Regulatory role of miR-146a in corneal epithelial wound healing via its inflammatory targets in human diabetic cornea

PurposeMiR-146a upregulated in limbus vs. central cornea and in diabetic vs. non-diabetic limbus has emerged as an important immune and inflammatory signaling mediator in corneal epithelial wound healing. Our aim was to investigate the potential inflammation-related miR-146a target genes and their roles in normal and impaired diabetic corneal epithelial wound healing.MethodsOur previous data from RNA-seq combined with quantitative proteomics of limbal epithelial cells (LECs) transfected with miR-146a mimic vs. mimic control were analyzed. Western blot and immunostaining were used to confirm the expression of miR-146a inflammatory target proteins in LECs and organ-cultured corneas. Luminex assay was performed on conditioned media at 6- and 20-h post-wounding in miR-146a mimic/inhibitor transfected normal and diabetic cultured LECs.ResultsOverexpression of miR-146a decreased the expression of pro-inflammatory TRAF6 and IRAK1 and downstream target NF-κB after challenge with lipopolysaccharide (LPS) or wounding. Additionally, miR-146a overexpression suppressed the production of downstream inflammatory mediators including secreted cytokines IL-1α, IL-1β, IL-6 and IL-8, and chemokines CXCL1, CXCL2 and CXCL5. These cytokines and chemokines were upregulated in normal but not in diabetic LEC during wounding. Furthermore, we achieved normalized levels of altered secreted cytokines and chemokines in diabetic wounded LEC via specific inhibition of miR-146a.ConclusionOur study documented significant impact of miR-146a on the expression of inflammatory mediators at the mRNA and protein levels during acute inflammatory responses and wound healing, providing insights into the regulatory role of miR-146a in corneal epithelial homeostasis in normal and diabetic conditions.     

葡萄膜恶性黑色素瘤转移相关的非编码RNA表达谱及竞争性内源RNA调控网络分析

目的:利用生物信息学方法分析与葡萄膜恶性黑色素瘤转移相关的非编码RNA,以及它们作为竞争性内源RNA的作用机制.方法:从癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库下载80例葡萄膜恶性黑色素瘤患者的RNA测序数据和临床资料,采用edgeR算法分析转移与非转移患者组织中差异表达(dif...     

不同病理类型的葡萄膜黑色素瘤中微小RNA差异表达谱分析

背景 目前研究证实微小RNA(miRNA)参与大多数人类肿瘤疾病的发生和发展,其作用类似于抑癌基因或癌基因.葡萄膜黑色素瘤(UM)是成人常见的眼部恶性肿瘤,其发生和转移机制仍未完全阐明.探讨UM组织中miRNA的差异表达情况有望为UM的靶向治疗提供依据. 目的 筛选不同病理类型的UM组织中特异性miRNA表达谱. 方法 收集于2013年3月至2015年10月在北京同仁医院手术局部切除并经常规组织病理学和免疫组织化学检测证实为梭型细胞型UM的标本4例和上皮细胞型UM标本4例,采用miRNA芯片分别检测2种UM组织中miRNA的表达,收集同期死于非肿瘤疾病的8个供体眼的正常葡萄膜组织作为对照,利用组间差异倍数筛选出差异≥2倍差异表达的miRNA;用在线软件预测差异表达miRNA的靶基因,采用生物信息学方法分析靶基因参与的信号功能通路.采用实时定量PCR法验证芯片检测结果.结果 收集的梭形细胞型和上皮细胞型UM标本经组织病理学检查均得到确诊,免疫组织化学检测梭形细胞型及上皮细胞型UM组织中HMB45、黑色素-A和S-100均呈阳性反应.与正常葡萄膜组织比较,在梭形细胞型UM组织中差异表达的miRNA有109个,其中29个上调,80个下调,上调的miRNA包括miR-146a-5p、miR-25-3p和miR-29b-1-5p,下调的miRNA包括miR-126-5 p、miR-183-5p和miR-96-5p;上皮细胞型UM中差异表达的miRNA有50个,其中23个上调,27个下调,上调的miRNA包括miR-155-5p、miR-210和miR-378 a-5p;下调的miRNA包括miR-199a-5p、miR-143-3p和miR-143-5p.在梭形细胞型和上皮细胞型UM组织中共同上调的miRNA为miR-132-3p、miR-21-5p、miR-34a-5p和miR-34b-5p,共同下调的miRNA为miR-125b-2-3p、miR-126-3p、miR-199a-3p和miR-214-3p.梭形细胞型和上皮细胞型UM组织中差异表达的miRNA所预测的靶基因分别参与癌症通路、丝裂原活化蛋白激酶(MAPK)信号通路、Wnt信号通路、细胞间黏附、胞吞作用、前列腺癌通路、结直肠癌通路和细胞黏附通路.结论 与正常葡萄膜组织相比,梭形细胞型UM和上皮细胞型UM组织中存在多种miRNA的差异表达,梭形细胞型UM和上皮细胞型UM组织之间也存在明显的miRNA差异表达,这些差异表达的miRNA可通过不同的信号转导通路参与调控UM的生物学行为.     

