缺氧环境下肾小管上皮细胞外泌体中自噬相关microRNA表达量的研究

Expression of autophagy related microRNA in human renal tubular epithelial exudate under hypoxia

目的观察缺氧环境下肾小管上皮细胞(RTEC)外泌体中自噬相关的微小核糖核酸(miRNA)表达量的变化。方法体外培养RTEC,常氧及缺氧分别处理RTEC,采用ExoQuick-TC提取外泌体,采用透射电镜、蛋白浓度定量鉴定并比较外泌体的特性,采用逆转录聚合酶链式反应(RT-PCR)检测RTEC外泌体中自噬相关miRNA-30b、miRNA-30c、miRNA-214、miRNA-183、miRNA-200a表达量。结果缺氧状态下肾小管细胞分泌的外泌体外观无明显变化,缺氧48 h外泌体/细胞蛋白比值明显上升,明显高于常氧组,随着缺氧时间延长外泌体自噬相关miRNA表达逐渐上升,缺氧组各时间段自噬相关miRNA-30b、miRNA-30c、miRNA-214、miRNA-183、miRNA-200a含量均高于常氧组,且随着缺氧时间延长含量逐渐上升。结论缺氧可改变肾小管细胞分泌外泌体的生物学功能,表现为自噬相关miRNA表达上调,这可能是慢性肾脏病患者重要脏器出现自噬行为的原因之一。

Objective To observe the expression of autophagy-related microRNAs in renal tubular epithelial exosomes under hypoxia. Methods Tubular epithelial cells were cultured in vitro and treated with normoxia and hypoxia respectively. ExoQuick-TC was used to extract the exosomes. The characteristics of the exosomes were quantitatively identified by transmission electron microscopy and protein concentration. RT-PCR was used to detect autophagy-related microRNA30 b, microRNA30 c, microRNA214 and miRNA-183,miRNA-200 a expression in the exosomes of tubular epithelial cells. Results Under hypoxic condition, the secretion of renal tubular cells in vitro did not change significantly, and the secretion of exosome/cell protein content significantly increased after 48 hours of hypoxia than that in normoxia group. With the prolongation of hypoxia time, the expression of autophagy-related exosome gradually increased. The levels of autophagy-related miRNA-30 b, miRNA-30 c, miRNA-214, miRNA-183 and miRNA-200 a in the hypoxia group were higher than those in the normoxic group, and gradually increased with the prolongation of hypoxia. Conclusion Hypoxia can alter the biological function of renal tubular cell secretory exosome, which is manifested by up-regulation of autophagy-related microRNA expression. This may be one of the reasons for the important organ autophagy in patients with chronic kidney disease.