rat, mouse, and human retinas (Fig 1D). Higher MALAT1 levels were detected in the diabetic retinas and ONT retinas compared with the corresponding controls (Fig 1D). Moreover, we found that MALAT1 was expressed in multiple retinal cells, including RPE, primary RGCs, Müller cells, and RF/6A cells (Liu et al, 2014). Notably, MALAT1 was located in the nuclei of these cells (Fig 1E). MALAT1 knockdown affects retinal reactive gliosis and RGC survival To reveal the role of MALAT1 in retinal neurodegeneration, an intravitreal injection of scrambled shRNA or MALAT1 shRNA adenovirus was performed in ONT mice. We previously designed three different shRNAs for MALAT1. All of them could obviously reduce MALAT1 expression levels (Appendix Fig S1). We used MALAT1 shRNA1 for the subsequent study due to its greatest gene knockdown efficiency. MALAT1 shRNA injection specially reduced MALAT1 but not other lncRNAs expression (Appendix Fig S1). MALAT1 shRNA injection also significantly reduced MALAT1 expression throughout the experiment (Fig 2A). Figure 2. MALAT1 knockdown affects retinal reactive gliosis and RGC survival in vivo Four-month-old male C57Bl/6J mice were received an intravitreous injection of scrambled (Scr) shRNA or MALAT1 shRNA viral vector for 1 or 2 weeks. The viral vector was injected once a week. MALAT1 levels were detected using qRT–PCRs [n = 5 independent experiments; analyzed by Mann–Whitney U-test; *P = 0.0082 (1 week), *P = 0.0103 (2 weeks)]. Four-month-old male C57Bl/6J mice received an intravitreous injection of scrambled (Scr) shRNA or MALAT1 (M) shRNA, or left untreated for 1 week, and then, ONT models were established. All shRNA vectors were injected once a week. The untreated group was taken as the control group (Wt). Each experimental group had six animals. Two weeks after ONT, retinal slices were immunolabeled for marker proteins, including vimentin, GFAP, NeuN, and TUBB3 [n = 3 independent experiments; analyzed by Mann–Whitney U-test; vimentin: *P = 0.0032 (ONT), *P = 0.0043 (ONT+Scr), *P = 0.0142 (ONT+M), #P = 0.0168; GFAP: *P = 0.0026 (ONT), *P = 0.0037 (ONT+Scr), *P = 0.0212 (ONT+M), #P = 0.0135; NeuN: *P = 0.0278 (ONT), *P = 0.0205 (ONT+Scr), *P = 0.0149 (ONT+M), #P = 0.0298; TUBB3: *P = 0.0239 (ONT), *P = 0.0198 (ONT+Scr), *P = 0.0101 (ONT+M), #P = 0.0324]. Scale bar, 100 μm. GCL, ganglion cell layer; INL, inner nuclear layer; RGC, retinal ganglion cell; ONL, outer nuclear layer; ONT, optic nerve transection; Scr, scrambled shRNA; M, MALAT1 shRNA. Data information: * indicates significant difference compared with Wt group. # indicates significant difference between the marked groups. Data are represented as mean ± SEM. Download figure Download PowerPoint RGC degeneration and reactive gliosis are two important features of retinal neurodegeneration (Barreto et al, 2011). We performed protein immunolabeling experiments to determine the effect of MALAT1 knockdown on retinal neurodegeneration. ONT significantly increased reactive gliosis as shown by increased vimentin and GFAP staining, whereas MALAT1 knockdown reversed this trend (Fig 2B). ONT caused a marked decrease in RGC number as shown by decreased NeuN and TUBB3 (markers of RGCs) staining. MALAT1 knockdown further decreased the number of NeuN- or TUBB3-positive RGCs (Fig 2B). Retinal slices were also immunolabeled with the marker proteins, including calretinin (ganglion cells and amacrine cells), calbindin (ganglion cells, amacrine, and horizontal cells), rhodopsin (Rod and cone photoreceptor), and protein kinase Cα (PKCα; bipolar cells). Compared with ONT retinas, MALAT1 knockdown further decreased the number of calretinin-labeled cells in the GCL, but did not further affect the number of calretinin-labeled cells in the INL (Appendix Fig S2). MALAT1 knockdown further decreased the number of calbindin-labeled cells in the GCL, but did not further affect the number of calbindin-labeled horizontal and amacrine cells (Appendix Fig S2). Rhodopsin and PKCα immunolabeling revealed that MALAT1 knockdown had no effect on photoreceptors and bipolar cells (Appendix Fig S2). Moreover, Western blots revealed that MALAT1 knockdown reduced vimentin, GFAP, NeuN, and TUBB3 expression levels (Appendix Fig S3). Collectively, these results indicate that MALAT1 knockdown affects retinal reactive gliosis and RGC survival. Retinal neurodegeneration is also implicated in the pathogenesis of diabetic retinopathy. We observed a similar scenario in diabetic rat retinas as shown in ONT retinas. MALAT1 knockdown affected reactive gliosis and RGC survival, but had a minor effect on the number of horizontal cells, amacrine cells, photoreceptors, and bipolar cells (Appendix Figs S4 and S5). Western blots also showed that MALAT1 knockdown reduced