Epigenetic Landscape Analysis of the Long Non-Coding RNA and Messenger RNA in a Mouse Model of Corneal Alkali Burns

PurposeCorneal alkali burns (CABs) are a common clinical ocular disease, presenting a poor prognosis. Although some long noncoding RNAs (lncRNAs) reportedly play a key role in epigenetic regulation associated with CABs, studies regarding the lncRNA signature in CABs remain rare and elusive.MethodsA CAB model was established in C57BL/6J mice and profiling of lncRNA expressions was performed by RNA-Seq. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related pathological pathways and candidate genes. RT-qPCR was used to verify the expression pattern of lncRNAs and related mRNAs, both in vitro and in vivo. Data were statistically analyzed by GraphPad Prism version 6.0.ResultsIn all, 4436 aberrantly expressed lncRNAs were identified in CAB mice when compared with control mice. In the top 13 aberrantly expressed lncRNAs, {"type":"entrez-nucleotide","attrs":{"text":"Bc037156","term_id":"22477581"}}Bc037156 and 4930511E03Rik were confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that mitogen-activated protein kinase (MAPK) signaling pathway was most enriched. Following 4930511E03Rik siRNA treated, Srgn, IL-1β and Cxcr2 were significant upregulated in corneal epithelial cells, corneal keratocytes, and bone marrow dendritic cells, with NaOH treatment. Moreover, after {"type":"entrez-nucleotide","attrs":{"text":"Bc037156","term_id":"22477581"}}Bc037156 siRNA treated, expression levels of IL-1β and Srgn were significantly downregulated in the three cell lines.ConclusionsOur study suggests that {"type":"entrez-nucleotide","attrs":{"text":"Bc037156","term_id":"22477581"}}Bc037156 and 4930511E03Rik may be involved in inflammation, immune response, and neovascularization by regulating Srgn, IL-1β, and Cxcr2 expression after CAB. These candidate lncRNAs and mRNAs may be the potential targets for the treatment strategy of the alkali injured cornea.     

Analysis of MicroRNA Expression in Tears of Patients with Herpes Epithelial Keratitis: A Preliminary Study

