ABSTRACT
BACKGROUND: LncRNA NNT-AS1 (NNT-AS1) has been extensively studied as the causative agent in propagation and progression of lung and bladder cancers, and cholangiocarcinoma. However, its significance in proliferation and inflammation of diabetic nephropathy is enigmatic. This study focuses on the molecular mechanisms followed by NNT-AS1 to establish diabetic nephropathy (DN) and its potential miRNA target. METHODS: Bioinformatics analysis to identify potential miRNA target of NNT-AS1 and smad4 transcription factor was conducted using LncBase and TargetScan, and was subsequently confirmed by luciferase reporter assay. Relative quantitative expression of NNT-AS1 in human glomerular mesangial cells (HGMCs) was detected through quantitative real-time PCR and WB analysis. Cell proliferation was detected through CCK-8 assay, whereas, ELISA was conducted to evaluate the expression of inflammatory cytokines. Following this, relative expression of miR-214-5p and smad4 were confirmed through qRT-PCR and western blot analysis. RESULTS: Results from the experiments manifested up-regulated levels of NNT-AS1 and smad4 in the blood samples of DN patients as well as in HGMCs, whereas, downregulated levels of miR-214-5p were measured in the HGMCs suggesting the negative correlation between NNT-AS1 and miR-214-5p. Potential binding sites of NNT-AS1 showed miR-214-5p as its direct target and NNT-AS1 as potential absorber for this microRNA, in turn increasing the expression of transcription factor smad4. CONCLUSION: The data suggests that NNT-AS1 can be positively used as a potential biomarker and indicator of DN and causes extracellular matrix (ECM) accumulation and inflammation of human mesangial cells.
Subject(s)
Cell Proliferation , Diabetic Nephropathies/physiopathology , Extracellular Matrix/metabolism , Inflammation/physiopathology , Mesangial Cells/cytology , NADP Transhydrogenase, AB-Specific/physiology , RNA, Long Noncoding/physiology , Blood Glucose/metabolism , Diabetic Nephropathies/blood , Down-Regulation , Humans , Mesangial Cells/metabolism , MicroRNAs/blood , MicroRNAs/genetics , Mitochondrial Proteins/blood , Mitochondrial Proteins/genetics , Mitochondrial Proteins/physiology , NADP Transhydrogenase, AB-Specific/blood , NADP Transhydrogenase, AB-Specific/genetics , RNA, Long Noncoding/blood , RNA, Long Noncoding/genetics , Smad4 Protein/blood , Smad4 Protein/genetics , Up-RegulationABSTRACT
Cerebral ischemia-reperfusion injury (CIRI) mainly arises from the clinical treatment of ischemic stroke, induced by the blood-brain barrier (BBB) disruption and infiltrated inflammation. The Sigma-1 receptor (Sigma-1R) is a novel target for neuroprotection, and the α2-receptor agonist pain medication dexmedetomidine displays a neuroprotective effect through activating Sigma-1R. The present study aims to investigate the potential therapeutic effect of dexmedetomidine in a mouse stroke model and hypoxia/reoxygenation(OGD/R)-induced brain endothelial dysfunction. First, we found that Sigma-1R was significantly upregulated in middle cerebral artery occlusion (MCAO) mice by the administration of dexmedetomidine. In vivo experiments revealed that dexmedetomidine ameliorated hyperpermeability of the blood-brain barrier (BBB), lowered the expression level of Occludin, and impaired brain function as measured by neurological scores in MCAO mice. In vitro assays show that dexmedetomidine alleviated OGD/R-caused cytotoxicity, hyperpermeability, abnormal expression of Occludin, and inflammatory factors in human brain microvascular endothelial cells (HBMVECs). Moreover, blockage of Sigma-1R by its antagonist BD1047 abolished the neuroprotective property of dexmedetomidine in both animal and cell culture experiments. On the basis of these findings, we conclude that dexmedetomidine therapy shows neuroprotection in MCAO mice. Mechanistically, dexmedetomidine alleviated hypoxia/reoxygenation-induced cerebral endothelial dysfunction by activating the Sigma-1R-mediated signaling pathway.
