摘要
目的研究组蛋白去乙酰化酶3(HDAC3)调控低氧诱导H9C2心肌细胞自噬的功能和机制。方法体外建立H9C2心肌细胞低氧模型,采用Western blot和免疫荧光染色技术检测自噬相关蛋白LC3B-Ⅱ、Beclin-1和P62的表达变化以及荧光活性。分别转染HDAC3过表达质粒和干扰RNA后,检测自噬调节分子mTOR和LC3B的蛋白表达和荧光活性变化。结果与对照组比较,低氧1、6、12 h后,H9C2心肌细胞中LC3B-Ⅱ和Beclin-1表达显著增加(P<0.05);P62蛋白表达先增高(1 h)后降低(6 h)(P<0.05)。低氧1 h和6 h后,HDAC3和mTOR蛋白表达明显增高(P<0.05);低氧12 h后HDAC3和mTOR蛋白表达显著减少。过表达HDAC3能够降低mTOR蛋白表达和荧光活性,增加LC3B的蛋白表达和荧光活性(P<0.05);下调HDAC3蛋白表达能够升高mTOR蛋白水平和荧光活性,减少了LC3B蛋白表达和荧光活性(P<0.05)。结论HDAC3负性调节mTOR蛋白,促进了低氧诱导的H9C2心肌细胞自噬。
Objective To investigate the function and mechanism of histone deacetylase 3(HDAC3)in modulating hypoxia-induced autophagy in H9C2 cardiac cells.Methods The hypoxia model of H9C2 was established in vitro.The expression and fluorescence activity of autophagy-related proteins LC3B-Ⅱ,Beclin-1,and P62 were detected by Western blot and immunofluorescence staining.After the transfection of HDAC3 plasmid or interfering RNA,the expression and fluorescence activity of autophagy regulatory molecule mTOR and autophagy-related protein LC3B were detected.Results Compared with the control group,the protein level of LC3B-Ⅱand Beclin-1 were augmented after hypoxia for 1 h,6 h,and 12 h in H9C2 cardiac cells(P<0.05),and the expression of p62 protein also increased first at 1 h and then decreased at 6 h(P<0.05).After hypoxia for 1 h and 6 h,the protein expression of HDAC3 and mTOR were amplified,while both of them declined at hypoxia for 12 h(P<0.05).The protein level and fluorescence activity of mTOR decreased,but the protein level and fluorescence activity of LC3B increased after overexpression of HDAC3(P<0.05).Downregulation of HDAC3,the expression and fluorescence activity of mTOR enhanced,while the protein level and fluorescence activity of LC3B reduced(P<0.05).Conclusion HDAC3 negatively regulates mTOR protein and promotes hypoxia-induced autophagy in H9C2 cardiac cells.
作者
王月涵
黄颖
Wang Yuehan;Huang Ying(Dept of Electrocardiographic and Cardiac Function,The First Affiliated Hospital of Anhui Medical University,Hefei 230022;Dept of Cardiology,The First Affiliated Hospital of Anhui Medical University,Hefei 230022)
出处
《安徽医科大学学报》
CAS
北大核心
2021年第10期1575-1579,共5页
Acta Universitatis Medicinalis Anhui
基金
安徽省自然科学基金(编号:1608085QH196)。
