长非编码RNA(lncRNA)富含核的丰富转录物1(NEAT1)经常被发现失调,这有助于糖尿病相关的并发症。本研究旨在探讨敲除对小鼠肾小球系膜细胞(MMC)活力的影响,凋亡,糖尿病肾病(DN)体外模型中的炎症和纤维化。首先在高糖条件下培养SV40MES13MMC细胞系,建立体外MMCDN细胞模型。将Lnc-NEAT1shRNA或阴性对照shRNA转染到MMCDN细胞中,然后测量细胞活力,凋亡,炎症,纤维化和microRNA(miR)-124表达,lnc-NEAT1的已知靶标,使用细胞计数试剂盒-8,流式细胞术,ELISA,蛋白质印迹[Capain1(capn1),β-连环蛋白(CTNNB1),裂解的半胱天冬酶3,裂解的多聚(ADP核糖)聚合酶,纤连蛋白和胶原蛋白]和逆转录定量PCR(Capn1,CTNNB1,lnc-NEAT1,纤连蛋白,胶原蛋白和miR-124),分别。在救援实验中,将miR-124和阴性对照抑制剂共转染到lnc-NEAT1下调细胞中,随后细胞活力,凋亡,炎症,纤维化,测量capn1和CTNNB1表达。Lnc-NEAT1表达在高糖处理的细胞中与正常葡萄糖处理的细胞和渗透对照细胞相比增加,表明lnc-NEAT1在MMCDN细胞模型中过表达。在MMCDN单元模型中,lncRNA-NEAT1敲低增强了细胞凋亡,但降低了细胞活力和上清液中炎症细胞因子的分泌(IL-1β,IL-8,单核细胞趋化蛋白1和TNF-α),除了减少裂解物中纤维化标志物纤连蛋白和胶原蛋白I的表达。Lnc-NEAT1敲低增加miR-124表达。此外,miR-124抑制剂的转染减少了细胞凋亡,但增加了细胞活力,lnc-NEAT1下调的MMCDN细胞的炎症和纤维化。miR-124抑制剂转染也增加了Capn1和CTNNB1的表达水平。一起来看,本研究的结果表明,lnc-NEAT1敲低能够减弱MMC的活力,通过调节miR-124表达和下游Capn1/β-catenin信号通路来实现炎症和纤维化。因此,Lnc-NEAT1可作为DN的潜在治疗靶点。
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 in vitro model of diabetic nephropathy (DN). The SV40 MES13 MMC cell line was first cultured with high glucose to establish an in vitro 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.