Paired Box Gene 6 Regulates Heme Oxygenase-1 Expression and Mitigates Hydrogen Peroxide-Induced Oxidative Stress in Lens Epithelial Cells.

PURPOSE: This study aimed to investigate the regulation of heme oxygenase-1 (HO-1) by paired box gene 6 (Pax6) and their roles in hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in lens epithelial cells (LECs) (SRA01/04, HLE-B3). METHODS: Lens anterior capsule membranes of mice of different ages were obtained to compare differences in the expression of Pax6 and HO-1 using Western blotting. Pax6-overexpressing plasmid and small interfering RNA were designed to overexpress and silence Pax6, respectively. Cobalt protoporphyrin (CoPP) was used to promote the expression of HO-1. Oxidative damage in LECs was induced by treatment with H2O2 (400 µM) for 24 h. Cell viability was measured using the Cell Counting Kit-8 assay. Intracellular reactive oxygen species (ROS) were detected using flow cytometry and immunofluorescence. Superoxide dismutase (SOD) level was measured using SOD Assay Kit and apoptotic cells were quantified using annexin V-fluorescein isothiocyanate/propidium iodide staining. RESULTS: Pax6 and HO-1 expression levels showed an age-dependent decrease in LECs of mouse. Overexpressing Pax6 upregulated HO-1 expression level. Silencing Pax6 downregulated the HO-1 expression level, resulting in increased generation of ROS, reduced SOD activity, decreased cell viability, and increased apoptotic cells of LECs under H2O2-induced oxidative stress. Overexpressing Pax6 and CoPP both mitigates H2O2-induced oxidative stress by increasing the expression of HO-1 of LECs. CONCLUSION: Pax6 and HO-1 expression levels showed an age-dependent decrease in LECs in mouse anterior capsules. Pax6 could regulate the expression of HO-1 in LECs. The decrease of Pax6 weakened the antioxidant ability of LECs under H2O2-induced oxidative stress by downregulating HO-1, which may be a potential mechanism for the formation of age-related cataract.

LncRNA MCM3AP-AS1 is downregulated in diabetic retinopathy and promotes cell apoptosis by regulating miR-211/SIRT1.

AIM: This study aimed to investigate the role of lncRNA MCM3AP-AS1 in diabetic retinopathy (DR). METHODS: Plasma MCM3AP-AS1 levels in DR patients (n = 80), T2DM patients (n = 80), and Controls (n = 80) were measured by qPCR and compared using ANOVA (one-way) and Tukey test. The expressions of lncRNA MCM3AP-AS1 and miR-211 in Human retinal pigment epithelial cells (hRPE) line ARPE-19 were detected by RT-qPCR. Western blot and annexin V-FITC staining were performed to investigate the role of MCM3AP-AS1/SIRT1 in ARPE-19 cell proliferation and apoptosis in vitro. RESULTS: We observed that MCM3AP-AS1 was downregulated in DR patients 25 comparing to T2D patients without significantly complications. Bioinformatics analysis showed that MCM3AP-AS1 might bind miR-211. However, no significant correlation between these two factors was observed in DR patients. Consistently, overexpression of MCM3AP-AS1 and miR-211 failed to affect the expression of each other in hRPE. Interestingly, MCM3AP-AS1 overexpression upregulated SIRT1, a target of miR-211. Moreover, MCM3AP-AS1 was downregulated in DR patients compared to type 2 diabetic mellitus patients without significant complications. In RPEs, high glucose treatment downregulated MCM3AP-AS1. Cell apoptosis analysis showed that MCM3AP-AS1 and SIRT1 overexpression decreased the apoptotic rate of RPEs, and miR-211 overexpression reduced the effect of MCM3AP-AS1 and SIRT1 overexpression. CONCLUSION: MCM3AP-AS1 is downregulated in DR and promotes cell apoptosis by regulating miR-211/SIRT1.

Plasma Levels and Diagnostic Significance of miR-335-3p and EGFR in Diabetic Retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is the common complication of diabetes, accounting for most blindness cases worldwide. MicroRNAs (miRs) are small non-coding RNAs and participate in the pathogenesis and develop-ment of various diseases, including DR. The present study aimed to investigate miR-335-3p and vascular endothelial growth factor (EGFR) roles in DR diagnosis and development. METHODS: A total of 104 healthy volunteers, 96 type 2 diabetes mellitus (T2DM) patients, and 102 DR cases were enrolled in this study. The clinicopathological information of all subjects were collected and analyzed using chisquared test. After collecting plasma from each participant, a ROC assay was conducted to determine the dis-criminative value of miR-335-3p and EGFR in DR diagnosis. The targeted relationship between miR-335-3p and EGFR was examined by dual-luciferase reporter assay and correlation analysis. After exposing APRE-19 cells to different concentrations of high glucose (HG), the DR in vitro cell model was constructed. The expression levels of miR-335-3p and EGFR were detected using RT-qPCR. The effects of miR-335-3p and EGFR on HG-treated APRE-19 cell viability were determined by CCK-8 assay. RESULTS: Clinicopathological information presented that BMI index, fasting blood glucose (FBP), 2h-BG, HbA1c, miR-335-3p, and EGFR levels were strongly associated with DR pathogenesis. MiR-335-3p was significantly decreased while EGFR was increased in DR patients and HG-treated APRE-19 cells. MiR-335-3p and EGFR presented high accuracy, sensitivity, and specificity in differentiating DR from healthy cases and T2DM patients; moreover, miR-335-3p and EGFR could also discriminate proliferative DR (PDR) cases from healthy controls. After confirming that miR-335-3p was negatively correlated with its target EGFR, we found miR-335-3p could increase the viability in HG-treated APRE-19 cells while silencing of EGFR could also reverse the inhibitory effects of HG conditions on APRE-19 cell viability. CONCLUSIONS: We concluded that plasma miR-335-3p and EGFR may be utilized as non-invasive biomarkers for screening DR cases, contributing to DR diagnosis and treatment.