PurposeHerpes epithelial keratitis (HEK) is the most common form of herpes simplex virus (HSV) eye involvement, and understanding the molecular mechanisms underlying HEK is important. We investigated the expression of microRNAs (miRNAs) in the tears of patients with HEK.MethodsTear samples from eight patients with HEK and seven age-matched controls were evaluated. Clinical ophthalmologic evaluation was performed, and an anterior segment photograph was obtained after fluorescence staining. Dendritic or geographic ulcer areas were measured using ImageJ software. The expression of 43 different miRNAs in tears was measured using real-time polymerase chain reaction and compared between patients with HEK and controls. Differences in miRNA expression between the dendritic and geographic ulcer groups and correlations involving miRNA expression and ulcer area were evaluated.ResultsOf the 43 miRNAs, 23 were upregulated in patients with HEK compared to normal controls. MiR-15b-5p, miR-16-5p, miR-20b-5p, miR-21-5p, miR-23b-3p, miR-25-3p, miR-29a-3p, miR-30a-3p, miR-30d-5p, miR-92a-3p, miR-124-3p, miR-127-3p, miR-132-3p, miR-142-3p, miR-145-5p, miR-146a-5p, miR-146b-5p, miR-155-5p, miR-182-5p, miR-183-5p, miR-221-3p, miR-223-3p, and miR-338-5p were significantly upregulated in patients with HEK. MiR-29a-3p exhibited significant differences between the dendritic and geographic ulcer groups. All 23 miRNAs with significant differences between patients with HEK and the control group were not significantly correlated with ulcer area.ConclusionsTwenty-three miRNAs were significantly upregulated in the tears of patients with HEK, and the expression of miRNAs may play important roles in herpes infection in relation to host immunity.     

Plasma miR-26a-5p is a biomarker for retinal neurodegeneration of early diabetic retinopathy

PurposeRetinal neurodegeneration is an early pathological change in diabetic retinopathy (DR). Early-stage retinal neurodegeneration is usually asymptomatic. This study aims to identify circulating microRNAs (miRNAs) as sensitive biomarkers for early retinal neurodegeneration.MethodsWe profiled the plasma miRNA expression in three mild nonproliferative diabetic retinopathy (NPDR) cases and three matched non-DR patients using RNA sequencing. The differential miRNAs were validated with qRT-PCR. The retinal nerve fibre layer (RNFL) thickness of the eyes was measured using spectral-domain Optical coherence tomography (SD-OCT). The association between differential miRNAs and RNFL thickness was analysed using the Pearson correlation analysis. Bioinformatics tools were used to predict potential targets of miRNA associated with RNFL thickness and investigate the functions of the potential target genes.ResultsRNA sequencing identified 69 differential miRNAs and eight of them were reported to be associated with DR. The qRT-PCR for these eight miRNAs validated the down-regulation of circulating miR-26a-5p and miR-126-5p in a larger validating cohort. A positive correlation between plasma miR-26a-5p level and the RNFL thickness of the superior quadrant of both eyes was identified in another cohort, including 33 mild NPDR cases, 33 matched non-DR patients and 20 healthy controls. Furthermore, 367 candidate targets of miR-26a-5p were predicted. The functional studies revealed that these target genes are profoundly involved in various cellular functions and signalling pathways.ConclusionsCirculating miR-26a-5p is a potential biomarker for early-stage retinal neurodegeneration and it may be involved in the development of DR via profoundly influencing the functions of retinal cells.Subject terms: Retina, Prognostic markers     

