Xiu-Miao Li

LncRNA-MIAT Regulates Microvascular Dysfunction by Functioning as a Competing Endogenous RNA

RATIONALE Pathological angiogenesis is a critical component of diseases, such as ocular disorders, cancers, and atherosclerosis. It is usually caused by the abnormal activity of biological processes, such as cell proliferation, cell motility, immune, or inflammation response. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of these biological processes. However, the role of lncRNA in diabetes mellitus-induced microvascular dysfunction is largely unknown. OBJECTIVE To elucidate whether lncRNA-myocardial infarction-associated transcript (MIAT) is involved in diabetes mellitus-induced microvascular dysfunction. METHODS AND RESULTS Using quantitative polymerase chain reaction, we demonstrated increased expression of lncRNA-MIAT in diabetic retinas and endothelial cells cultured in high glucose medium. Visual electrophysiology examination, TUNEL staining, retinal trypsin digestion, vascular permeability assay, and in vitro studies revealed that MIAT knockdown obviously ameliorated diabetes mellitus-induced retinal microvascular dysfunction in vivo, and inhibited endothelial cell proliferation, migration, and tube formation in vitro. Bioinformatics analysis, luciferase assay, RNA immunoprecipitation, and in vitro studies revealed that MIAT functioned as a competing endogenous RNA, and formed a feedback loop with vascular endothelial growth factor and miR-150-5p to regulate endothelial cell function. CONCLUSIONS This study highlights the involvement of lncRNA-MIAT in pathological angiogenesis and facilitates the development of lncRNA-directed diagnostics and therapeutics against neovascular diseases.

Aberrant expression of long noncoding RNAs in early diabetic retinopathy.

PURPOSE Long noncoding RNAs (lncRNAs) are broadly classified as transcripts longer than 200 nucleotides. lncRNA-mediated biology has been implicated in a variety of cellular processes and human diseases. Diabetic retinopathy (DR) is one of the leading causes of blindness. However, little is known about the role of lncRNAs in DR The goal of this study aimed to identify lncRNAs involved in early DR and characterize their roles in DR pathogenesis. METHODS We established a mouse model of streptozotocin (STZ)-induced diabetes, and performed lncRNA expression profiling of retinas using microarray analysis. Based on the Pearson correlation analysis, an lncRNA/mRNA coexpression network was constructed. Gene ontology (GO) enrichment and KEGG analysis of lncRNAs-coexpressed mRNAs was conducted to identify the related biological modules and pathologic pathways. Real-time PCR was conducted to detect the expression pattern of lncRNA in the clinical samples and the RF/6A cell model of hyperglycemia. RESULTS Approximately 303 lncRNAs were aberrantly expressed in the retinas of early DR, including 214 downregulated lncRNAs and 89 upregulated lncRNAs. GO analysis indicated that these lncRNAs-coexpressed mRNAs were targeted to eye development process (ontology: biological process), integral to membrane (ontology: cellular component), and structural molecule activity (ontology: molecular function). Pathway analysis indicated that lncRNAs-coexpressed mRNAs were mostly enriched in axon guidance signaling pathway. In addition, MALAT1, a conserved lncRNA, was significantly upregulated in an RF/6A cell model of hyperglycemia, in the aqueous humor samples, and in fibrovascular membranes of diabetic patients. CONCLUSIONS lncRNAs are involved in the pathogenesis of DR through the modulation of multiple pathogenetic pathways. MALAT1, a conserved lncRNA, may become a potential therapeutic target for the prognosis, diagnosis, and treatment of DR.

Epigallocatechin-gallate (EGCG) regulates autophagy in human retinal pigment epithelial cells: A potential role for reducing UVB light-induced retinal damage

Autophagy is an intracellular catabolic process involved in protein and organelle degradation via the lysosomal pathway that has been linked in the pathogenesis of age-related macular degeneration (AMD). UVB irradiation-mediated degeneration of the macular retinal pigment epithelial (RPE) cells is an important hallmark of AMD, which is along with the change in RPE autophagy. Thus, pharmacological manipulation of RPE autophagy may offer an alternative therapeutic target in AMD. Here, we found that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, plays a regulatory role in UVB irradiation-induced autophagy in RPE cells. UVB irradiation results in a marked increase in the amount of LC3-II protein in a dose-dependent manner. EGCG administration leads to a significant reduction in the formation of LC3-II and autophagosomes. mTOR signaling activation is required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation is significantly impaired by rapamycin administration. Moreover, EGCG significantly alleviates the toxic effects of UVB irradiation on RPE cells in an autophagy-dependent manner. Collectively, our study reveals a novel role of EGCG in RPE autophagy. EGCG may be exploited as a potential therapeutic reagent for the treatment of pathological conditions associated with abnormal autophagy.

