Wang Yao, Xiao Shumin, Zhou Saijun, Zhang Rui, Liu Hongyan, Lin Yao, Yu Pei
NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, China.
Front Physiol. 2020 Oct 19;11:552483. doi: 10.3389/fphys.2020.552483. eCollection 2020.
The underlying mechanisms by which diabetes and dyslipidemia contribute to diabetic nephropathy (DN) are not fully understood. In this study, we aimed to investigate the role of high glucose (HG) on intracellular cholesterol accumulation in glomerular endothelial cells (GEnCs) and its potential mechanism.
Oil red O staining, RT-qPCR, Western blotting, and immunocytofluorescence analyses were used to determine cholesterol accumulation and the expressions of LXRs and ABCA1 in GEnCs under high cholesterol (HC) and/or HG conditions, and the effect of these treatments was compared to that of low glucose without adding cholesterol. LncRNA microarrays were used to identify a long non-coding RNA (LncRNA OR13C9), of which levels increased in cells treated with the LXR agonist, GW3965. Fluorescence hybridization (FISH) was conducted to confirm subcellular localization of LncOR13C9 and a bioinformatics analysis was used to identify competing endogenous RNA (ceRNA) regulatory networks between LncOR13C9 and microRNA-23a-5p (miR-23a-5p). Gain and loss of function, rescue assay approaches, and dual-luciferase reporter assay were conducted to study interactions between LncOR13C9, miR-23a-5p, and ABCA1.
We showed that HG could decrease the response ability of GEnCs to cholesterol load, specifically that HG could downregulate LXRs expression in GEnCs under cholesterol load and that the decrease in LXRs expression suppressed ABCA1 expression and increased cholesterol accumulation. We focused on the targets of LXRs and identified a long non-coding RNA (LncOR13C9) that was downregulated in GEnCs grown in HG and HC conditions, compared with that grown in HC conditions. We speculated that LncRNAOR13C9 was important for LXRs to increase cholesterol efflux via ABCA1 under HC. Furthermore, using gain of function, loss of function, and rescue assay approaches, we showed that LncOR13C9 could regulate ABCA1 by inhibiting the action of miR-23a-5p in the LXR pathway. Furthermore, dual-luciferase reporter assay was conducted to study the interaction of LncOR13C9 with miR-23a-5p.
Overall, our study identified the LXRs/LncOR13C9/miR23A-5p/ABCA1 regulatory network in GEnCs, which may be helpful to better understand the effect of HG on cholesterol accumulation in GEnCs under cholesterol load and to explore new therapeutic tools for the management of DN patients.
糖尿病和血脂异常导致糖尿病肾病（DN）的潜在机制尚未完全明确。在本研究中，我们旨在探讨高糖（HG）对肾小球内皮细胞（GEnCs）内胆固醇蓄积的作用及其潜在机制。
采用油红O染色、RT-qPCR、蛋白质免疫印迹及免疫细胞荧光分析，检测高胆固醇（HC）和/或HG条件下GEnCs中胆固醇蓄积情况以及肝脏X受体（LXRs）和三磷酸腺苷结合盒转运体A1（ABCA1）的表达，并将这些处理的效果与未添加胆固醇的低糖处理效果进行比较。利用长链非编码RNA（LncRNA）芯片鉴定出一种长链非编码RNA（LncRNA OR13C9），其在LXR激动剂GW3965处理的细胞中表达水平升高。进行荧光原位杂交（FISH）以确认LncOR13C9的亚细胞定位，并采用生物信息学分析鉴定LncOR13C9与微小RNA-23a-5p（miR-23a-5p）之间的竞争性内源RNA（ceRNA）调控网络。采用功能获得和缺失、拯救实验方法以及双荧光素酶报告基因实验，研究LncOR13C9、miR-23a-5p和ABCA1之间的相互作用。
我们发现HG可降低GEnCs对胆固醇负荷的反应能力，具体而言，HG可下调胆固醇负荷条件下GEnCs中LXRs的表达，而LXRs表达的降低抑制了ABCA1的表达并增加了胆固醇蓄积。我们聚焦于LXRs的靶点，鉴定出一种长链非编码RNA（LncOR13C9），与在HC条件下生长的细胞相比，其在HG和HC条件下生长的GEnCs中表达下调。我们推测LncRNAOR13C9对于LXRs在HC条件下通过ABCA1增加胆固醇流出至关重要。此外，通过功能获得、缺失和拯救实验方法，我们发现LncOR13C9可通过抑制miR-23a-5p在LXR途径中的作用来调节ABCA1。此外，进行双荧光素酶报告基因实验以研究LncOR13C9与miR-23a-5p的相互作用。
总体而言，我们的研究在GEnCs中鉴定出LXRs/LncOR13C9/miR23A-5p/ABCA1调控网络，这可能有助于更好地理解HG对胆固醇负荷条件下GEnCs中胆固醇蓄积的影响，并为探索DN患者的新治疗手段提供帮助。