miR-34a-5p通过调节Nrf2-Keap1信号通路介导年龄相关性白内障氧化应激的实验研究

目的　探讨miR-34a-5p通过调节Nrf2-Keap1信号通路介导年龄相关性白内障的氧化应激。方法　检测年龄相关性白内障患者和正常透明晶状体前囊组织及人晶状体上皮细胞氧化应激模型中miR-34a-5p和预测靶基因Nrf2表达及人晶状体上皮细胞内活性氧（reactive oxygen species，ROS）活性。将miR-34a-5p模拟物、模拟物对照、miR-34a-5p抑制剂和抑制剂对照分别转染人晶状体上皮细胞，然后用400 μmol·L^-1 H₂O₂作用8 h后检测miR-34a-5p、Nrf2 mRNA和Nrf2蛋白表达，并检测人晶状体上皮细胞增殖活性。结果　正常晶状体前囊组织中miR-34a-5p的相对表达量为1.03±0.29，Nrf2 mRNA的相对表达量为1.31±0.39；年龄相关性白内障组织中miR-34a-5p的相对表达量为2.69±0.99，Nrf2 mRNA的相对表达量为0.64±0.25。与正常组织相比，年龄相关性白内障晶状体组织中miR-34a-5p表达显著升高，Nrf2表达显著降低。Nrf2蛋白在年龄相关性白内障晶状体组织中表达也显著降低（均为P<0.001）。在人晶状体上皮细胞氧化应激模型中，miR-34a-5p表达显著升高，靶基因Nrf2表达显著降低，内源性ROS水平显著升高（均为P<0.001）。miR-34a-5p模拟物转染人晶状体上皮细胞后，miR-34a-5p表达显著升高，Nrf2表达显著降低，内源性ROS水平显著升高，细胞增殖活性显著降低（均为P<0.001），然而，miR-34a-5p抑制剂转染人晶状体上皮细胞后，miR-34a-5p表达显著降低，Nrf2表达显著升高，内源性ROS水平显著降低，细胞增殖活性显著升高（均为P<0.001）。双荧光素酶报告分析证实，Nrf2是miR-34a-5p的直接靶点。结论　MiR-34a-5p通过调节Nrf2-Keap1信号通路增加晶状体上皮细胞氧化应激，抑制晶状体上皮细胞的增殖，从而参与年龄相关性白内障的发生发展过程。     

microRNA-182抑制人角膜上皮细胞增殖和迁移

目的：通过体内动物实验与体外细胞实验来阐明microRNA-182（miR-182）在角膜上皮损伤修复中的功能。方法：实验研究。体内实验选取5 只C57BL/6J野生型小鼠,通过机械刮伤法构建小鼠角膜上皮损伤修复模型作为实验组,对侧眼作为对照组,通过实时定量RT-PCR法检测miR-182 在损伤修复过程（ 损伤后48 h）的表达。体外实验采用脂质体介导的方法将miR-182和随机序列寡核苷酸链转染入人角膜上皮细胞（HCECs）,通过细胞增殖实验（MTS法）和细胞克隆形成实验检测细胞增殖和生长能力,流式细胞术检测细胞周期,细胞划痕实验检测细胞迁移能力。MiR-182 表达量及细胞实验各参数2 组间比较采用独立样本t检验。结果：体内实验中,与对照组相比,实验组中5 个样本量的miR-182 在角膜上皮损伤修复过程中表达水平显著下调（t=147.6、79.2、136.8、41.3、89.8,均P<0.001）。体外实验中,miR-182组相对细胞数目为（81±5）%,与阴性对照组（100%）相比显著减少（t=6.6,P=0.003）,同时miR-182组细胞克隆数明显少于阴性对照组。细胞周期检测结果显示实验组处于G1期细胞数量比例为（49±7）%,明显高于阴性对照组的（35±4）%（t=-3.0,P=0.041）。此外,细胞迁移实验结果显示miR-182组HCECs细胞迁移的距离为（99±12）μm,而阴性对照组的迁移距离为（213±14）μm（t=10.8,P=0.001）,迁移速度明显变慢。结论：miR-182通过抑制角膜上皮细胞的增殖与迁移,从而对角膜上皮损伤修复起到负调控的作用。     

Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway

PurposePrevious work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H₂) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism.MethodsMNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H₂. The effect of H₂ was examined using morphological and functional assays.ResultsIt was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na⁺/K⁺-ATPase, whereas H₂ improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H₂ could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway.ConclusionsThis study suggests that topical application of H₂ could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.     