Long non-coding RNAs: new players in ocular neovascularization

AbstractPathological neovascularization are the most prevalent causes of moderate or severe vision loss. Long non-coding RNAs (lncRNAs) have emerged as a novel class of regulatory molecules involved in numerous biological processes and complicated diseases. However, the role of lncRNAs in ocular neovascularization is still unclear. Here, we constructed a murine model of ocular neovascularization, and determined lncRNA expression profiles using microarray analysis. We identified 326 or 51 lncRNAs that were significantly either up-regulated or down-regulated in the vaso-obliteration or neovascularization phase, respectively. Based on Pearson correlation analysis, lncRNAs/mRNAs co-expression networks were constructed. GO enrichment analysis of lncRNAs-co-expressed mRNAs indicated that the biological modules were correlated with chromosome organization, extracellular region and guanylate cyclase activator activity in the vaso-obliteration phase, and correlated with cell proliferation, extracellular region and guanylate cyclase regulator activity in the neovascularization phase. KEGG pathway analysis indicated that MAPK signaling was the most significantly enriched pathway in both phases. Importantly, Vax2os1 and Vax2os2 were not only dynamically expressed in the vaso-obliteration and neovascularization phases, but also significantly altered in the aqueous humor of patients with neovascular age-related macular degeneration (AMD), suggesting a potential role of lncRNAs in the regulation of ocular neovascularization. Taken together, this study provided novel insights into the molecular pathogenesis of ocular neovascularization. The intervention of dysregulated lncRNA could become a potential target for the prevention and treatment of ocular vascular diseases.

3H-1,2-dithiole-3-thione protects retinal pigment epithelium cells against Ultra-violet radiation via activation of Akt-mTORC1-dependent Nrf2-HO-1 signaling.

Excessive UV radiation and reactive oxygen species (ROS) cause retinal pigment epithelium (RPE) cell injuries. Nrf2 regulates transcriptional activation of many anti-oxidant genes. Here, we tested the potential role of 3H-1,2-dithiole-3-thione (D3T) against UV or ROS damages in cultured RPE cells (both primary cells and ARPE-19 line). We showed that D3T significantly inhibited UV-/H2O2-induced RPE cell death and apoptosis. UV-stimulated ROS production was dramatically inhibited by D3T pretreatment. D3T induced Nrf2 phosphorylation in cultured RPE cells, causing Nrf2 disassociation with KEAP1 and its subsequent nuclear accumulation. This led to expression of antioxidant response elements (ARE)-dependent gene heme oxygenase-1 (HO-1). Nrf2-HO-1 activation was required for D3T-mediated cytoprotective effect. Nrf2 shRNA knockdown or S40T dominant negative mutation as well as the HO-1 inhibitor Zinc protoporphyrin (ZnPP) largely inhibited D3T’s RPE cytoprotective effects against UV radiation. Yet, exogenous overexpression Nrf2 enhanced D3T’s activity in RPE cells. Further studies showed that D3T activated Akt/mTORC1 in cultured RPE cells. Akt-mTORC1 inhibitors, or Akt1 knockdown by shRNA, not only inhibited D3T-induced Nrf2-HO-1 activation, but also abolished the RPE cytoprotective effects. In vivo, D3T intravitreal injection protected from light-induced retinal dysfunctions in mice. Thus, D3T protects RPE cells from UV-induced damages via activation of Akt-mTORC1-Nrf2-HO-1 signaling axis.

miRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells

Activation of NF-E2-related factor 2 (Nrf2) signaling could protect cells from ultra violet (UV) radiation. We aim to provoke Nrf2 activation via downregulating its inhibitor Keap1 by microRNA-141 (“miR-141”). In both human retinal pigment epithelium cells (RPEs) and retinal ganglion cells (RGCs), forced-expression of miR-141 downregulated Keap1, causing Nrf2 stabilization, accumulation and nuclear translocation, which led to transcription of multiple antioxidant-responsive element (ARE) genes (HO1, NOQ1 and GCLC). Further, UV-induced reactive oxygen species (ROS) production and cell death were significantly attenuated in miR-141-expressing RPEs and RGCs. On the other hand, depletion of miR-141 via expressing its inhibitor antagomiR-141 led to Keap1 upregulation and Nrf2 degradation, which aggravated UV-induced death of RPEs and RGCs. Significantly, Nrf2 shRNA knockdown almost abolished miR-141-mediated cytoprotection against UV in RPEs. These results demonstrate that miR-141 targets Keap1 to activate Nrf2 signaling, which protects RPEs and RGCs from UV radiation.

Regulation of autophagy by high glucose in human retinal pigment epithelium.

