LncRNA CASC2 Alleviates Renal Interstitial Inflammation and Fibrosis through MEF2C Downregulation-Induced Hinderance of M1 Macrophage Polarization.

INTRODUCTION: Long noncoding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) alleviates the progression of diabetic nephropathy by inhibiting inflammation and fibrosis. This study investigated how CASC2 impacts renal interstitial fibrosis (RIF) through regulating M1 macrophage (M1) polarization. METHOD: Nine-week-old mice underwent unilateral ureteral obstruction (UUO) establishment. Macrophages were induced toward M1 polarization using lipopolysaccharide (LPS) in vitro and cocultured with fibroblasts to examine how M1 polarization influences RIF. LnCeCell predicted that CASC2 interacted with myocyte enhancer factor 2 C (MEF2C), which was validated by dual-luciferase reporter assay. CASC2/MEF2C overexpression was achieved by lentivirus-expressing lncRNA CASC2 injection in vivo or CASC2 and MEF2C transfection in vitro. Renal injury was evaluated through biochemical analysis and hematoxylin-eosin/Masson staining. Macrophage infiltration and M1 polarization in the kidney and/or macrophages were detected by immunofluorescence, flow cytometry, and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR). Expressions of CASC2, MEF2C, and markers related to inflammation/M1/fibrosis in the kidney/macrophages/fibroblasts were analyzed by qRT-PCR, fluorescence in situ hybridization, enzyme-linked immunosorbent assay, and/or Western blot. RESULT: In the kidneys of mice, CASC2 was downregulated and macrophage infiltration was promoted time-dependently from days 3 to 14 post-UUO induction; CASC2 overexpression alleviated renal histological abnormalities, hindered macrophage infiltration and M1 polarization, downregulated renal function markers serum creatinine and blood urea nitrogen and inflammation/M1/fibrosis-related makers, and offset UUO-induced MEF2C upregulation. LncRNA CASC2 overexpression inhibited fibroblast fibrosis and M1 polarization in cocultured fibroblasts with LPS-activated macrophages. Also, CASC2 bound to MEF2C and inhibited its expression in LPS-activated macrophages. Furthermore, MEF2C reversed the inhibitory effects of lncRNA CASC2 overexpression. CONCLUSION: CASC2 alleviates RIF by inhibiting M1 polarization through directly downregulating MEF2C expression. CASC2 might represent a promising value of future investigations on treatment for RIF.

The protective effects of lncRNA ZFAS1/miR-421-3p/MEF2C axis on cerebral ischemia-reperfusion injury.

LncRNA ZNFX1 antisense RNA 1 (ZFAS1) could improve neuronal damage and inhibit inflammation and apoptosis. We conducted an in-depth exploration on the protective mechanism of ZFAS1 in cerebral ischemia-reperfusion injury. Overexpressed or silenced plasmids of ZFAS1 were transfected into the cells to analyze the effects of oxygen-glucose deprivation/reperfusion (OGD/R) treatment on the viability, apoptosis and related gene expressions of Neuro-2a cell by performing MTT assay, flow cytometry, qRT-PCR, and Western blot. Bioinformatic analysis, qRT-PCR, dual-luciferase reporter assay and RNA immunoprecipitation were used to screen and verify the miRNA(s) which could competitively bind with ZFAS1 and downstream mRNA(s) targeted by the miRNA(s). The effects of ZFAS1 and the above target miRNA(s) or gene(s) on the apoptosis of OGD/R-injured cells, apoptosis-related proteins, inflammatory factors and p65/IκBα pathway were further verified via the rescue test. The results from the middle cerebral artery occlusion (MCAO) mouse model in vivo were consistent with those from the cellular experiments. The expression of lncRNA ZFAS1 in OGD/R-injured cells was inhibited, and the up-regulation of ZFAS1 protected Neuro-2a cells. MiR-421-3p was predicted to be the target miRNA of ZFAS1 and could offset the protective effect of ZFAS1 overexpression on OGD/R-injured cells following its up-regulation. MEF2C, which was the downstream target gene of miR-421-3p, reversed the OGD/R-induced enhanced cell damage caused by miR-421-3p mimic when MEF2C was overexpressed. In in vivo studies, ZFAS1 overexpression reduced brain tissue infarction, apoptosis and gene regulation caused by MCAO, while miR-421-3p mimic had the opposite effect. Collectively, the regulation of lncRNA ZFAS1/miR-421-3p/MEF2C axis showed protective effects on cerebral ischemia-reperfusion injury.