Pigment epithelium-derived factor (PEDF) plays anti-inflammatory roles in the pathogenesis of dry eye disease

PurposeTo investigate the expression of pigment epithelium-derived factor (PEDF) in ocular surface in dry eye disease (DED) and its anti-inflammatory roles and mechanisms, clinically and by experiments in vivo and in vitro.MethodsA cross-sectional study was conducted to detect the expression of PEDF in tears of dry eye patients by enzyme-linked immunosorbent assay (ELISA). Using dry eye mouse model and human corneal epithelial cells (hCECs) stimulated by hyperosmolarity or inflammatory cytokines, expression of PEDF in corneal epithelial cells, stroma and conjunctiva was quantified by real-time polymerase chain reaction, ELISA and Western blot. Next, either dry eye mice or hyperosmotic hCECs were treated with recombinant PEDF or neutralizing antibodies, and the expressions of inflammatory cytokines and immune cells were detected. Finally, Western blot was performed on MAPK and NF-κB to investigate the signaling pathways by which PEDF played its roles.ResultsConcentrations of PEDF were increased in tears of dry eye patients. Increased PEDF was observed in corneal epithelial cells (CECs) rather than corneal stroma or conjunctiva in dry eye mice. Furthermore, hCECs exposed to hyperosmolarity showed upregulation of PEDF. In vivo and in vitro studies showed that PEDF suppressed the expression of inflammatory cytokines including IL-1β, IL-6, TNF-α and IL-17A, as well as the percentage of Th17 cells in DED. Further investigation showed that PEDF inhibited the phosphorylation of MAPK p38 and JNK in hyperosmotic hCECs.ConclusionsCECs derived PEDF is increased in DED. PEDF plays anti-inflammatory and immunoregulatory roles in the pathogenesis of DED.     

Labial Mucosa Stem Cells: Isolation,Characterization, and Their Potential for Corneal Epithelial Reconstruction

PurposeThe purpose of this study was to characterize labial mucosa stem cells (LMSCs) and to investigate their potential for corneal epithelial reconstruction in a rabbit model of total limbal stem cell deficiency (LSCD).MethodsRabbit LMSCs (rLMSCs) and human (hLMSCs) LMSCs were derived from labial mucosa and characterized in terms of their proliferation activity by the evaluation of proliferation index (PI) and colony forming efficiency (CFE), cell senescence, and differentiation abilities. The expression of various limbus-specific, stem cell-specific, and epithelial markers was assessed via immunocytochemistry. Flow cytometry was used to evaluate mesenchymal and hematopoietic cell surface markers expression. Chromosomal stability of the derived cells was examined using the conventional GTG-banding technique. To assess the impact of LMSCs on corneal epithelial reconstruction, rLMSCs were seeded onto a decellularized human amniotic membrane (dHAM), thereafter their regeneration potential was examined in the rabbit model of total LSCD.ResultsBoth rLMSCs and hLMSCs showed high proliferation and differentiation abilities, entered senescence at later passages, and expressed different stem cell-specific (ABCB5, ALDH3A1, ABCG2, and p63α), mesenchymal (vimentin), and epithelial (CK3/12, CK15) markers. Cell surface antigen expression was similar to other described mesenchymal stem cells. No clonal structural chromosome abnormalities (CSCAs) and the low percentage of non-clonal structural chromosome abnormalities (NSCAs) were observed. Transplantation of rLMSCs promoted corneal epithelial reconstruction and enhanced corneal transparency.ConclusionsLMSCs have significant proliferation and differentiation abilities, display no detrimental chromosome aberrations, and demonstrate considerable potential for corneal repair.     

