Zhong Ling, Liao Guangneng, Wang Xiaojiao, Li Lan, Zhang Jie, Chen Younan, Liu Jingping, Liu Shuyun, Wei Lingling, Zhang Wengeng, Lu Yanrong
Key Lab of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
Department of Clinical and Experimental Medicine, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan 610072, China.
Exp Biol Med (Maywood). 2018 Nov;243(15-16):1233-1242. doi: 10.1177/1535370218819726. Epub 2019 Jan 6.
Microvesicles (MVs) from mesenchymal stem cells (MSCs) have been reported as a new communicated way between cells. This study evaluated the influence and underlying mechanism of MVs-shuttled miR-451a on renal fibrosis and epithelial mesenchymal transformation (EMT) in diabetic nephropathy (DN) with hyperuricemia. MVs were isolated from MSCs-cultured medium by gradient ultracentrifugation. The level of miR-451a in MSCs and MVs was analyzed by qPCR. The changes of miR-451a, E-cadherin, α-SMA, P15INK4b (P15), and P19INK4d (P19) were measured in hyperglycosis and hyperuricemia-induced cell (HK-2) and mouse models. The changes of cell cycle were analyzed by flow cytometry. The ability of proliferation and viability was measured by BrdU and CCK8, respectively. Dual-luciferase reporter assays were conducted to determine the target binding sites. The renal function and histological changes of mice were analyzed. MVs showed the same surface markers as MSCs but much higher miR-451a expression (4.87 ± 2.03 fold higher than MSCs). miR-451a was decreased to 26% ± 11% and 6.7% ± 0.82% in injured HK-2 cells and kidney, respectively. MV-miR-451a enhanced the HK2 cells proliferation and viability in vitro, and decreased the morphologic and functional injury of kidney in vivo. Moreover, infusion of MV-miR-451a reduced the level of α-SMA and raised E-cadherin expression. These effects were responsible for the improved arrested cell cycle and down-regulation of P15 and P19 via miR-451a targeting their 3′-UTR sites. This study demonstrated that MSC–MV-miR-451a could inhibit cell cycle inhibitors P15 and P19 to restart the blocked cell cycle and reverse EMT in vivo and in vitro, and thus miR-451a is potentially a new target for DN therapy.
The mechanism of MSCs repairing the injured kidney in diabetic nephropathy is not yet clear. In the research, MVs showed the same surface markers as MSCs but much higher MiR-451a expression. miR-451a was decreased in both injured HK-2 cells and kidneys. MV-miR-451a stimulated the cell proliferation and viability in vitro and promoted structural and functional improvements of injured kidney in vivo. Infusion of MV-miR-451a ameliorated EMT by reducing α-SMA and increasing E-cadherin. These effects relied on the improved cell cycle arrest and the down-regulation of P15 and P19 via miR-451a binding to their 3′-UTR region. This study demonstrated that MSC–MV-miR-451a could specifically inhibit cell cycle inhibitors to restart the blocked cell cycle and reverse EMT in vivo and in vitro. Therefore, miR-451a may be a new target for DN therapy.
据报道，间充质干细胞（MSC）来源的微泡（MV）是细胞间一种新的通讯方式。本研究评估了MV穿梭的miR-451a对伴有高尿酸血症的糖尿病肾病（DN）肾纤维化和上皮-间质转化（EMT）的影响及潜在机制。通过梯度超速离心从MSC培养基中分离MV。采用qPCR分析MSC和MV中miR-451a的水平。在高糖和高尿酸血症诱导的细胞（HK-2）及小鼠模型中检测miR-451a、E-钙黏蛋白、α-平滑肌肌动蛋白（α-SMA）、P15INK4b（P15）和P19INK4d（P19）的变化。通过流式细胞术分析细胞周期变化。分别采用BrdU和CCK8检测增殖能力和活力。进行双荧光素酶报告基因检测以确定靶结合位点。分析小鼠的肾功能和组织学变化。MV显示出与MSC相同的表面标志物，但miR-451a表达水平高得多（比MSC高4.87±2.03倍）。在受损的HK-2细胞和肾脏中，miR-451a分别降至26%±11%和6.7%±0.82%。MV-miR-451a在体外增强HK2细胞增殖和活力，并在体内减轻肾脏的形态和功能损伤。此外，输注MV-miR-451a可降低α-SMA水平并提高E-钙黏蛋白表达。这些作用是通过miR-451a靶向P15和P19的3′-UTR位点改善细胞周期阻滞以及下调P15和P19所致。本研究表明，MSC-MV-miR-451a可在体内外抑制细胞周期抑制剂P15和P19，重启受阻的细胞周期并逆转EMT，因此miR-451a可能是DN治疗的新靶点。
MSC修复糖尿病肾病中受损肾脏的机制尚不清楚。在本研究中，MV显示出与MSC相同的表面标志物，但miR-451a表达水平高得多。在受损的HK-2细胞和肾脏中miR-451a均降低。MV-miR-451a在体外刺激细胞增殖和活力，并在体内促进受损肾脏的结构和功能改善。输注MV-miR-451a通过降低α-SMA和增加E-钙黏蛋白改善EMT。这些作用依赖于通过miR-451a与P15和P19的3′-UTR区域结合改善细胞周期阻滞以及下调P15和P19。本研究表明，MSC-MV-miR-451a可在体内外特异性抑制细胞周期抑制剂，重启受阻的细胞周期并逆转EMT。因此，miR-451a可能是DN治疗的新靶点。
