Objective: To explore the mechanism by which ghrelin regulates insulin sensitivity through modulation of miR-455-5p in hepatic cells. Methods: HepG2 cells were treated with or without DAG (1 μM). Glucose consumption,...Objective: To explore the mechanism by which ghrelin regulates insulin sensitivity through modulation of miR-455-5p in hepatic cells. Methods: HepG2 cells were treated with or without DAG (1 μM). Glucose consumption, intracellular glycogen content, phosphorylation of PI3K and Akt stimulated by insulin, expression of miR-455-5p, as well as IGF-1R protein level were analyzed. In addition, bioinformatic analysis, dual luciferase reporter assay, miR- 455-5p mimic or inhibitor treatment was conducted to investigate the molecular mechanisms. Results: High glucose treatment upregulated miR-455-5p expression but reduced glucose consumption and glycogen content. DAG reversed the effect of high glucose on glucose metabolism, increased protein level of IGF-1R and phosphorylation of PI3K/Akt stimulated by insulin, as well as downregulated miR-455-5p expression. Bioinformatic analysis indicated IGF-1R was the target of miR-455-5p. Dual luciferase reporter assay, as well as transfection with miR-455-5p mimic/inhibitor confirmed that DAG activated IGF-1R/PI3K/Akt signaling via inhibiting miR-455-5p. Conclusion: DAG improves insulin resistance via miR-455-5p- mediated activation of IGF-1R/PI3K/Akt system, suggesting that suppression of miR-455-5p or activation of DAG may be potential targets for T2DM therapy.展开更多
目的:揭示miR-455-5p对呼吸道合胞病毒(RSV)感染致气道上皮细胞炎症反应的作用机制。方法:qRT-PCR检测30例健康体检儿童(健康组)、RSV感染轻症组(n=41)和重症组(n=31)患儿血清miR-455-5p水平。将16HBE细胞分为Control组、NC-agomir组、m...目的:揭示miR-455-5p对呼吸道合胞病毒(RSV)感染致气道上皮细胞炎症反应的作用机制。方法:qRT-PCR检测30例健康体检儿童(健康组)、RSV感染轻症组(n=41)和重症组(n=31)患儿血清miR-455-5p水平。将16HBE细胞分为Control组、NC-agomir组、miR-455-5p-agomir组、NC-antagomir组、miR-455-5p-antagomir组。使用Lipofectamine 3000转染16HBE细胞后培养48 h后,分为Blank组、NC组(转染了NC-agomir的细胞)、RSV+NC-agomir组、RSV+miR-455-5p-agomir组,用RSV病毒液感染RSV+NC-agomir组和RSV+miR-455-5p-agomir组16HBE细胞,Blank组和NC组16HBE细胞正常培养。用CCK-8法和EdU法检测细胞增殖、TUNEL法检测细胞凋亡、ELISA法检测上清液中TNF-α、IL-6、IL-8水平,qRT-PCR检测miR-455-5p和SOCS3的m RNA水平,Western blot检测SOCS3、IFN-α、STAT1、STAT2、p-STAT1和p-STAT2的蛋白水平。结果:与健康组相比,轻症组和重症组患儿的血清miR-455-5p水平降低(P<0.05)。与轻症组相比,重症组的血清miR-455-5p水平降低(P<0.05)。与Control组和NC-agomir组相比,miR-455-5p-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率升高(P<0.05),TUNEL阳性率降低(P<0.05),上清液中的TNF-α、IL-6和IL-8水平降低(P<0.05),SOCS3 m RNA和蛋白水平降低(P<0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平升高(P<0.05)。与Control组和NC-antagomir组相比,miR-455-5p-antagomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低(P<0.05),TUNEL阳性率升高(P<0.05),上清液中的TNF-α、IL-6和IL-8水平升高(P<0.05),SOCS3 m RNA和蛋白水平升高(P<0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平降低(P<0.05)。与Blank组和NC组相比,RSV+NC-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低(P<0.05),TUNEL阳性率升高(P<0.05),上清液中的TNF-α、IL-6和IL-8水平升高(P<0.05),SOCS3 m RNA和蛋白水平升高(P<0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平降低(P<0.05)。与RSV+NC-agomir组相比,RSV+miR-455-5p-agomir组的miR-455-5p水平、相对细胞活力和EdU阳性率升高(P<0.05),TUNEL阳性率降低(P<0.05),上清液中的TNF-α、IL-6和IL-8水平降低(P<0.05),SOCS3m RNA和蛋白水平降低(P<0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平升高(P<0.05)。结论:miR-455-5p在RSV感染患儿血清中下调,上调miR-455-5p通过抑制SOCS3的转录和表达从而激活RSV感染的16HBE细胞中IFN-α介导的抗病毒反应。展开更多