Hyaluronan Facilitates Corneal Epithelial Wound Healing in Diabetic Rats

We investigated the effect of hyaluronan on corneal epithelial wound healing in rats affected by diabetes. Furthermore, because hyaluronan is thought to affect corneal epithelial wound healing through the mechanism of binding of hyaluronan to provisional fibronectin in the wounded area, we compared the localization of fibronectin immunohistochemically during corneal epithelial wound healing in diabetic and non-diabetic rats. Streptozotocin was used to induce diabetes in half the rats. Two weeks after treatment, the whole corneal epithelium of diabetic and untreated rats was debrided. The rats were divided into groups (seven or eight rats per group), and hyaluronan eye drops at concentrations of 0.03, 0.1, or 0.3%, chondroitin sulfate (3%), or phosphate buffered saline (PBS) was given in eye drops 6 times a day for 4 days, starting immediately after debridement. The area of the corneal epithelial wound was measured immediately after debridement and at 12, 18, 24, 30, 48, 72, and 96 hours afterwards. Although the healing process was similar in non-diabetic and diabetic rats, the healing rate in diabetic rats was slower than that in normal controls. In both diabetic and non-diabetic rats, hyaluronan increased the healing rate in a dose-dependent manner; the difference was significant compared with the PBS-treated group, at hyaluronan doses of 0.1% and 0.3%. However, chondroitin sulfate did not affect corneal epithelial wound closure, regardless of whether the rats were diabetic or not; the healing rates were identical to those of PBS-treated diabetic and non-diabetic controls. In both diabetic and non-diabetic corneas, fibronectin was localized in the corneal subepithelial region, and in streaks between collagen fibers of the stroma. One day after debridement, a layer of fibronectin immunofluorescence was clearly visible on the surface of the denuded stroma. As healing progressed staining of fibronectin diminished at the interface between the new epithelium and the stroma. These changes in localization of fibronectin during corneal epithelial wound healing were similar in both diabetic and non-diabetic rats. Our results demonstrate that hyaluronan facilitates corneal epithelial wound healing in diabetic rats, and suggest that one possible mechanism of its stimulatory effect lies in its binding to a provisional fibronectin matrix, in both diabetic and non-diabetic rats.     

lncRNA KCNQ1OT1通过miR-19a-3p/TSHZ3影响高糖诱导的人视网膜上皮细胞增殖与凋亡

目的：观察长链非编码RNA(lncRNA)KCNQ1OT1通过miR-19a-3p/TSHZ3影响高糖(HG)诱导的人视网膜上皮细胞(ARPE-19)增殖、凋亡与氧化应激情况。

方法：运用细胞计数试剂盒8(CCK-8)检测5、15、45、135mmol/L HG刺激的ARPE-19的细胞存活率。将ARPE-19细胞分为NC组、45mmol/L HG组、si-NC+45mmol/L HG组、si-lncRNA KCNQ1OT1+45mmol/L HG组、miR-NC+45mmol/L HG组、miR-19a-3p mimics+45mmol/L HG组、si-con+45mmol/L HG组、si-TSHZ3+45mmol/LHG组、pcDNA+si-lncRNA KCNQ1OT1+45mmol/L HG组、pcDNA-TSHZ3+si-lncRNA KCNQ1OT1+45mmol/L HG组。运用CCK-8检测细胞存活率,qRT-PCR检测lncRNA KCNQ1OT1、miR-19a-3p和TSHZ3 mRNA表达,Western Blot检测TSHZ3、活化-含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-caspase-3)、B细胞淋巴瘤/白血病-2(Bcl-2)相关X蛋白(Bax)蛋白质的表达,酶联免疫吸附测定法(ELISA)检测氧化应激指标活性氧(ROS)、丙二醛(MDA)水平。双荧光素酶活性检测lncRNA KCNQ1OT1和miR-19a-3p、miR-19a-3p和TSHZ3之间的靶向结合。

结果：15、45、135mmol/L HG抑制ARPE-19细胞的存活率,后续选择细胞存活率约50%的HG浓度45mmol/L。45mmol/L HG提高ARPE-19细胞中lncRNA KCNQ1OT1、TSHZ3 mRNA、TSHZ3蛋白表达水平、凋亡率、Cleaved-caspase-3、Bax蛋白表达、ROS和MDA水平,降低miR-19a-3p表达水平、细胞存活率(P<0.05)。lncRNA KCNQ1OT1、TSHZ3低表达或miR-19a-3p高表达提高HG诱导的ARPE-19细胞的存活率,降低凋亡率、Cleaved-caspase-3、Bax蛋白表达、ROS和MDA水平(P<0.05)。lncRNA KCNQ1OT1靶向miR-19a-3p,miR-19a-3p靶向TSHZ3,lncRNA KCNQ1OT13通过miR-19a-3p调控TSHZ3的表达。lncRNA KCNQ1OT1低表达对HG诱导的ARPE-19细胞存活率、凋亡以及氧化应激的影响被TSHZ3过表达所逆转。

