THE IMPACT OF HSA-MIR-146A-5P ON HUMAN RETINAL ENDOTHELIAL CELLS IN DIABETIC CONDITIONS

Abstract

Background: Diabetic retinopathy (DR) is a microvascular complication of diabetes that leads to visual impairment in the adult population with effective available therapy. Micro-Ribonucleic acids (miRNAs) are small (?22 nucleotides) expressed RNA molecules that do not code for protein. They affect the expression of the target gene at the post-transcriptional level, hence, affecting several cellular processes. Clinical and experimental studies demonstrated that miRNAs are implicated in the pathogenesis of diabetic retinopathy. We identified hsa-miR-146a-5p is differentially expressed in human retinal microvascular endothelial cells (HRECs) under hyperglycemic conditions treated with adiponectin using RNA-seq for miRNA and qRT-PCR. Aim and objectives: To explore the impact of hsa-miR-146a-5p in hyperglycemia condition in vitro using HRECs and evaluate whether miR-146a-5p could mimic adiponectin's actions and effects in ameliorating the hyperglycemic effect in HRECS in vitro. Methods: HRECs were treated with 25 mM glucose, then transfected with a low dose of 10 nM or high dose of 50 nM of miR-146a-5p mimic to overexpress the miR-146a-5p. Moreover, we transfected HRECs using 10 nM of miR-146a-5p inhibitory to silence the expression. Apoptosis and oxidative stress were evaluated. Total RNA was extracted from different HRECs-treated groups, and the gene expression of miR-146a-5p, TRAF6, TNF-, ICAM-1, PPAR-a and antioxidant enzymes were evaluated using qRT-PCR. Further, we utilized RT-PCR profile array technology to explore the gene panel for inflammatory, cytokine receptor molecules and angiogenesis in HRECs under hyperglycemic conditions transfected with 50nM miR-146a-5p mimic. Bioinformatic analysis tools were used to explore the impact of 50 nM miR-146a-5p mimic transfection on inflammatory, oxidation, and angiogenesis pathways. Results and discussion: Transfection with miR-146a-5p inhibitory and 10 nM of miR-146a-5p mimic upregulated the expression of TNF-?, ICAM-1, and ROS production in HRECs, indicating a pro-inflammatory effect. Transfection of HRECs with 50nM miR-146a-5p mimic revealed anti-inflammatory impact and significantly reduced the expression of TRAF-6, TNF-?, and ICAM-1, whereas PPAR- ? expression was not affected. Pathway analysis demonstrated a pleiotropic effect of miR-146a-5p miRNA on gene expression via different pathways such as Il-17 signaling pathway, IL-8 Signaling, Toll-like Receptor Signaling, PPAR Signaling, and IL-6 Signaling, and also via inhibiting several transcription factors such as HMGB1, Jun, STAT4, NFKB1, and RELA. Conclusion: These results propose that miR-146a-5p could be involved in the amelioration of hyperglycemia-induced endothelial inflammation. High expression of miR-146a-5p and its anti-inflammatory, anti-oxidant, and anti-angiogenesis impact could be a potentially promising therapeutic and preventive option for DR patients.