Regulatory Role Of miR-146a Research Articles

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.

MiR-146a suppresses STAT3/VEGF pathways and reduces apoptosis through IL-6 signaling in primary human retinal microvascular endothelial cells in high glucose conditions

microRNA (miRNA) play critical roles in the pathological processes of diabetic retinopathy, including inflammatory responses, insulin signaling, and angiogenesis.In addition to their regulatory functions on gene expression, miRNA is considered as a potential therapeutic target, as well as a diagnostic marker for many diseases. Our understanding on the pathological mechanisms underlying diabetic retinopathy is still incomplete and additional investigations are required to develop novel therapeutic strategies. The aim of this study was to investigate our hypothesis that miR-146a plays a role in suppressing pro-inflammatory pathways, involving STAT3 and VEGF, through regulating IL-6 signaling to reduce apoptosis of human retinal endothelial cells (REC) in high glucose conditions. Human REC were cultured in normal (5mM) glucose or high glucose medium (25mM) for 3days. We performed transfections on REC with miRNA mimics (hsa-miR-146a-5p). Overexpression of miR-146a reduced IL-6 levels, STAT3 phosphorylation, and VEGF levels in REC cultured in high glucose. Cellular apoptosis was decreased in REC overexpressing miR-146a, as demonstrated by the inhibition of DNA fragmentation. More importantly, we demonstrated that the regulatory role of miR-146a on STAT3/VEGF and apoptosis was mediated by IL-6 receptor signaling in REC.Overall, we report that miR-146a suppressed IL-6 signaling, leading to reduced levels of STAT3 and VEGF in REC in high glucose conditions, leading to decreased apoptosis. The outcome suggests that miR-146a is a potential molecular target for inhibiting inflammation and apoptosis in the diabetic retina through the suppression of the IL-6-mediated STAT3/VEGF pathway.

MiR-146a suppresses cellular immune response during Japanese encephalitis virus JaOArS982 strain infection in human microglial cells.

BackgroundJapanese encephalitis virus (JEV) is the causative agent of Japanese encephalitis which is more prevalent in South and Southeast Asia. JEV is a neurotropic virus which infiltrates into the brain through vascular endothelial cells. JEV infects neurons and microglial cells which causes neuronal damage and inflammation. However, JEV also evades the cellular immune response to survive in host cells. Viruses are known to modulate the expression of microRNAs, which in turn modulate cellular immune response by targeting expression of antiviral genes. The aim of this study is to understand the anti-inflammatory role of miR-146a during JEV infection, which facilitates immune evasion.MethodsHuman brain microglial cells (CHME3) were infected by JEV: JaOArS982 and P20778 strain, and expression of miR-146a were analyzed. Overexpression and knockdown studies of miR-146a were done to see the effect on NF-κB pathway and antiviral Jak-STAT pathway. Regulatory role of miR-146a on expression of interferon-stimulated genes was determined by real-time PCR and luciferase assays.ResultsJEV infection elevated the expression of miR-146a in JaOArS982 strain which caused downregulation of TRAF6, IRAK1, IRAK2, and STAT1 genes. Exogenous overexpression of miR-146a led to suppression of NF-κB activation and abrogation of Jak-STAT pathway upon JEV infection which led to downregulation of interferon-stimulated genes (IFIT-1 and IFIT-2) and facilitated viral replication. JEV infection initially upregulated cytokine production and activated STAT1 activity but STAT1 levels reduced at later time point, which led to the downregulation of interferon-stimulated genes.ConclusionUpregulation of miR-146a by JEV JaOArS982 strain leads to suppression of NF-κB activity and disruption of antiviral Jak-STAT signaling which helps the virus to evade the cellular immune response. This effect of JEV infection on miR-146a expression was found to be strain specific.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0249-0) contains supplementary material, which is available to authorized users.

MiR-146a regulates monocyte hyper-reactivity to LPS and Poly I : C in patients with primary biliary cirrhosis

Objective To investigate whether the hyper-reactivity of monocytes from the patients with primary biliary cirrhosis (PBC) to LPS and Poly I ∶ C was associated with miR-146a.Methods Peripheral blood mononuclear cells from PBC patients and healthy controls were stimulated with 10 μg/ml of LPS or Poly I ∶ C.The levels of IL-1 β,IL-6,TNF-α and IFN-α in the culture medium were detected by ELISA.The relative expressions of miR-146a,miR-21,let-7e,miR-155 and miR-125b were detected by RT-PCR.The regulatory role of miR-146a on the production of inflammatory factors in LPS or Poly I ∶ C stimulated THP-1 cells was studied by gain-and-loss function assay.Results The up-regulation of miR146a,which was induced by both LPS or Poly I ∶ C,was impaired in the monocytes from PBC patients.The production of LPS or Poly I ∶ C induced inflammatory factor,could be enhanced by miR-146a down-regulation.Conclusion The hyper-reactivity of monocytes from PBC patients to LPS and Poly I ∶ C was associated with miR-146a. Key words: miR-146a; Primary biliary cirrhosis; Innate immunity; Inflammatory factors