结论：lncRNA KCNQ1OT13低表达通过miR-19a-3p/TSHZ3,促进HG诱导的ARPE-19,并抑制其凋亡和氧化应激。     

Inhibition of miR-665-3p Enhances Autophagy and Alleviates Inflammation in Fusarium solani-Induced Keratitis

PurposeAccumulated evidence has shown that microRNAs (miRNAs) are closely related with the regulation of autophagy, which plays vital roles in fungal keratitis (FK). Microarray data showed elevated expression of miR-665-3p in mouse corneal tissues after infection with Fusarium solani (F. solani). Here, we investigated the effect of miR-665-3p in regulating autophagy in experimental F. solani keratitis and determined the potential molecular mechanisms involved.MethodsIn this article, we established an in vivo mouse model of FK and an in vitro model of corneal stromal cells by inoculating with F. solani. We divided them into the following six groups: control, chloroquine (CQ), rapamycin (Rapa), miR-665-3p antagomir (ant-665), miR-665-3p agomir (miR-665), and the negative control group (miR-NC). The levels of autophagy were detected by electron microscopy, Western blotting, and immunofluorescence. Then, we used a dual-luciferase reporter assay to determine the binding of miR-665-3p to the autophagy-related gene (ATG)5 3''UTR. Detection of IL-1β protein levels and hematoxylin and eosin (H&E) staining of corneal tissues were used to observe the effect of miR-665-3p on inflammation in FK.ResultsHere, we showed that inhibition of miR-665-3p expression in FK upregulated autophagy and alleviated inflammation. Nevertheless, the opposite results were found by overexpressing miR-665-3p. Additionally, ATG5 was a direct target gene for miR-665-3p.ConclusionsTogether, our data demonstrated that miR-665-3p might be involved in F. solani keratitis of mice by regulating autophagic pathways and inflammation.     

Abnormal deposition of laminin and type IV collagen at corneal epithelial basement membrane during wound healing in diabetic rats

PURPOSE: To understand the pathophysiology of the corneal basement membrane in diabetes, we compared the localization of laminin and type IV collagen in the epithelial basement membrane during corneal epithelial wound healing in diabetic and nondiabetic rats. METHODS: Streptozotocin was used to induce diabetes in half the rats. Two weeks later, the whole corneal epithelium was debrided. Diabetic and healthy rats (3-5 per group) were sacrificed before debridement and 1, 3, and 7 days and 1 month afterwards. The localization of laminin and type IV collagen was observed in cryosections by epifluorescence microscopy. RESULTS: In unwounded corneas of both diabetic and normal rats, laminin and type IV collagen were localized in the corneal epithelial basement. The intensity of fluorescence, however, was clearly stronger in the diabetic rats. In normal rats, wounding initially removed laminin and type IV collagen, but during healing these two proteins reappeared beneath the resurfacing corneal epithelium. Although similar results were observed in diabetic rats, the expression of laminin and type IV collagen was delayed, and their deposition was fragmented and irregular. CONCLUSIONS: These results suggest that delayed corneal epithelial wound healing in diabetes might involve delayed reappearance and abnormal reformation of epithelial basement membrane proteins.     

