Email Alert | RSS

北京大学学报（医学版） ›› 2019, Vol. 51 ›› Issue (5): 805-812. doi: 10.19723/j.issn.1671-167X.2019.05.003

• 论著 • 上一篇    下一篇

环状RNA circ-SOD2对肠上皮屏障和溃疡性结肠炎的作用

王婷婷1,2,韩影1,高芳芳1,叶磊1,张育军1,△()   

  1. 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
  • 收稿日期:2019-01-02 出版日期:2019-10-18 发布日期:2019-10-23
  • 通讯作者: 张育军 E-mail:zhangyujun@pkuph.edu.cn
  • 基金资助:
    北京大学人民医院研究与发展基金(RDX2018-05)

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

Ting-ting WANG1,2,Ying HAN1,Fang-fang GAO1,Lei YE1,Yu-jun ZHANG1,△()   

  1. 1. The Central Laboratory, Peking University People’s Hospital, Beijing, 100044, China
    2. Department of Gastroenterology, Peking University People’s Hospital, Beijing, 100044, China
  • Received:2019-01-02 Online:2019-10-18 Published:2019-10-23
  • Contact: Yu-jun ZHANG E-mail:zhangyujun@pkuph.edu.cn
  • Supported by:
    Supported by the Peking University People’s Hospital Research and Development Funds Project(RDX2018-05)

RICH HTML

 48   

PDF (PC)

298

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)。结论:溃疡性结肠炎中环状RNA表达出现明显异常,其中在UC中表达上调的circ-SOD2可减弱肠上皮屏障,进而可能导致溃疡性结肠炎发生。

关键词: 环状RNA, circ-SOD2, 肠上皮屏障, 溃疡性结肠炎

Abstract:

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-α and 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 mesenchyme and inflammatory cells. The overexpression of circ-SOD2 in Caco2 cells resulted in a decrease of transepithelial electrical resistance (TEER), an increase of the FITC-dextran permeability and the downregulation of epithelial barrier related molecule CLDN-8 (P<0.05). Conclusion: The dysregulation of circRNAs existed in UC inflamed colorectal mucosa, among which, the upregulated circ-SOD2 weakened the intestinal epithelial barrier and thus might promote the occurrence of ulcerative colitis.

Key words: circRNAs, circ-SOD2, Intestinal epithelial barrier, Ulcerative colitis

中图分类号: 

  • R574.62

图1

UC的环状RNA表达谱分析"

表1

差异显著的10个circRNA"

Rank circRNA identity Raw data (group) Fold change Host gene
Top 5 upregulated circRNAs
1 hsa_circ_0004662 598.9 3.4993729 SOD2
2 hsa_circ_0057090 1 399.5 3.0880182 PDK1
3 hsa_circ_0000992 1 122.6 2.8650179 PRKD3
4 hsa_circ_0002211 1 872.4 2.4547362 DDX17
5 hsa_circ_0006006 1 099.4 2.1420162 PDK1
Top 5 downregulated circRNAs
6 hsa_circ_0003915 434 3.3094226 SATB2
7 hsa_circ_0007422 553.8 2.8208505 SATB2
8 hsa_circ_0007919 430.8 2.4014379 ABR
9 hsa_circ_0001727 1 737.2 2.3361569 ZKSCAN1
10 hsa_circ_0008267 460.5 2.2679094 LINC00969

图2

circ-SOD2在UC肠黏膜组织及炎症细胞模型中的表达改变"

图3

circ-SOD2的来源、细胞定位及过表达载体构建"

图4

circ-SOD2过表达后对Caco2细胞屏障的影响"