Combination therapy with semaglutide and rosiglitazone as a synergistic treatment for diabetic retinopathy in rodent animals.

OBJECTIVE: To investigate the protective efficacies and potent mechanisms of combination therapy with semaglutide and rosiglitazone (RSG) on the high-glucose incubated human ARPE-19 cells and diabetic retinopathy (DR) model rats. MAIN METHODS: The CCK-8 methods were used to evaluate the protective effects of semaglutide and RSG alone or combination on the cell viability of high-glucose treated ARPE-19 cells. After the DR rat model was established, the effects of combined treatment on general indexes, retinal morphological changes, retinal Müller cells as well as PI3K/Akt/MTOR related factors of DR model rats were investigated. RESULTS: The CCK-8 assay showed obviously enhanced protective efficacies of combination therapy with semaglutide and RSG on the ARPE-19 with oxidative stress induced by high-glucose with combination index all below 1.5 demonstrating obvious synergistic effects. Combined incubation could also effectively decrease the expression of inflammatory factors, including TNF-α, IL-1ß, IL-6, and the increase of ROS content in ARPE cell culture supernatant induced by high-glucose. Combined use of the antioxidant, PI3K/Akt and mTOR inhibitors, we further demonstrated that combined incubation of semaglutide and RSG could effectively by reduce high glucose-induced inflammatory injury inhibiting ROS/PI3K/Akt/mTOR signaling. Furthermore, chronic combination treatment effectively improved the histopathological characteristics and down-regulated the GFAP expression in Müller cells as well as PI3K/Akt/MTOR signaling pathway-related factors in retina which was better than any monomer treatment group. CONCLUSIONS: Combined semaglutide with RSG exhibited synergistically protective efficacies on retinal cells by decreasing the GFAP expression, inhibiting oxidative stress and PI3K/Akt/MTOR signaling-transduction in DR model rats.

miR-652 Promotes Proliferation and Migration of Uveal Melanoma Cells by Targeting HOXA9.

BACKGROUND Dysregulation of the microRNA (miRNA) network is a typical feature of many cancers. However, the key specific miRNAs involved in uveal melanoma carcinogenesis are largely unknown. MATERIAL AND METHODS RT-qPCR was used to detected miR-652 expression in uveal melanoma tissues and cell lines. miR-652 inhibitor was transfected into uveal melanoma cells to decrease miR-652 expression and determine the biological role of miR-652 by CCK-8 and wound healing assays. Bioinformatic analysis and dual luciferase reporter assay were used to predict and validate the target gene of miR-652. HOXA9 siRNA was transfected into cells to confirm that miR-652 relies on regulation of HOXA9 to regulate cell proliferation and migration. RESULTS RT-qPCR showed that miR-652 was overexpressed in uveal melanoma cell lines (MUM-2B, MEL270) compared with melanocyte cells (ARPE-19). Overexpression of miR-652 was also observed in uveal melanoma compared to paired non-tumor tissues. Downregulation of miR-652 inhibited the cell proliferation ability and migration ability of uveal melanoma cells. Using bioinformatic analysis, HOXA9 was found to be a potential target gene of miR-652. The direct regulation of HOXA9 by miR-652 was experimentally validated in uveal melanoma cells by dual luciferase assay and Western blotting. We also observed that miR-652 promoted HIF-1alpha signaling via repression of HOXA9 in uveal melanoma cells. Silencing of HOXA9 attenuated the miR-652 inhibitor decreased cell growth rate and decreased migration ability in uveal melanoma cells. CONCLUSIONS Our data demonstrate an oncogenic role of miR-652 in uveal melanoma, showing that miR-652 may be a useful biomarker for prediction of prognosis for patients with uveal melanoma.

LncRNA TP73-AS1 down-regulates miR-139-3p to promote retinoblastoma cell proliferation.

Our study aimed to investigate the role of long non-coding RNAs (lncRNA) TP73-AS1 in retinoblastoma (Rb). In the present study, we found that TP73-AS1 was up-regulated, while miR-139-3p was down-regulated in Rb. TP73-AS1 and miR-139-3p were inversely correlated in Rb tissues. In cells of Rb cell lines, overexpression of miR-139-3p failed to affect TP73-AS1, while TP73-AS1 overexpression caused the down-regulated miR-139-3p TP73-AS1 overexpression caused promoted proliferation of Rb cells but showed no significant effects on cell migration and invasion. miR-139-3p overexpression played an opposite role and attenuated the effects of TP73-AS1 overexpression. Therefore, lncRNA TP73-AS1 may down-regulate miR-139-3p to promote Rb cell proliferation.