Prophylactic Knockdown of the miR-183/96/182 Cluster Ameliorates Pseudomonas aeruginosa–Induced Keratitis

PurposePreviously, we demonstrated that miR-183/96/182 cluster (miR-183C) knockout mice exhibit decreased severity of Pseudomonas aeruginosa (PA)-induced keratitis. This study tests the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis indicative of a therapeutic potential.MethodsEight-week-old miR-183C wild-type and C57BL/6J inbred mice were used. Locked nucleic acid–modified anti-miR-183C or negative control oligoribonucleotides with scrambled sequences (NC ORNs) were injected subconjunctivally 1 day before and then topically applied once daily for 5 days post-infection (dpi) (strain 19660). Corneal disease was graded at 1, 3, and 5 dpi. Corneas were harvested for RT-PCR, ELISA, immunofluorescence (IF), myeloperoxidase and plate count assays, and flow cytometry. Corneal nerve density was evaluated in flatmounted corneas by IF staining with anti-β-III tubulin antibody.ResultsAnti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1β at 1 dpi, which was decreased at 5 dpi; fewer polymorphonuclear leukocytes (PMNs) at 5 dpi; lower viable bacterial plate count at both 1 and 5 dpi; increased percentages of MHCII⁺ macrophages (Mϕ) and dendritic cells (DCs), consistent with enhanced activation/maturation; and decreased severity of PA keratitis. Anti-miR-183C treatment in the cornea of naïve mice resulted in a transient reduction of corneal nerve density, which was fully recovered one week after the last anti-miR application. miR-183C targets repulsive axon-guidance receptor molecule Neuropilin 1, which may mediate the effect of anti-miR-183C on corneal nerve regression.ConclusionsProphylactic miR-183C knockdown is protective against PA keratitis through its regulation of innate immunity, corneal innervation, and neuroimmune interactions.     

外周血液中miRNA表达与老年性黄斑变性相关性研究

目的　检测微小核糖核酸（microribonucleicacids，miRNA）在老年性黄斑变性（age-related macular degeneration，AMD）患者中的表达，并探讨miRNA的表达量与AMD病程之间的关系。方法　选取2014年1月至2016年11月于同济大学附属第十人民医院眼科门诊就诊的AMD患者6例为试验组，并选取同期6名正常人为对照组，通过基因芯片技术检测两组血液中miRNA的表达量。扩大样本的病例对照研究中共纳入126例AMD患者和140名正常人，检测其血液样本中miRNA的表达，比较两组人群间miRNA的表达量差异。结果　通过基因芯片技术，在试验组与对照组间共检测出216个miRNA存在表达差异（均为P<0.05），与对照组相比，试验组中111个miRNA表达量上升，105个miRNA表达量下降，差异均有统计学意义（均为P<0.05）。扩大样本的病例对照研究结果表明，在AMD患者中，miR-27a-3p、miR-29b-3p、miR-195-5p的表达量显著上升，同时，湿性AMD患者血液中miR-27a-3p的表达量高于干性AMD患者，差异均有统计学意义（均为P<0.05）。结论　AMD患者外周血中miRNA表达量水平有明显变化，miR-27a-3p、miR-29b-3p、miR-195-5p可能成为AMD血清学诊断和预后的标志物。     

miR-204调控眼科疾病的研究进展

微小RNA(miRNA)是一类广泛存在于真核生物体内、长度为20～ 25个核苷酸、具有调控功能的非编码单链RNA,参与机体的各种生命进程,包括细胞的生长、分化、增生、凋亡和自噬.miRNA-204-5p(miR-204-5p)是由位于染色体9q21.12上的TRPM3大内含子6表达.研究发现,miR-204在角膜损伤愈合过程中起着十分重要的作用,亦能够保持静止状态下血-视网膜屏障的稳定,并且在人小梁网细胞中,miR-204与细胞的凋亡、生存能力以及炎症介质的表达有着重要联系.这些研究都表明miR-204在眼部呈多维表达,提示miR-204很可能是不同眼部疾病的关键miRNA.本文从miRNA的生物合成,miR-204与糖尿病性角膜病变、视网膜色素上皮细胞、人小梁网细胞、年龄相关性白内障、糖尿病视网膜病变、视网膜母细胞瘤的关系,以及miR-204与自噬的相关研究等几个方面,就miR-204调控眼科疾病的研究进展进行综述,为探寻眼部难治性疾病的防治方法寻找新的靶点.