Abstract：
Diabetic nephropathy (DN) is a serious microvascular complication of diabetes characterized by structural and functional changes of kidneys. Human renal tubular epithelial (HK-2) cells are important for kidney recovery post injury and usually used for establishment of DN cell models. The study explored the role of microRNA (miR)-133a-3p in DN cell model and animal model. A cell model for DN was established via high glucose (HG) stimulation to HK-2 cells. Cell viability and apoptotic rate were measured by cell counting kit 8 and flow cytometry. Polymerase chain reaction was performed to quantify levels of miR-133a-3p and targets. Luciferase reporter assay was conducted to verify the binding of miR-133a-3p and MAML1. After establishment of a mouse model of DN, levels of renal function indicators were measured by biochemical analysis. Hematoxylin-eosin and periodic acid-schiff staining of kidney samples were performed to analyze histological changes. Western blotting was conducted to quantify levels of apoptotic markers, MAML1, and factors related to Notch signaling. Results showed that HG induced HK-2 cell apoptosis and the reduction of cell viability. MiR-133a-3p was lowly expressed in HG-stimulated HK-2 cells. Overexpressed miR-133a-3p improved HK-2 cell injury by increasing cell viability and hampering apoptosis under HG condition. In addition, miR-133a-3p directly targets MAML1 3\'-untranslated region. MAML1 overexpression countervailed the repressive impact of miR-133a-3p on cell apoptosis in the context of HG. Moreover, miR-133a-3p inhibited the activity of Notch pathway by downregulating MAML1. MiR-133a-3p inhibits DN progression in mice, as evidenced by reduced fasting blood glucose level, improved levels of renal function parameters, and alleviation of kidney atrophy. In conclusion, miR-133a-3p improves HG-induced HK-2 cell injury and inhibits DN progression by targeting MAML1 and inactivating Notch signaling.
摘要：
糖尿病肾病（DN）是糖尿病的严重微血管并发症，其特征是肾脏的结构和功能改变。人肾小管上皮（HK-2）细胞对于损伤后肾脏的恢复很重要，通常用于建立DN细胞模型。本研究探讨微小RNA(miR)-133a-3p在DN细胞模型和动物模型中的作用。通过高糖(HG)刺激HK-2细胞建立DN的细胞模型。通过细胞计数试剂盒8和流式细胞术测量细胞活力和凋亡率。进行聚合酶链反应以定量miR-133a-3p和靶标的水平。进行荧光素酶报告基因测定以验证miR-133a-3p与MAML1的结合。建立DN小鼠模型后，生化分析检测肾功能指标水平。对肾脏样本进行苏木精-伊红和高碘酸-希夫染色以分析组织学变化。进行蛋白质印迹以定量凋亡标志物的水平，MAML1和与Notch信号相关的因素。结果表明，HG诱导HK-2细胞凋亡和细胞活力降低。MiR-133a-3p在HG刺激的HK-2细胞中低表达。在HG条件下，过表达的miR-133a-3p通过增加细胞活力和阻碍细胞凋亡来改善HK-2细胞损伤。此外,miR-133a-3p直接靶向MAML13'非翻译区。MAML1过表达抵消了miR-133a-3p在HG背景下对细胞凋亡的抑制作用。此外，miR-133a-3p通过下调MAML1抑制Notch通路的活性。MiR-133a-3p抑制小鼠DN进展，空腹血糖水平降低证明了这一点，改善肾功能参数水平，和减轻肾脏萎缩。总之,miR-133a-3p通过靶向MAML1和灭活Notch信号来改善HG诱导的HK-2细胞损伤并抑制DN进展。
