Zhao Li, Chen Huaqian, Zeng Yan, Yang Kun, Zhang Ren, Li Zhengdong, Yang Tao, Ruan Hualing
Department of Nephrology, Affliated Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, P.R.China.
Department of Endocrinology, Affliated Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, P.R.China.
Endocr J. 2021 Oct 28;68(10):1155-1164. doi: 10.1507/endocrj.EJ20-0739. Epub 2021 May 11.
Diabetic nephropathy (DN), a frequent diabetes complication, has complex pathogenesis. Circular RNAs (circRNAs) circ_0000712 has been reported to be upregulated in kidney tissues and high glucose (HG)-inducted Mesangial cells (MCs). This study is designed to explore the role and mechanism of circ_0000712 in the HG-inducted MCs injury in DN. Circ_0000712, microRNA-879-5p (miR-879-5p), and SRY-Box Transcription Factor 6 (SOX6) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell apoptosis was examined by flow cytometry assay. Protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), fibronectin (FN), collagen type I (Col. I), collagen type IV (Col. IV), and SOX6 were assessed by western blot assay. Levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) generation, Lactate Dehydrogenase (LDH) activity, and Superoxide Dismutase (SOD) activity were detected by the corresponding kits. The binding relationship between miR-879-5p and circ_0000712 or SOX6 was predicted by starBase and Targetscan, and then verified by a dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. Circ_0000712 and SOX6 were highly expressed, and miR-879-5p was decreased in db/db DN mice and HG-inducted SV40-MES13 cells. Furthermore, circ_0000712 deficiency repressed HG-caused apoptosis, inflammation, oxidative stress, and fibrosis in SV40-MES13 cells. Mechanically, circ_0000712 could regulate SOX6 expression by sponging miR-879-5p. Circ_0000712 knockdown could hinder HG-inducted SV40-MES13 cell injury through targeting the miR-879-5p/SOX6 axis, implying a possible circRNA-targeted therapy for DN.
糖尿病肾病（DN）是一种常见的糖尿病并发症，其发病机制复杂。据报道，环状RNA（circRNA）circ_0000712在肾组织和高糖（HG）诱导的系膜细胞（MCs）中上调。本研究旨在探讨circ_0000712在DN中HG诱导的MCs损伤中的作用及机制。通过实时定量聚合酶链反应（RT-qPCR）检测circ_0000712、微小RNA-879-5p（miR-879-5p）和SRY盒转录因子6（SOX6）的水平。采用流式细胞术检测细胞凋亡。通过蛋白质免疫印迹法检测B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、纤连蛋白（FN）、I型胶原（Col. I）、IV型胶原（Col. IV）和SOX6的蛋白水平。采用酶联免疫吸附测定（ELISA）检测白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和肿瘤坏死因子α（TNF-α）的水平。使用相应试剂盒检测活性氧（ROS）生成、乳酸脱氢酶（LDH）活性和超氧化物歧化酶（SOD）活性。通过starBase和Targetscan预测miR-879-5p与circ_0000712或SOX6之间的结合关系，然后通过双荧光素酶报告基因和RNA免疫沉淀（RIP）实验进行验证。在db/db DN小鼠和HG诱导的SV40-MES13细胞中，circ_0000712和SOX6高表达，而miR-879-5p表达降低。此外，circ_0000712缺陷可抑制HG诱导的SV40-MES13细胞凋亡、炎症、氧化应激和纤维化。机制上，circ_0000712可通过海绵吸附miR-879-5p来调节SOX6表达。敲低circ_0000712可通过靶向miR-879-5p/SOX6轴来抑制HG诱导的SV40-MES13细胞损伤，这意味着circRNA靶向治疗DN具有潜在可能性。