TGF-ß3/Smad3 Contributes to Isoflurane Postconditioning Against Cerebral Ischemia-Reperfusion Injury by Upregulating MEF2C.

Isoflurane postconditioning alleviates cerebral ischemic-reperfusion injury (CIRI), but the underlying mechanism has not been fully clarified. We previously demonstrated that the transforming growth factor beta-1 (TGF-ß1)/Smads signaling pathway is involved in the neuroprotective effect of isoflurane postconditioning. TGF-ß3 has a highly homologous sequence relative to that of TGF-ß1. In this study, we explored the roles of the TGF-ß3/Smad3 signaling pathway and myocyte enhancer factor 2C (MEF2C) in neuroprotection induced by isoflurane postconditioning. A CIRI rat model was established by middle cerebral artery occlusion for 1.5 h, followed by 24 h of reperfusion. Isoflurane postconditioning led to lower infarct volumes and neurologic deficit scores, more surviving neurons, and less damaged and apoptotic neurons as compared with those of CIRI rats. Moreover, isoflurane postconditioning upregulated the expressions of TGF-ß3, p-Smad3, and MEF2C. However, the neuroprotective effect was reversed by pirfenidone, a TGF-ß3/Smad3 signaling pathway inhibitor. Also, pirfenidone treatment downregulated the expression of MEF2C. These results indicate that the TGF-ß3/Smad3 signaling pathway contributes to the neuroprotection of isoflurane postconditioning after CIRI and is possibly related to MEF2C.

Cardiac reprogramming factor Gata4 reduces postinfarct cardiac fibrosis through direct repression of the profibrotic mediator snail.

OBJECTIVE: The administration of a variety of reprogramming factor cocktails has now been shown to reprogram cardiac fibroblasts into induced cardiomyocyte-like cells. However, reductions in ventricular fibrosis observed after reprogramming factor administration seem to far exceed the extent of induced cardiomyocyte-like cell generation in vivo. We investigated whether reprogramming factor administration might primarily play a role in activating antifibrotic molecular pathways. METHODS: Adult rat cardiac fibroblasts were infected with lentivirus encoding the transcription factors Gata4, Mef2c, or Tbx5, all 3 vectors, or a green fluorescent protein control vector. Gene and protein expression assays were performed to identify relevant antifibrotic targets of these factors. The antifibrotic effects of these factors were then investigated in a rat coronary ligation model. RESULTS: Gata4, Mef2c, or Tbx5 administration to rat cardiac fibroblasts in vitro significantly downregulated expression of Snail and the profibrotic factors connective tissue growth factor, collagen1a1, and fibronectin. Of these factors, Gata4 was shown to be the one responsible for the downregulation of the profibrotic factors and Snail (mRNA expression fold change relative to green fluorescent protein for Snail, Gata4: 0.5 ± 0.3, Mef2c: 1.3 ± 1.0, Tbx5: 0.9 ± 0.5, Gata4, Mef2c, or Tbx5: 0.6 ± 0.2, P < .05). Chromatin immunoprecipitation quantitative polymerase chain reaction identified Gata4 binding sites in the Snail promoter. In a rat coronary ligation model, only Gata4 administration alone improved postinfarct ventricular function and reduced the extent of postinfarct fibrosis. CONCLUSIONS: Gata4 administration reduces postinfarct ventricular fibrosis and improves ventricular function in a rat coronary ligation model, potentially as a result of Gata4-mediated downregulation of the profibrotic mediator Snail.