vimentin, GFAP, NeuN, and TUBB3 expression levels in the retinas of diabetic rats (Appendix Fig S6). MALAT1 knockdown affects Müller glia activation in vivo Müller glia is the major glial component of the retina. Its activation protects the retina from a wide variety of pathological stimuli such as trauma, ischemia, and degeneration via releasing neurotrophic factors (Barres, 2008; Mamczur et al, 2015). We found that MALAT1 knockdown reduced the expression of neurotrophic factors, including GDNF, NT-4, BDNF, and NGF in the ONT retinas (Fig 3A), and reduced the expression of neurotrophic factors, including GDNF, CNTF, and BDNF in the diabetic retinas (Appendix Fig S7). Figure 3. MALAT1 knockdown affects Müller glia activation in vivo A. Four-month-old male C57Bl/6J mice were received an intravitreous injection of scrambled (Scr) shRNA or MALAT1 shRNA, or left untreated for 1 week, and then, ONT models were established. All shRNA vectors were injected once a week. Each experimental group had six animals. Two weeks after ONT, the effect of MALAT1 knockdown on neurotrophic factor expression was determined. Data are shown as the relative change compared with ONT group [n = 3 independent experiments; analyzed by Mann–Whitney U-test; *P = 0.0143 (GDNF), *P = 0.0207 (NT-4), *P = 0.0109 (BDNF), *P = 0.0383 (NGF)]. GNDF, glial cell line-derived neurotrophic factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; CNTF, ciliary neurotrophic factor; BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor. B. Four-month-old male C57Bl/6J mice received an intravitreous injection of scrambled (Scr) shRNA or MALAT1 shRNA, or left untreated for 1 week, and then, ONT models were established. 5-bromo-2′-deoxyuridine (BrdU, 50 mg/kg) was injected at day 7 after ONT. Meanwhile, BDNF (1 μg/μl) was intraocularly injected into ONT, ONT+Scr shRNA, or ONT+MALAT1 shRNA retinas. These mice were killed at day 14 after ONT. BrdU-labeled cells in the INL were double labeled (arrowheads) with glutamine synthetase (GS). Each experimental group had five animals (n = 3 independent experiments; analyzed by Mann–Whitney U-test; P = 0.015). Scale bar, 100 μm. C, D. Four-month-old mice received an intravitreous injection of scrambled (Scr) shRNA or MALAT1 shRNA, or left untreated for 1 week, and then, ONT models were established for additional 2 weeks. Each experimental group had five animals. Immunohistochemical analysis was performed to detect nestin (C; n = 3 independent experiments; analyzed by Mann–Whitney U-test; P = 0.021) or vimentin expression (D; n = 3 independent experiments; analyzed by Mann–Whitney U-test; P = 0.009). Scale bar, 50 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; DAPI, 4′,6-diamidino-2-phenylindole. Data information: * indicates significant difference compared with ONT group. Data are represented as mean ± SEM. Download figure Download PowerPoint Müller glia has the capability to dedifferentiate and reenter the proliferation cycle under the stress condition (Pekny & Nilsson, 2005; Bringmann et al, 2009). We also examined whether MALAT1 knockdown affected the regenerative ability of Müller cells. We labeled proliferating retinal cells using BrdU reagent. Intravitreous injection of MALAT1 shRNA significantly reduced the number of BrdU-labeled cells in the ONT and diabetic retinas. Moreover, BrdU-labeled staining cells were overlapped with glutamine synthetase (GS) staining, suggesting that MALAT1 knockdown affects Müller glia proliferation (Fig 3B and Appendix Fig S7). The reactivation of stem and progenitor properties of glial cells also promoted us to determine the effect of MALAT1 knockdown on the expression of progenitor markers, such as nestin and vimentin. Immunofluorescent staining and Western blots showed that MALAT1 knockdown significantly reduced nestin and vimentin levels in the ONT retinas (Fig 3C and D, and Appendix Fig S8). We also observed that MALAT1 knockdown decreased nestin and vimentin expression in the diabetic retinas (Appendix Figs S7 and S8). MALAT1 knockdown regulates Müller cell function in vitro The above-mentioned results showed that MALAT1 mainly acted on Müller cells and RGCs. We then studied the mechanistic aspects and functional significance of MALAT1 alteration in vitro. We designed two different MALAT1 siRNAs and revealed that MALAT1 siRNA transfection significantly reduced MALAT1 levels in Müller cells (Appendix Fig S9). We selected one MALAT1 siRNA with greater silencing efficiency for the subsequent experiments. Müller cells were treated with H2O2 to mimic oxidative stress. MTT assays showed that H2O2 treatment significantly decreased Müller cell viability. MALAT1 knockdown further decreased Müller cell viability (Fig 4A). We then determined whether MALAT1 regulates the