Zhao Na, Du Likun, Ma Yingli, Wang Yang, Ma Jian, Fang Zhaohui
Department of Endocrinology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, P.R. China.
School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, P.R. China.
Exp Ther Med. 2022 Jun 9;24(2):507. doi: 10.3892/etm.2022.11434. eCollection 2022 Aug.
Long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) has been frequently found to be dysregulated, which contributes to diabetes-related complications. The present study aimed to explore the effect of knockdown on mouse mesangial cell (MMC) viability, apoptosis, inflammation and fibrosis in an model of diabetic nephropathy (DN). The SV40 MES13 MMC cell line was first cultured with high glucose to establish an MMC DN cell model. Lnc-NEAT1 shRNA or the negative control shRNA were transfected into MMC DN cells, followed by the measurement of cell viability, apoptosis, inflammation, fibrosis and microRNA (miR)-124 expression, a known target of lnc-NEAT1, using Cell Counting Kit-8, flow cytometry, ELISA, western blotting [Capain1 (capn1), β-catenin (CTNNB1), cleaved caspase 3, cleaved poly-(ADP ribose) polymerase, fibronectin and Collagen] and reverse transcription-quantitative PCR (Capn1, CTNNB1, lnc-NEAT1, fibronectin, collagen and miR-124), respectively. In rescue experiments, the miR-124 and negative control inhibitor were co-transfected into lnc-NEAT1-downregulated cells, following which cell viability, apoptosis, inflammation, fibrosis, capn1 and CTNNB1 expression were measured. Lnc-NEAT1 expression was increased in high glucose-treated cells compared with that in normal glucose-treated cells and osmotic control cells, suggesting that lnc-NEAT1 is overexpressed in the MMC DN cell model. In the MMC DN cell model, lncRNA-NEAT1 knockdown enhanced cell apoptosis but reduced cell viability and the secretion of inflammatory cytokines in the supernatant (IL-1β, IL-8, monocyte chemotactic protein 1 and TNF-α), in addition to reducing the expression of fibrosis markers fibronectin and collagen I in the lysates. Lnc-NEAT1 knockdown increased miR-124 expression. Furthermore, transfection with the miR-124 inhibitor reduced cell apoptosis but increased cell viability, inflammation and fibrosis in lnc-NEAT1-downregulated MMC DN cells. miR-124 inhibitor transfection also increased the expression levels of Capn1 and CTNNB1. Taken together, the findings of the present study demonstrated that lnc-NEAT1 knockdown was able to attenuate MMC viability, inflammation and fibrosis by regulating miR-124 expression and the Capn1/β-catenin signaling pathway downstream. Therefore, Lnc-NEAT1 may serve as a potential therapeutic target for DN.
长链非编码RNA（lncRNA）核富集丰富转录本1（NEAT1）经常被发现表达失调，这与糖尿病相关并发症有关。本研究旨在探讨在糖尿病肾病（DN）模型中敲低lncRNA对小鼠系膜细胞（MMC）活力、凋亡、炎症和纤维化的影响。首先将SV40 MES13 MMC细胞系用高糖培养以建立MMC DN细胞模型。将Lnc-NEAT1短发夹RNA（shRNA）或阴性对照shRNA转染到MMC DN细胞中，随后分别使用细胞计数试剂盒-8、流式细胞术、酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法[组织蛋白酶1（capn1）、β-连环蛋白（CTNNB1）、裂解的半胱天冬酶3、裂解的聚（ADP核糖）聚合酶、纤连蛋白和胶原蛋白]以及逆转录定量聚合酶链反应（RT-qPCR）（Capn1、CTNNB1、lnc-NEAT1、纤连蛋白、胶原蛋白和miR-124）检测细胞活力、凋亡、炎症、纤维化以及lnc-NEAT1的已知靶标微小RNA（miR）-124的表达。在拯救实验中，将miR-124和阴性对照抑制剂共转染到lnc-NEAT1表达下调的细胞中，随后检测细胞活力、凋亡、炎症、纤维化、Capn1和CTNNB1的表达。与正常葡萄糖处理的细胞和渗透压对照细胞相比，高糖处理的细胞中Lnc-NEAT1表达增加，表明lnc-NEAT1在MMC DN细胞模型中过表达。在MMC DN细胞模型中，敲低lncRNA-NEAT1可增强细胞凋亡，但降低细胞活力以及上清液中炎性细胞因子（白细胞介素-1β、白细胞介素-8、单核细胞趋化蛋白1和肿瘤坏死因子-α）的分泌，此外还可降低裂解物中纤维化标志物纤连蛋白和I型胶原蛋白的表达。敲低Lnc-NEAT1可增加miR-1中的表达。此外，用miR-124抑制剂转染可降低lnc-NEAT1表达下调的MMC DN细胞的凋亡，但增加细胞活力、炎症和纤维化。转染miR-124抑制剂还可增加Capn1和CTNNB1的表达水平。综上所述，本研究结果表明，敲低lnc-NEAT1能够通过调节miR-124表达以及下游的Capn1/β-连环蛋白信号通路来减弱MMC的活力、炎症和纤维化。因此，Lnc-NEAT有可能成为DN的潜在治疗靶点。
