环状RNA circ-SOD2对肠上皮屏障和溃疡性结肠炎的作用  

Effects of circular RNA circ-SOD2 on intestinal epithelial barrier and ulcerative colitis

作　　者：王婷婷[1,2] 韩影 高芳芳[1] 叶磊[1] 张育军 WANG Ting-ting;HAN Ying;GAO Fang-fang;YE Lei;ZHANG Yu-jun(The Central Laboratory,Peking University People’s Hospital,Beijing,100044,China;Department of Gastroenterology,Peking University People’s Hospital,Beijing,100044,China)

机构地区：[1]北京大学人民医院中心实验室,北京100044 [2]北京大学人民医院消化内科,北京100044

出　　处：《北京大学学报（医学版）》2019年第5期805-812,共8页Journal of Peking University:Health Sciences

基　　金：北京大学人民医院研究与发展基金(RDX2018-05)~~

摘　　要：目的:探索溃疡性结肠炎(ulcerative colitis,UC)中环状RNA的表达谱改变,寻找在溃疡性结肠炎中表达发生明显异常的环状RNA,并探讨其对肠上皮细胞屏障功能的影响。方法:挑选5对UC患者炎症结直肠黏膜组织和正常黏膜组织进行环状RNA,芯片检测,筛选溃疡性结肠炎中表达发生改变的环状RNA。使用实时荧光定量PCR在30例UC患者炎症结直肠黏膜组织和正常黏膜组织中进一步验证表达发生明显上调的环状RNA circ-SOD2。使用炎症因子(LPS、TNF-α和IL1-β)刺激肠上皮细胞系Caco2、NMC460,检测circ-SOD2的表达改变。使用荧光原位杂交(fluorescence in situ hybridization,FISH)实验检测circ-SOD2在UC肠黏膜组织中的细胞定位。构建并合成circ-SOD2过表达载体,将其转染至Caco2细胞后检测Caco2细胞的跨上皮细胞电阻、FITC-右旋糖酐透过率,使用Western blotting检测上皮细胞屏障相关蛋白的表达改变。结果:环状RNA芯片检测后,通过限定条件(差异倍数>1.5,P<0.05),在溃疡性结肠炎受累的结直肠黏膜中共筛选出111个上调、153个下调的circRNA。限定筛选条件:(1)在circRNA表达谱芯片中原始信号值(raw data)>100;(2)差异表达倍数>2倍;(3)差异有统计学意义(P<0.05),筛选出10个差异明显的circRNA,并最终锁定在UC炎症肠黏膜组织中上调倍数最高的circ-SOD2。进一步扩大样本量在30对溃疡性结肠炎患者炎症和正常肠黏膜组织中使用荧光实时定量PCR技术进行验证,发现circ-SOD2表达明显上调(P<0.001);使用LPS、TNF-α、IL1-β刺激Caco2、NCM460细胞后,发现circ-SOD2在刺激后1~7 h的不同时间点均表达上调。荧光原位杂交实验表明circ-SOD2主要表达于肠黏膜组织的肠上皮细胞,而在间质和炎症细胞中表达较少。在Caco2细胞中过表达circ-SOD2后,跨上皮细胞电阻明显下降,FITC-右旋糖酐透过率明显上升,上皮细胞屏障分子闭合蛋白(claudin-8,CLDN-8)表达明显下降(P<0.05)。�Objective:To explore the expression profiling of circRNAs in ulcerative colitis(UC)and then determine the significantly changed circRNA and its influences on intestinal epithelial barrier.Methods:In this study,we selected 5 pairs of inflamed and normal colorectal mucosa tissues from UC patients to perform circRNAs microarray and identified the differentially expressed circRNAs in the UC inflamed colorectal mucosa tissues,and quantitative real-time PCR was used to identify the expression change of circ-SOD2 in 30 UC patients’inflamed and normal colorectal mucosa tissues.We detected the expression of circ-SOD2 in Caco2 and NCM460 cells after being treated with inflammatory factors(LPS,TNF-α,IL1-β).Fluorescence in situ hybridization(FISH)was used to determine the cellular location of circ-SOD2 in the UC colorectal mucosal tissues.The circ-SOD2 overexpression vector was constructed and produced and then transfected into Caco2 cells to examine the cells’trans-epithelial electrical resistance(TEER),permeability of FITC-dextran and the alterations of epithelial barrier related molecules.Results:We found 264 circRNAs(111 increased and 153 decreased)differentially expressed in the inflamed colon mucosa compared with normal colon mucosa using a P-value<0.05 and a>1.5-fold change cutoff.To validate the circRNA microarray results,we selected some circRNAs to perform qRT-PCR based on the following criteria:(1)circRNAs raw data>100 in each sample,(2)fold-change>2,(3)P<0.05.We identified 10 dysregulated circRNA,among them,circ-SOD2 was upregulated with maximum fold-change in the UC inflamed colorectal mucosa tissues.Then we identified circ-SOD2 was upregulated significantly through quantitative real-time PCR(qRT-PCR)in expanded 30 paired colorectal mucosa tissues(P<0.001).After treatments with LPS,TNF-αand IL1-β,circ-SOD2 was upregulated in Caco2 and NCM460 cells at different points from 1 to 7 h.Fluorescence in situ hybridization(FISH)indicated that circ-SOD2 located in intestinal epithelium mostly and few in mesenchym

关 键 词：环状RNA circ-SOD2 肠上皮屏障 溃疡性结肠炎 

分 类 号：R574.62[医药卫生—消化系统]