[1] Magro FGP, Eliakim R, Ardizzone S , et al. Third European evidence-based consensus on diagnosis and management of ulcerative colitis. Part 1: definitions, diagnosis, extra-intestinal manifestations, pregnancy, cancer surveillance, surgery, and ileo-anal pouch disorders[J]. J Crohns Colitis, 2017,11(6):649-670.
[2] Ng SC, Shi HY, Hamidi N , et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies[J]. Lancet, 2017,390(10114):2769-2778.
[3] Costello CM, Mah N, Hasler R , et al. Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays[J]. PLoS Med, 2005,2(8):e199.
[4] Ventham NT, Kennedy NA, Nimmo ER , et al. Beyond gene discovery in inflammatory bowel disease: the emerging role of epigenetics[J]. Gastroenterology, 2013,145(2):293-308.
[5] Jeck WR, Sorrentino JA, Wang K , et al. Circular RNAs are abundant, conserved, and associated with ALU repeats[J]. RNA, 2013,19(2):141-157.
[6] Salzman J, Chen RE, Olsen MN , et al. Cell-type specifc features of circular RNA expression[J]. PLoS Genet, 2013,9(9):e1003777.
[7] Han D, Li J, Wang H , et al. Circular RNA circMTO1 acts as the sponge of microRNA-9 to suppress hepatocellular carcinoma progression[J]. Hepatology, 2017,66(4):1151-1164.
[8] Guarnerio J, Bezzi M, Jeong JC , et al. Oncogenic role of fusion-circRNAs derived from cancer-associated chromosomal translocations[J]. Cell, 2016,165(2):289-302.
[9] Liu Q, Zhang X, Hu X , et al. Circular RNA related to the chondrocyte ECM regulates MMP13 expression by functioning as a MiR-136 ‘Sponge’ in human cartilage degradation[J]. Sci Rep, 2016,6:22572.
[10] Iparraguirre L, Munoz-Culla M, Prada-Luengo I , et al. Circular RNA profiling reveals that circular RNAs from ANXA2 can be used as new biomarkers for multiple sclerosis[J]. Hum Mol Genet, 2017,26(18):3564-3572.
[11] Qiao YQ, Cai CW, Shen J , et al. Circular RNA expression alterations in colon tissues of Crohn’s disease patients[J]. Mol Med Rep, 2019,19(5):4500-4506.
[12] Yuan G, Chen T, Zhang H , et al. Comprehensive analysis of differential circular RNA expression in a mouse model of colitis-induced colon carcinoma[J]. Mol Carcinog, 2018,57(12):1825-1834.
[13] Piwecka M, Glazar P, Hernandez-Miranda LR , et al. Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function[J]. Science, 2017,357(6357):8526.
[14] Min M, Peng L, Yang Y , et al. MicroRNA-155 is involved in the pathogenesis of ulcerative colitis by targeting FOXO3a[J]. Inflamm Bowel Dis, 2014,20(4):652-659.
[15] Wang H, Chao K, Ng SC , et al. Pro-inflammatory miR-223 mediates the cross-talk between the IL23 pathway and the intestinal barrier in inflammatory bowel disease[J]. Genome Biol, 2016,17:58.
[16] He C, Yu T, Shi Y , et al. MicroRNA 301A promotes intestinal inflammation and colitis-associated cancer development by inhi-biting BTG1[J]. Gastroenterology, 2017,152(6):1434-1448.
[17] Qu S, Yang X, Li X , et al. Circular RNA: a new star of non-coding RNAs[J]. Cancer Lett, 2015,365(2):141-148.
[18] Glažar P, Papavasileiou P, Rajewsky N . circBase: a database for circular RNAs[J]. RNA, 2014,20(11):1666-1670.
[19] Kramer MC, Liang D, Tatomer DC , et al. Combinatorial control of drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins[J]. Genes Dev, 2015,29(20):2168-2182.
[20] Gerasimenko TN, Senyavina NV, Anisimov NU , et al. A model of cadmium uptake and transport in Caco-2 cells[J]. Bull Exp Biol Med, 2016,161(1):187-192.
[21] Mirza AH, Berthelsen CH, Seemann SE , et al. Transcriptomic landscape of lncRNAs in inflammatory bowel disease[J]. Genome Med, 2015,7(1):39.
[22] Wu F, Huang Y, Dong F , et al. Ulcerative colitis-associated long noncoding RNA, BC012900, regulates intestinal epithelial cell apoptosis[J]. Inflamm Bowel Dis, 2016,22(4):782-795.
[23] Hammond SM . An overview of microRNAs[J]. Adv Drug Deliv Rev, 2015,87:3-14.
[1] 刘艳霞，杨雪松，付卫，姚宏伟. 单核苷酸多态性位点rs6983267与溃疡性结肠炎及大肠癌的相关性[J]. 北京大学学报(医学版), 2016, 48(6): 994-999.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] 田增民, 陈涛, Nanbert ZHONG, 李志超, 尹丰, 刘爽. 神经干细胞移植治疗遗传性小脑萎缩的临床研究（英文稿）[J]. 北京大学学报（医学版）, 2009, 41(4): 456 -458 .
[2] 郭岩, 谢铮. 用一代人时间弥合差距——健康社会决定因素理论及其国际经验[J]. 北京大学学报（医学版）, 2009, 41(2): 125 -128 .
[3] 成刚, 钱振华, 胡军. 艾滋病项目自愿咨询检测的技术效率分析[J]. 北京大学学报（医学版）, 2009, 41(2): 135 -140 .
[4] 卢恬, 朱晓辉, 柳世庆, 郑杰, 邱晓彦. 白细胞介素2促进宫颈癌细胞系HeLaS3免疫球蛋白G的表达[J]. 北京大学学报（医学版）, 2009, 41(2): 158 -161 .
[5] 袁惠燕, 张苑, 范田园. 离子交换型栓塞微球及其载平阳霉素的制备与性质研究[J]. 北京大学学报（医学版）, 2009, 41(2): 217 -220 .
[6] 徐莉, 孟焕新, 张立, 陈智滨, 冯向辉, 释栋. 侵袭性牙周炎患者血清中抗牙龈卟啉单胞菌的IgG抗体水平的研究[J]. 北京大学学报（医学版）, 2009, 41(1): 52 -55 .
[7] 董稳, 刘瑞昌, 刘克英, 关明, 杨旭东. 氯诺昔康和舒芬太尼用于颌面外科术后自控静脉镇痛的比较[J]. 北京大学学报（医学版）, 2009, 41(1): 109 -111 .
[8] 祁琨, 邓芙蓉, 郭新彪. 纳米二氧化钛颗粒对人肺成纤维细胞缝隙连接通讯的影响[J]. 北京大学学报（医学版）, 2009, 41(3): 297 -301 .
[9] 李宏亮*, 安卫红*, 赵扬玉, 朱曦. 妊娠合并高脂血症性胰腺炎行血液净化治疗1例[J]. 北京大学学报（医学版）, 2009, 41(5): 599 -601 .
[10] 李伟军, 邢晓芳, 曲立科, 孟麟, 寿成超. PRL-3基因C104S位点突变体和CAAX缺失体的构建及表达[J]. 北京大学学报（医学版）, 2009, 41(5): 516 -520 .
ISSN 1671-167X
CN 11-4691/R
主管单位：中华人民共和国教育部
主办单位：北京大学
地址： 北京市海淀区学院路38号 北京大学医学部 《北京大学学报（医学版）》
邮编：100191
电话：010-82801551
Email： xbbjb2@bjmu.edu.cn

《北京大学学报（医学版）》
微信公众号
版权所有 © 2018 《北京大学学报（医学版）》编辑部