基金Changshu Science and Technology Plan(Social Development)Project(No.CS202130)Key Project of Changshu No.2 People’s Hospital(No.CSEY2021007)。
文摘Objective: To explore the mechanism by which ghrelin regulates insulin sensitivity through modulation of miR-455-5p in hepatic cells. Methods: HepG2 cells were treated with or without DAG (1 μM). Glucose consumption, intracellular glycogen content, phosphorylation of PI3K and Akt stimulated by insulin, expression of miR-455-5p, as well as IGF-1R protein level were analyzed. In addition, bioinformatic analysis, dual luciferase reporter assay, miR- 455-5p mimic or inhibitor treatment was conducted to investigate the molecular mechanisms. Results: High glucose treatment upregulated miR-455-5p expression but reduced glucose consumption and glycogen content. DAG reversed the effect of high glucose on glucose metabolism, increased protein level of IGF-1R and phosphorylation of PI3K/Akt stimulated by insulin, as well as downregulated miR-455-5p expression. Bioinformatic analysis indicated IGF-1R was the target of miR-455-5p. Dual luciferase reporter assay, as well as transfection with miR-455-5p mimic/inhibitor confirmed that DAG activated IGF-1R/PI3K/Akt signaling via inhibiting miR-455-5p. Conclusion: DAG improves insulin resistance via miR-455-5p- mediated activation of IGF-1R/PI3K/Akt system, suggesting that suppression of miR-455-5p or activation of DAG may be potential targets for T2DM therapy.
文摘目的:揭示miR-455-5p对呼吸道合胞病毒(RSV)感染致气道上皮细胞炎症反应的作用机制。方法:qRT-PCR检测30例健康体检儿童(健康组)、RSV感染轻症组(n=41)和重症组(n=31)患儿血清miR-455-5p水平。将16HBE细胞分为Control组、NC-agomir组、miR-455-5p-agomir组、NC-antagomir组、miR-455-5p-antagomir组。使用Lipofectamine 3000转染16HBE细胞后培养48 h后,分为Blank组、NC组(转染了NC-agomir的细胞)、RSV+NC-agomir组、RSV+miR-455-5p-agomir组,用RSV病毒液感染RSV+NC-agomir组和RSV+miR-455-5p-agomir组16HBE细胞,Blank组和NC组16HBE细胞正常培养。用CCK-8法和EdU法检测细胞增殖、TUNEL法检测细胞凋亡、ELISA法检测上清液中TNF-α、IL-6、IL-8水平,qRT-PCR检测miR-455-5p和SOCS3的m RNA水平,Western blot检测SOCS3、IFN-α、STAT1、STAT2、p-STAT1和p-STAT2的蛋白水平。结果:与健康组相比,轻症组和重症组患儿的血清miR-455-5p水平降低(P<0.05)。与轻症组相比,重症组的血清miR-455-5p水平降低(P<0.05)。与Control组和NC-agomir组相比,miR-455-5p-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率升高(P<0.05),TUNEL阳性率降低(P<0.05),上清液中的TNF-α、IL-6和IL-8水平降低(P<0.05),SOCS3 m RNA和蛋白水平降低(P<0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平升高(P<0.05)。与Control组和NC-antagomir组相比,miR-455-5p-antagomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低(P<0.05),TUNEL阳性率升高(P<0.05),上清液中的TNF-α、IL-6和IL-8水平升高(P<0.05),SOCS3 m RNA和蛋白水平升高(P<0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平降低(P<0.05)。与Blank组和NC组相比,RSV+NC-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低(P<0.05),TUNEL阳性率升高(P<0.05),上清液中的TNF-α、IL-6和IL-8水平升高(P<0.05),SOCS3 m RNA和蛋白水平升高(P<0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平降低(P<0.05)。与RSV+NC-agomir组相比,RSV+miR-455-5p-agomir组的miR-455-5p水平、相对细胞活力和EdU阳性率升高(P<0.05),TUNEL阳性率降低(P<0.05),上清液中的TNF-α、IL-6和IL-8水平降低(P<0.05),SOCS3m RNA和蛋白水平降低(P<0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平升高(P<0.05)。结论:miR-455-5p在RSV感染患儿血清中下调,上调miR-455-5p通过抑制SOCS3的转录和表达从而激活RSV感染的16HBE细胞中IFN-α介导的抗病毒反应。
