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miR-4262/SIRT1轴在儿童炎症性肠病中发挥重要作用的潜在机制。

Mechanism underlying the significant role of the miR-4262/SIRT1 axis in children with inflammatory bowel disease.

作者信息

Deng Xiaozhi, Shang Lihong, Du Min, Yuan Lan, Xiong Lijing, Xie Xiaoli

机构信息

Department of Children's Gastroenterology, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan 611731, P.R. China.

出版信息

Exp Ther Med. 2020 Sep;20(3):2227-2235. doi: 10.3892/etm.2020.8918. Epub 2020 Jun 19.

Abstract

Inflammatory bowel disease (IBD) is a term used to describe chronic and recurrent gastrointestinal disease. In total, >2 million individuals worldwide have been diagnosed with IBD, including ulcerative colitis (UC), Crohn's disease (CD) and indeterminate colitis. There is accumulating evidence to indicate that microRNAs (miRNAs or miRs) are involved in the development and progression of IBD. miR-4262, an underlying promoter in tumor diseases, has been reported to regulate inflammatory responses. However, the potential mechanisms underlying the role of miR-4262 in IBD remain unknown. The present study attempted to explore the role and mechanisms of miR-4262 in IBD. Firstly, reverse transcription-quantitative PCR (RT-qPCR) was used to detect the expression of miR-4262 in 30 IBD colonic mucosa tissues, 30 normal tissues, 2% dextran sulfate sodium (DSS)-treated Caco-2 cells and normal cells. It was demonstrated that the expression levels of miR-4262 in IBD colonic mucosa tissues and 2% DSS-stimulated Caco-2 cells were markedly higher compared with those in the control groups. Target gene prediction databases and dual-luciferase reporter assays were then used, and sirtuin 1 (SIRT1) was identified as a target gene of miR-4262. Furthermore, the levels of SIRT1 in 2% DSS-stimulated Caco-2 cells and IBD colonic mucosa tissues were suppressed compared with the corresponding control groups. In addition, it was observed that miR-4262 negatively regulated SIRT1 expression in Caco-2 cells. Thereafter, Caco-2 cells were treated with inhibitor control, miR-4262 inhibitor, control-siRNA or SIRT1-siRNA for 48 h, followed by 2% DSS treatment for 4 days. The secretion of inflammatory factors was analyzed via ELISA and RT-qPCR. MTT assay, flow cytometry and western blot analysis were performed to assess cell viability, apoptosis and NF-κB signaling pathway-related protein levels, respectively. The results indicated that DSS enhanced the inflammatory response, suppressed cell viability and promoted cell apoptosis, and this was decreased following transfection with an miR-4262 inhibitor. In addition, 2% DSS upregulated p-p65 expression and enhanced the ratio of p-p65/p65, while the miR-4246 inhibitor exerted an opposite effect. All the effects of miR-4262 inhibitor on Caco-2 cells were eliminated following transfection with SIRT1-siRNA. It was thus concluded that miR-4262 may serve a role in the progression of IBD via targeting SIRT1, and miR-4262/SIRT1 may represent a potential target for the diagnosis and treatment of IBD.

摘要

炎症性肠病(IBD)是用于描述慢性复发性胃肠道疾病的术语。全球共有超过200万人被诊断患有IBD,包括溃疡性结肠炎(UC)、克罗恩病(CD)和未定型结肠炎。越来越多的证据表明,微小RNA(miRNA或miR)参与了IBD的发生和发展。miR-4262是肿瘤疾病中的一种潜在启动子,据报道可调节炎症反应。然而,miR-4262在IBD中作用的潜在机制仍不清楚。本研究试图探讨miR-4262在IBD中的作用及机制。首先,采用逆转录定量PCR(RT-qPCR)检测30例IBD结肠黏膜组织、30例正常组织、2%葡聚糖硫酸钠(DSS)处理的Caco-2细胞和正常细胞中miR-4262的表达。结果表明,与对照组相比,IBD结肠黏膜组织和2%DSS刺激的Caco-2细胞中miR-4262的表达水平明显更高。随后使用靶基因预测数据库和双荧光素酶报告基因检测,确定沉默调节蛋白1(SIRT1)为miR-4262的靶基因。此外,与相应对照组相比,2%DSS刺激的Caco-2细胞和IBD结肠黏膜组织中SIRT1的水平受到抑制。另外,观察到miR-4262在Caco-2细胞中负向调节SIRT1的表达。此后,将Caco-2细胞用抑制剂对照、miR-4262抑制剂、对照小干扰RNA(siRNA)或SIRT1-siRNA处理48小时,随后用2%DSS处理4天。通过酶联免疫吸附测定(ELISA)和RT-qPCR分析炎症因子的分泌。分别进行MTT法、流式细胞术和蛋白质免疫印迹分析以评估细胞活力、细胞凋亡和核因子κB(NF-κB)信号通路相关蛋白水平。结果表明,DSS增强了炎症反应,抑制了细胞活力并促进了细胞凋亡,而用miR-4262抑制剂转染后这种情况有所减轻。此外,2%DSS上调了磷酸化p65(p-p65)的表达并提高了p-p65/p65的比值,而miR-4262抑制剂则发挥相反作用。在用SIRT1-siRNA转染后,miR-4262抑制剂对Caco-2细胞的所有作用均被消除。因此得出结论,miR-4262可能通过靶向SIRT1在IBD的进展中发挥作用,且miR-4262/SIRT1可能是IBD诊断和治疗的潜在靶点。

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