Background: Autophagy is a self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress. Retinal pigment epithelium (RPE) works as the outer blood retina barrier and is vulnerable to energy stress-induced injury. However, the effect of high glucose treatment on autophagy is still unclear in RPE. Methods: Transmission electron microscopy was used to detect the generation of autophagosome. Small interfering RNA (siRNA) and MTT was used to determine the effect of autophagy on cell viability. Western blots and immunohistochemistry were used to detect the expression pattern of autophagic markers, including LC3 and p62. Results: High glucose treatment results in a significant increase in the generation of autophagosome and altered expression of LC3 and p62. High glucose-induced autophagy is independent of mTOR signaling, but is mainly regulated via ROS-mediated ER stress signaling. Conclusion: In the scenario of high glucose-induced oxidative stress, autophagy may be required for the removal of damaged proteins, and provide a default mechanism to prevent high glucose-induced injury in RPE.

Silencing Of Circular RNA-ZNF609 Ameliorates Vascular Endothelial Dysfunction.

Vascular dysfunction is a hallmark of ischemic, cancer, and inflammatory diseases, contributing to disease progression. Circular RNAs (circRNAs) are endogenous non-coding RNAs, which have been reported to be abnormally expressed in many human diseases. In this study, we used retinal vasculature to determine the role of circular RNA in vascular dysfunction. We revealed that cZNF609 was significantly up-regulated upon high glucose and hypoxia stress in vivo and in vitro. cZNF609 silencing decreased retinal vessel loss and suppressed pathological angiogenesis in vivo. cZNF609 silencing increased endothelial cell migration and tube formation, and protected endothelial cell against oxidative stress and hypoxia stress in vitro. By contrast, transgenic overexpression of cZNF609 showed an opposite effects. cZNF609 acted as an endogenous miR-615-5p sponge to sequester and inhibit miR-615-5p activity, which led to increased MEF2A expression. MEF2A overexpression could rescue cZNF609 silencing-mediated effects on endothelial cell migration, tube formation, and apoptosis. Moreover, dysregulated cZNF609 expression was detected in the clinical samples of the patients with diabetes, hypertension, and coronary artery disease. Intervention of cZNF609 expression is promising therapy for vascular dysfunction.

Long non-coding RNA-MIAT promotes neurovascular remodeling in the eye and brain

Although nervous and vascular systems are functionally different, they usually share similar mechanisms for function maintenance. Neurovascular dysfunction has became the pathogenesis of several vascular and nervous disorders. Here we show that long non-coding RNA-MIAT is aberrantly expressed under neurovascular dysfunction condition. MIAT is shown as a regulator of vascular dysfunction, including retinal angiogenesis, corneal angiogenesis, and vascular permeability. MIAT is also shown as a regulator of retinal neurodegeneration under diabetic condition. Mechanistically, MIAT regulates neural and vascular cell function via MIAT/miR-150-5p/VEGF network. The eye is a valuable model to study central nervous system (CNS) disorders. We show that MIAT knockdown leads to cerebral microvascular degeneration, progressive neuronal loss and neurodegeneration, and behavioral deficits in a CNS neurovascular disorder, Alzheimers disease. MIAT may represent a pharmacological target for treating neurovascular-related disorders.

Identification and Characterization of Circular RNAs as a New Class of Putative Biomarkers in Diabetes Retinopathy

Purpose To reveal the expression profile and clinical significance of circular RNAs (circRNAs) in diabetic retinopathy (DR). Methods Circular RNA microarrays were performed to identify DR-related circRNAs. Gene ontology (GO) enrichment and KEGG analysis was performed to determine the biologic modules and signaling pathway. TargetScan and miRana program was used to predict circRNA/miRNA interaction. Quantitative PCR assays were performed to detect circRNA expression pattern in clinical samples. Ki67 staining, Transwell, tube formation, and spheroid sprouting assays were performed to investigate the role and mechanism of circRNA in endothelial angiogenic function. Results A total of 529 circRNAs were aberrantly expressed in diabetic retinas. The host genes of differentially expressed circRNAs were targeted to ATP binding (biologic process); extracellular exosome (cellular component); and intracellular signal transduction (molecular function). Circ_0005015 was verified to be upregulated in the plasma, vitreous sample, and fibrovascular membranes of DR patients. Circ_0005015 facilitated retinal endothelial angiogenic function via regulating endothelial cell proliferation, migration, and tube formation. Circ_0005015 acted as miR-519d-3p sponge to inhibit miR-519d-3p activity, leading to increased MMP-2, XIAP, and STAT3 expression. Conclusions circRNAs are involved in DR pathogenesis, and thus serve as potential biomarkers of DR diagnosis.