development of H2O2-induced apoptosis using Hoechst 33342, Calcein-AM/PI, and JC-1 staining. The combination of MALAT1 knockdown and H2O2 treatment resulted in higher apoptotic percentage than H2O2 treatment alone, as shown by increased apoptotic nuclei (condensed or fragmented) (Fig 4B), more PI-positive cells (dying or dead cells) (Fig 4C), and decreased mitochondrial depolarization (Fig 4D). MALAT1 knockdown accelerated the shift of fluorescence emission from green to red (Fig 4E). Ki67 staining showed that MALAT1 knockdown significantly reduced Müller cell proliferation (Fig 4F). MALAT1 knockdown also reduced Müller cell viability, accelerated cell apoptosis, and suppressed cell proliferation in response to hypoxia stress and excitatory toxicity of glutamate (Appendix Figs S10 and S11). Müller cells are usually activated against pathogenic stimuli. GFAP up-regulation is the most sensitive response upon stress (Bringmann et al, 2009). We found that MALAT1 knockdown significantly inhibited GFAP up-regulation upon high glucose and oxidative stress (Appendix Fig S12), suggesting a critical role of MALAT1 in Müller glia activation in vitro. Collectively, these results suggest that MALAT1 regulates Müller cell function in vitro. Figure 4. MALAT1 knockdown affects Müller cell function in vitroMüller cells were transfected with scrambled siRNA (Scr), MALAT1 siRNA, or left untreated (Wt) and then exposed to H2O2 (50 μm) for 48 h. The group without any treatment was taken as the control (Ctrl) group. A. Cell viability was detected using MTT method [n = 5 independent experiments; analyzed by two-sided Student's t-test; *P = 0.0245 (Wt), *P = 0.0221 (Scr), *P = 0.0158 (M), #P = 0.0402]. B. Apoptotic cells were analyzed using Hoechst staining and quantitated [n = 5 independent experiments; analyzed by two-sided Student's t-test; *P = 0.0105 (Wt), *P = 0.0173 (Scr), *P = 0.0086 (M), #P = 0.0365]. Scale bar, 20 μm. C. Dead or dying cells were analyzed using calcein-AM/PI staining from three independent experiments. Green, live cells; red, dead, or dying cell. Scale bar, 50 μm. D, E. Müller cells were incubated with JC-1 probe at 37°C for 30 min, centrifuged, washed, transferred to a 96-well plate (100,000 cells per well), assayed using a fluorescence plate reader [n = 5 independent experiments; analyzed by two-sided Student's t-test; *P = 0.0239 (Wt), *P = 0.0146 (Scr), *P = 0.0132 (M), #P = 0.0373], and observed using a fluorescence microscope. Red fluorescence, JC-1 aggregates; green fluorescence, JC-1 monomers (E). Scale bar, 50 μm. F. Ki67 staining and quantification analysis was performed to detect Müller cell proliferation [n = 5 independent experiments; analyzed by two-sided Student's t-test; *P = 0.0319 (Wt), *P = 0.0252 (Scr), *P = 0.0107 (M), #P = 0.0205]. Scale bar, 20 μm. DAPI, blue; Ki67, red. Data information: * indicates significant difference compared with Ctrl group. # indicates significant difference between the marked groups. Data are represented as mean ± SEM. Download figure Download PowerPoint Direct and indirect effect of MALAT1 on RGC protection in vitro Since FISH experiment showed that MALAT1 was also expressed in RGCs, we thus investigated whether MALAT1 alteration affected RGC function. MALAT1 shRNA transfection significantly reduced MALAT1 levels in the primary RGCs (Appendix Fig S13). H2O2 treatment significantly reduced the viability of RGCs. MALAT1 knockdown further reduced RGC viability (Fig 5A). The combination of MALAT1 knockdown and H2O2 treatment resulted in higher apoptotic percentage than H2O2 treatment alone (Fig 5B–E). Ki67 staining showed that MALAT1 knockdown significantly decreased RGC proliferation (Fig 5F). MALAT1 also regulated RGC viability, apoptosis development, and cell proliferation in response to hypoxia stress (Appendix Fig S14) and excitatory toxicity of glutamate stress (Appendix Fig S15). These results suggest that MALAT1 is an important regulator of RGC function in vitro. Figure 5. MALAT1 knockdown affects RGC function upon oxidative stress in vitroPrimary RGCs were transfected with scrambled (Scr) shRNA, MALAT1 shRNA, or left untreated and then exposed to H2O2 (50 μm) for 48 h. The group without any treatment was taken as the control (Ctrl) group. A. Cell viability was determined using MTT method [n = 5 independent experiments; analyzed by two-sided Student's t-test; *P = 0.0285 (Wt), *P = 0.0246 (Scr), *P = 0.0132 (M), #P = 0.0311]. B. Apoptotic cells were analyzed using Hoechst staining and quantitated [n = 5 independent experiments; analyzed by two-sided Student's t-test; *P = 0.0211 (Wt), *P = 0.0283 (Scr), *P = 0.0076 (M), #P = 0.0289]. Scale bar, 50 μm. C. Dead or dying cells were analyzed using calcein-AM/PI staining from three independent experi