Department of Nephrology, the First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China.
Yunnan Key Laboratory of Laboratory Medicine, the First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China.
J Cell Physiol. 2019 Aug;234(10):17505-17513. doi: 10.1002/jcp.28373. Epub 2019 Feb 27.
We aimed to explore the regulatory relationship among the long noncoding RNA H19, micorRNA-675 (miR-675), the vitamin D (VD) receptor (VDR), and the early growth response protein 1 (EGR1) in the pathogenesis of diabetic nephropathy (DN) among patients with diabetes mellitus (DM).
Expression levels of H19, miR-675, VDR, and EGR in patients or CIHP-1/HEK 293 cells were measured via quantitative reverse-transcription polymerase chain reaction and western blot analysis. Computational analysis and luciferase assays were performed to determine EGR1 as a target gene of miR-675.
The relative expression of miR-675 was higher in the presence of H19, whereas the expression of both VDR and EGR1 messenger RNA was decreased in the presence of H19 or miR-675. However, relative expression of H19 and miR-675 was increased, whereas VDR expression was suppressed upon the treatment of 1,25-dihydroxyvitamin D3 or EGR1. VDR was identified as a target gene of miR-675. The H19 promoter and EGR1 increased the luciferase activity of cells transfected with wild-type VDR. Compared with DM patients free of DN, the levels of H19 and miR-675 were increased in the DN(+) group, whereas the levels of VDR and EGR1 were decreased.
In summary, the above results indicate the presence of a negative feedback loop in the pathological mechanism of DN, where H19 downregulates the expression of VDR by upregulating the expression of miR-675, whereas reduced VDR expression subsequently reduced the expression of EGR1. Moreover, reduced EGR1 expression inhibits H19 expression, thus forming a negative feedback loop required to maintain the homeostasis of VDR and to reduce the incidence of DN.
本研究旨在探讨长链非编码 RNA H19、微小 RNA-675（miR-675）、维生素 D（VD）受体（VDR）和早期生长反应蛋白 1（EGR1）在糖尿病患者糖尿病肾病（DN）发病机制中的调控关系。
采用实时定量逆转录聚合酶链反应和 Western blot 分析检测患者或 CIHP-1/HEK 293 细胞中 H19、miR-675、VDR 和 EGR1 的表达水平。通过计算分析和荧光素酶报告基因实验，确定 EGR1 是 miR-675 的靶基因。
在 H19 存在的情况下，miR-675 的相对表达量升高，而在 H19 或 miR-675 存在的情况下，VDR 和 EGR1 信使 RNA 的表达均降低。然而，1,25-二羟维生素 D3 或 EGR1 处理后，H19 和 miR-675 的相对表达量增加，而 VDR 表达受到抑制。VDR 被鉴定为 miR-675 的靶基因。H19 启动子和 EGR1 增加了转染野生型 VDR 的细胞的荧光素酶活性。与无 DN 的 DM 患者相比，DN（+）组中 H19 和 miR-675 的水平升高，而 VDR 和 EGR1 的水平降低。
综上所述，上述结果表明在 DN 的病理机制中存在负反馈环，其中 H19 通过上调 miR-675 的表达而下调 VDR 的表达，而 VDR 表达降低随后降低 EGR1 的表达。此外，降低的 EGR1 表达抑制 H19 的表达，从而形成维持 VDR 内稳态和降低 DN 发生率所需的负反馈环。
