【摘要】 目的:探究miR-29a/HMGB1信号通路在高糖高脂(hyperglycemia and hyperlipidemia,HGHL)诱导的H9C2细胞纤维化过程中的作用。方法:使用含葡萄糖(33 mmol/L)和棕榈酸酯(500μmol/L)的DMEM培养基干预H9C2细胞24 h用于后续实验。共有8个实验分组,分别为NC组、HGHL组、miR-NC组、mimics组、inhibitor组、pc-HMGB1组、si-HMGB1组和miR-29a mimics+pc-HMGB1组。流式细胞术检测各组H9C2细胞的凋亡率。Western blot实验检测各组H9C2细胞内转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)、基质金属蛋白酶9(MMP-9)、过氧化物酶体增殖剂激活受体γ(PPARγ)和高迁移率族蛋白B1(HMGB1)的表达量。RT-qPCR检测各组细胞中miR-29a以及TGF-β1、CTGF、MMP-9、PPARγ、HMGB1 mRNA的表达水平。划痕实验检测各组H9C2细胞的迁移能力。结果:HGHL干预后,H9C2细胞的凋亡率显著增加(P <0.05),细胞迁移能力显著增强(P <0.05),细胞内TGF-β1、CTGF和MMP-9 mRNA表达水平显著增加(P<0.05),PPARγ mRNA的表达水平显著降低(P<0.05),相应蛋白的表达量也随mRNA的变化发生改变(P<0.05),此外H9C2细胞内miR-29a的表达水平也显著降低(P<0.05)。转染miR-29a mimics后,H9C2细胞因HGHL干预引起的凋亡率增加受到显著抑制(P<0.05),细胞的迁移能力也受到显著抑制(P<0.05),细胞内TGF-β1、CTGF和MMP-9蛋白表达量和mRNA表达水平相较于HGHL组显著降低(P<0.05),PPARγ蛋白表达量和mRNA表达水平显著增加(P<0.05)。转染miR-29a inhibitor后促进了HGHL诱导的H9C2细胞纤维化过程。miR-29a负调控HMGB1蛋白及其mRNA在H9C2细胞内的表达,双荧光素酶报告基因实验结果显示HMGB1是miR-29a的下游靶基因。转染si-HMGB1与转染miR-29a mimics对HGHL诱导的H9C2细胞纤维化作用类似。同时转染miR-29a mimics与pc-HMGB1对HGHL诱导的H9C2心肌细胞纤维化无显著影响。结论:HGHL干预后显著增加H9C2细胞的凋亡率,增强其迁移能力和纤维化的过程。同时HGHL干预显著下调miR-29a在细胞内的表达水平,miR-29a通过负调控HMGB1在细胞内的表达进而影响HGHL诱导的H9C2细胞纤维化。更多还原

【Abstract】 AIM: To investigate the role of miR-29a/HMGB1 signaling pathway in fibrosis H9C2 cells induced by HGHL. METHODS:DMEM medium containing glucose( 33 mmol/L)and palmitate( 500 μmol/L) was used to intervene in H9C2 cells for 24 h for subsequent experiments.There were 8 experimental groups, namely NC group, HGHL group, miR-NC group, mimics group, inhibitor group, pc-HMGB1 group, siHMGB1 group,and miR-29a mimics + pc-HMGB1 group. Flow cytometry was used to detect the apoptosis rate of H9C2 cells in each group. The Western blot experiment detected the expression of TGF-β1,CTGF,MMP-9,PPARγ,and HMGB1 in H9C2 cells of each group. RT-qPCR detected the expression levels of miR-29a,TGF-β1,CTGF,MMP-9,PPARγ,HMGB1 mRNA in each group of cells. The scratch test was used to detect the migration ability of H9C2 cells in each group. RESULTS: After HGHL intervention,the apoptosis rate of H9C2 cells was significantly increased( P <0.05),and the cell migration ability was significantly enhanced( P <0.05). The expression level of TGF-β1,CTGF,and MMP-9 mRNA in cells increased significantly( P <0.05),but the expression level of PPARγ mRNA decreased significantly( P < 0.05),and the expression of corresponding proteins also changed with the changes in mRNA( P <0.05). Besides,the expression level of miR-29a in H9C2 cells was also significantly reduced( P < 0.05). After the transfection of miR-29a mimics,the increase in apoptosis rate of H9C2 cells caused by HGHL intervention was significantly inhibited( P < 0.05),and the cell migration ability was also significantly inhibited( P < 0.05).Compared with the HGHL group,TGF-β1,CTGF,and MMP-9 protein expression and mRNA expression levels in H9C2 cells were significantly lower( P<0.05),and PPARγ protein expression and mRNA expression levels were significantly increased( P <0.05). Transfection of miR-29a inhibitor promoted the fibrosis process of H9C2 cells induced by HGHL. miR-29a negatively regulated the expression of HMGB1 protein and its mRNA in H9C2 cells. The results of dual-luciferase reporter gene experiments showed that HMGB1 was a downstream target gene of miR-29a. Transfection of si-HMGB1 and miR-29a mimics had similar effects on H9C2 cell fibrosis induced by HGHL. Simultaneous transfection of miR-29a mimics and pc-HMGB1 had no significant effect on H9C2 cardiomyocyte fibrosis induced by HGHL.CONCLUSION: HGHL intervention can significantly increase the apoptosis rate of H9C2 cells,enhance their migration ability,and the process of fibrosis. At the same time,HGHL intervention can significantly down-regulate the expression level of miR-29a in cells,miR-29a negatively regulates the expression of HMGB1 in cells and then affects HGHL-induced H9C2 cell fibrosis.更多还原