Huang Cong, Zheng Yi, Chen Yuanzhen, Cheng Yuchang, Jiang Ying, Cai Miaoyan, Song Dan
Department of Nephrology, Shenzhen Guangming New District People's Hospital Shenzhen, China.
Central Laboratory, Shenzhen Guangming New District People's Hospital Shenzhen, China.
Int J Clin Exp Pathol. 2019 Jan 1;12(1):344-355. eCollection 2019.
Diabetic nephropathy (DN) is a leading cause of end-stage renal disease worldwide. microRNAs (miRNAs) have been reported to play essential roles in DN progression. However, the mechanism of miR-216a-5p on DN progression is still unclear.
A DN model was established in human mesangial cells (HMC) by high glucose treatment. Cell proliferation was investigated using the cell counting kit-8 (CCK-8) assay. The cell cycle was measured through a propidium iodide (PI) cell cycle kit with flow cytometry. The interaction between miR-216a-5p and forkhead boxO1 (FoxO1) was probed by a bioinformatics analysis and luciferase activity assay. The expression of miR-216a-5p was detected using a quantitative real-time polymerase chain reaction (qRT-PCR). The abundances of FoxO1 and cell cycle-related cyclinD1, cyclin-dependent kinase 4 (CDK4), CDK6 and p27 were examined by qRT-PCR and Western blots (WB).
miR-216a-5p was up-regulated while FoxO1 was down-regulated in DN tissues. Moreover, miR-216a-5p promoted cell proliferation by regulating the cell cycle in high glucose-treated HMC cells. Notably, FoxO1 was a direct target and negatively correlated with miR-216a-5p. In addition, miR-216a induced cyclinD1, CDK4 and CDK6 but inhibited p27 expressions at the mRNA and protein levels. Furthermore, FoxO1 restoration reversed the regulatory effect of miR-216a on the cell cycle by regulating cyclinD1, CDK4, CDK6 and p27 abundances at the mRNA and protein levels.
miR-216a-5p is ectopic in DN and it promotes cell proliferation through regulating the cell cycle by targeting FoxO1 in high glucose-stimulated HMC cells, indicating it may serve as a novel biomarker for DN treatment.
糖尿病肾病（DN）是全球终末期肾病的主要病因。据报道，微小RNA（miRNA）在DN进展中起重要作用。然而，miR-216a-5p在DN进展中的机制仍不清楚。
通过高糖处理在人系膜细胞（HMC）中建立DN模型。使用细胞计数试剂盒-8（CCK-8）检测法研究细胞增殖。通过碘化丙啶（PI）细胞周期试剂盒和流式细胞术测量细胞周期。通过生物信息学分析和荧光素酶活性检测探究miR-216a-5p与叉头框O1（FoxO1）之间的相互作用。使用定量实时聚合酶链反应（qRT-PCR）检测miR-216a-5p的表达。通过qRT-PCR和蛋白质印迹法（WB）检测FoxO1以及细胞周期相关的细胞周期蛋白D1、细胞周期蛋白依赖性激酶4（CDK4）、CDK6和p27的丰度。
在DN组织中，miR-216a-5p上调而FoxO1下调。此外，miR-216a-5p通过调节高糖处理的HMC细胞的细胞周期来促进细胞增殖。值得注意的是，FoxO1是miR-216a-5p的直接靶标，且与miR-216a-5p呈负相关。此外，miR-216a在mRNA和蛋白质水平上诱导细胞周期蛋白D1、CDK4和CDK6的表达，但抑制p27的表达。此外，恢复FoxO1可通过在mRNA和蛋白质水平上调节细胞周期蛋白D1、CDK4、CDK6和p27的丰度来逆转miR-216a对细胞周期的调节作用。
miR-216a-5p在DN中异位表达，并且在高糖刺激的HMC细胞中通过靶向FoxO1调节细胞周期来促进细胞增殖，表明它可能作为DN治疗的新型生物标志物。
