D-半乳糖诱导的心肌细胞自噬下降与microRNA的表达变化有关

The decreasing of autophagy in cardiomyocytes induced by D-galactose is related to the changes of microRNA expression

目的探讨microRNA(miRNA)和衰老心肌细胞自噬之间的关系。方法使用0、4、8、16、20 g/L D-半乳糖刺激大鼠心肌细胞H9c2衰老,刺激3、6、9天后,β-半乳糖苷酶染色法检测细胞衰老水平;电镜观察D-半乳糖刺激衰老大鼠心肌细胞自噬体形成;分别采用实时荧光定量PCR(qRT-PCR)及蛋白质印迹法(Western blot)检测衰老大鼠心肌细胞与正常H9c2细胞中miRNA-30a、miRNA-126、miRNA-204及自噬蛋白LC3B II/LC3B I表达水平。结果与对照组比较,随着药物浓度的升高及刺激天数的增加,细胞染色逐渐加深,在D-半乳糖浓度为8 g/L刺激9天时,细胞染色最明显;电镜结果显示,D-半乳糖诱导衰老心肌细胞自噬体减少;Western blot结果显示,衰老心肌细胞LC3B II/LC3B I水平降低;实时荧光定量PCR结果显示,诱导衰老组心肌细胞中,miRNA-30a、miRNA-126表达水平明显下降,miRNA-204表达水平明显上升。结论 D-半乳糖诱导的衰老心肌细胞自噬水平降低的过程中,存在miRNA-30a、miRNA-126表达水平的降低以及miRNA-204表达水平的增加,miRNA的差异表达可能是衰老心肌细胞自噬水平变化的重要机制。

Aim To investigate the relationship between microRNA （miRNA） and autophagy in aged myocardial cells induced by D-galactose. Methods The D-galactose with 0, 4, 8, 16, 20 g/L was used to induce the rat myocardial cells to senescence respectively,and being stimulated 3, 6, 9 days later, the aging level of H9c2 was detected by the beta-galactosidase staining; the formation of autophagosomes in aging rat cardiomyocytes induced by D-galactose was detected by electron microscope; the mRNA expression level of miRNA-30a, miRNA-126, miRNA-204 and protein expression level of LC3B Ⅱ/LC3B Ⅰ in aging rat cardiomyocytes and H9C2 cells was detected by quantitative real-time（qRT-PCR） and Western blot methods respectively. Results Compared with the control group, the cell staining was gradually deepened with the increase of drug concentra tion and the stimulation days, and when D-galactose concentration was 8 g/L for 9 days, cell staining was the most obvious ; electron microscopy showed that the autophagy in aging cardiomyocytes induced by D-galactose was decreased; Western blot results showed that the level of LC3B Ⅱ/LC3BⅠ in aging eardiomyocytes was reduced; qRT-PCR results showed that in the aging cardio- myocytes group the expression levels of miRNA-30a and miRNA-126 were decreased and miRNA-204 was significantly increased. Conclusion In the process of the autophagy level decreasing in senescent cardiomyocytes induced by D-galactose, there exists a reducing in miRNA-30a, miRNA-126 expression level and an increasing in miRNA-204 expression level, the differential expression of miRNAs may be an important mechanism of autophagy in senescent cardiomyocytes.