ABSTRACT
INTRODUCTION: Obesity has been believed to be closely linked with many kinds of diseases including atherosclerosis, hypertension, cerebrovascular thrombosis, and diabetes. Ghrelin and Homeobox transcript antisense RNA (HOTAIR) were believed to be involved in the regulation of myocardial injury. METHODS: The obesity mice model was established through feeding mice (C57BL/6J, male, eight-week-old) with high-fat diet and palmitate (PA)-induced cardiomyocyte injury. RNA and protein levels were detected with Quantitative real-time PCR and Western blotting. The levels of TG, TCH, LDL, CK-MB, cTnl, and BNP in the serum or cell medium supernatant were measured using ELISA kits. The ROS level was detected with the DCFH-DA method. Binding sites between different targets were identified using detection of dual luciferase reporter assay. Cell apoptosis was analyzed by flow cytometry. RNA-binding protein immunoprecipitation and chromatin immunoprecipitation were used to detect the binding of DNMT3B with HOTAIR or miR-196b promoter. RESULTS: The expression of HOTAIR was downregulated, and miR-196b was upregulated in the obese myocardial injury. Ghrelin attenuated PA-induced cardiomyocyte injury by increasing HOTAIR. HOTAIR regulated the expression of miR-196b by recruiting DNMT3B to induce methylation of the miR-196b gene promoter. The binding site between miR-196b and IGF-1 was identified. DISCUSSION/CONCLUSION: We demonstrated that ghrelin attenuated PA-induced cardiomyocyte injury by regulating the HOTAIR/miR-196b/IGF-1 signaling pathway. Our findings might provide novel thought for the prevention and treatment of obesity-induced myocardial injury by targeting HOTAIR/miR-196b.
Subject(s)
Ghrelin , MicroRNAs , RNA, Long Noncoding , Animals , Epigenesis, Genetic , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Obesity/complications , Obesity/genetics , RNA, Long Noncoding/geneticsABSTRACT
BACKGROUND: Obesity is associated with the impairment of cardiac fitness and consequent ventricular dysfunction and heart failure. Ghrelin has been largely documented to be cardioprotective against ischaemia/reperfusion injury. However, the role of ghrelin in obesity-induced myocardial injury is largely unknown. This study sought to determine the cardiac effect of ghrelin against obesity-induced injury and the underlying mechanisms. METHODS: The effect of ghrelin was evaluated in a mouse model of obesity and a palmitic acid (PA)-treated cardiomyocyte cell line with or without ghrelin transfection. Gene and protein expression levels were determined by real-time PCR and western blot, respectively. Cell apoptosis was measured by flow cytometry analysis. RESULTS: In the present study, we found that both a high-fat diet (HFD) and PA treatment caused myocardial injury by increasing apoptosis and the expression of inflammatory cytokines. Overexpression of ghrelin reversed the effects induced by HFD or PA treatment. Knockdown of lncRNA H19 or overexpression of miR-29a abrogated the cardioprotective effects of ghrelin against apoptosis and inflammation. We also found that IGF-1 was a target gene of miR-29a and that H19 regulated IGF-1 expression via miR-29a. Overexpression of IGF-1 partially reversed the apoptosis and inflammation promoting effects of miR-29a. CONCLUSIONS: Our findings suggested that ghrelin protected against obesity-induced myocardial injury by regulating the H19/miR-29a/IGF-1 signalling axis, providing further evidence for the clinical application of ghrelin.
Subject(s)
Ghrelin/genetics , Heart Injuries/genetics , Insulin-Like Growth Factor I/genetics , MicroRNAs/genetics , RNA, Long Noncoding/genetics , Animals , Apoptosis/genetics , Cell Line , Disease Models, Animal , Gene Expression Regulation/genetics , Heart Injuries/etiology , Heart Injuries/pathology , Humans , Mice , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Obesity/complications , Obesity/genetics , Obesity/pathology , Signal Transduction/genetics , TransfectionABSTRACT
Shikonin derivatives, which are the active components of the medicinal plant Lithospermum erythrorhizon, exhibit many biological effects including apoptosis induction through undefined mechanisms. We recently discovered that orphan nuclear receptor Nur77 migrates from the nucleus to the mitochondria, where it binds to Bcl-2 to induce apoptosis. Here, we report that certain shikonin derivatives could modulate the Nur77/Bcl-2 apoptotic pathway by increasing levels of Nur77 protein and promoting its mitochondrial targeting in cancer cells. Structural modification of acetylshikonin resulted in the identification of a derivative 5,8-diacetoxyl-6-(1'-acetoxyl-4'-methyl-3'-pentenyl)-1,4-naphthaquinones (SK07) that exhibited improved efficacy and specificity in activating the pathway. Unlike other Nur77 modulators, shikonins increased the levels of Nur77 protein through their posttranscriptional regulation. The apoptotic effect of SK07 was impaired in Nur77 knockout cells and suppressed by cotreatment with leptomycin B that inhibited Nur77 cytoplasmic localization. Furthermore, SK07 induced apoptosis in cells expressing the COOH-terminal half of Nur77 protein but not its NH(2)-terminal region. Our data also showed that SK07-induced apoptosis was associated with a Bcl-2 conformational change and Bax activation. Together, our results show that certain shikonin derivatives act as modulators of the Nur77-mediated apoptotic pathway and identify a new shikonin-based lead that targets Nur77 for apoptosis induction.
