Chang Jing, Fang Zhan, Wang Dan, He Haiyan, Sun Fang, Teng Jian, Yang Lina
Department of Nephrology, Yantaishan Hospital, Yantai,Shandong,264000, China.
Department of Nephrology, Yantai Yuhuangding Hospital,Yantai,Shandong，264000, China.
J Cardiovasc Pharmacol. 2022 Aug 19. doi: 10.1097/FJC.0000000000001234.
Diabetic nephropathy (DN) is the most common complication of diabetes mellitus. Although G protein subunit beta 4 (GNB4)-derived circular RNA (circ-GNB4; hsa_circ_0068087) is a promising candidate biomarker in diabetes mellitus, whether circ-GNB4 participates in DN occurrence and development remains unknown. Herein, we focused on DN-associated human renal mesangial cells (HRMCs) injury, and HRMCs were exposed in high glucose (HG) condition. Using quantitative polymerase chain reaction and western blotting, we found that circ-GNB4 and early growth response factor 1 (EGR1) were upregulated, whereas microRNA (miR)-23c was downregulated in DN patients' sera and HG-stimulated HRMCs. HG-induced injuries were measured by MTS method, western blotting, enzyme-linked immunosorbent assay and other special assay kits. Consequently, HG could inhibit superoxide dismutase activity, but induce cell proliferation and levels of malondialdehyde, Fibronectin, Collagen I, Collagen IV, interleukin-6, interleukin-1β, and tumor necrosis factor-α. However, HG-induced these injuries were overall suppressed by silencing circ-GNB4 or overexpressing miR-23c. Moreover, miR-23c knockdown could counteract the effect of circ-GNB4 deficiency, and EGR1 restoration abrogated miR-23c overexpression role in HG-stimulated HRMCs. Notably, circ-GNB4 could target miR-23c and EGR1 was targeted by miR-23c, as confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. Moreover, EGR1 expression was positively modulated by circ-GNB4 via miR-23c. Collectively, circ-GNB4 might be a novel mechanism of DN-induced HRMCs injury, and there was a circ-GNB4/miR-23c/EGR1 pathway underlying the proliferation, extracellular matrix accumulation, inflammation and oxidative stress. This study suggested circ-GNB4 as a potential target to interfere the development of DN.
糖尿病肾病（DN）是糖尿病最常见的并发症。尽管G蛋白亚基β4（GNB4）衍生的环状RNA（circ-GNB4；hsa_circ_0068087）是糖尿病中有前景的候选生物标志物，但circ-GNB4是否参与DN的发生和发展仍不清楚。在此，我们聚焦于与DN相关的人肾小球系膜细胞（HRMCs）损伤，并将HRMCs暴露于高糖（HG）条件下。通过定量聚合酶链反应和蛋白质印迹法，我们发现circ-GNB4和早期生长反应因子1（EGR1）上调，而微小RNA（miR）-23c在DN患者血清和HG刺激的HRMCs中下调。通过MTS法、蛋白质印迹法、酶联免疫吸附测定和其他特殊检测试剂盒测量HG诱导的损伤。结果，HG可抑制超氧化物歧化酶活性，但诱导细胞增殖以及丙二醛、纤连蛋白、I型胶原、IV型胶原、白细胞介素-6、白细胞介素-1β和肿瘤坏死因子-α的水平。然而，通过沉默circ-GNB4或过表达miR-23c可总体上抑制HG诱导的这些损伤。此外，miR-23c敲低可抵消circ-GNB4缺乏的作用，而EGR1恢复可消除miR-23c过表达在HG刺激的HRMCs中的作用。值得注意的是，双荧光素酶报告基因测定和RNA免疫沉淀证实，circ-GNB4可靶向miR-23c，且EGR1被miR-23c靶向。此外，circ-GNB4通过miR-23c正向调节EGR1表达。总的来说，circ-GNB4可能是DN诱导的HRMCs损伤的新机制，并且在增殖、细胞外基质积累、炎症和氧化应激方面存在circ-GNB4/miR-23c/EGR1通路。本研究表明circ-GNB4是干扰DN发展的潜在靶点。
