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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (5): 805-812. doi: 10.19723/j.issn.1671-167X.2019.05.003

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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)

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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

CLC Number: 

  • R574.62

Figure 1

The analysis of circRNA expression profile of UC A, circRNAs’ heat map, red strips represent the upregulated circRNAs and green strips represent the downregulated circRNAs in UC inflamed mucosa tissues; B, scatter plot, the two green lines above and below represent the critical value of 1.5 fold-change of circrnas expression in UC inflammatory intestinal mucosal tissues and normal tissues, the point beyond the critical line represents the circRNAs with abnormal expression change of >1.5 times; C, the top 5 upregulated and downregulated circRNAs in UC inflamed mucosa tissues, red strips represent the upregulated circRNAs and green strips represent the downregulated circRNAs."

Table 1

Top 10 differentially expressed 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

Figure 2

The expression change of circ-SOD2 in UC intestinal mucosa tissues and inflamed epithelial cells A, circ-SOD2 was significantly upregulated in UC inflamed colorectal mucosa tissues (n=30); B, the relation between circ-SOD2 expression and UC patients’ Mayo endoscopy subscore; C and D, circ-SOD2 was up-regulated in Caco2 and NCM460 cells stimulated by inflammatory factors (n=3). *P<0.05, # P<0.01, ★ P<0.001, ns, no significance."

Figure 3

The resource and cellular localization of circ-SOD2 and construction of circ-SOD2 overexpression vector A, circ-SOD2 is derived from the back-splicing of exon 3 and exon 5 of SOD2; B, circ-SOD2 is highly expressed in intestinal epithelial cells of UC colorectal mucosa tissues and the expression of circ-SOD2 in inflammatory tissues was significantly higher than that in normal tissues. Circ-SOD2 was labeled with cy3 red fluorescence, the nuclei were stained with DAPI, and the magnification was ×400; C, The schema graph of circ-SOD2 overexpression vector; D, circ-SOD2 expression was significantly up-regulated after the over-expression vector was transfected into Caco2 cells (n=3), *P<0.001."

Figure 4

The effect of circ-SOD2 overexpression on epithelial barrier of Caco2 cells A, the transepithelial electrical resistance (TEER) of Caco2 cells decreased significantly after circ-SOD2 overexpression; B, the FITC-dextran permeability increased in circ-SOD2 over-expressed Caco2 cells; C, the overexpression of circ-SOD2 caused that the expression of CLDN-8, the barrier molecule of Caco2 cells, was down-regulated, while the expression of ZO-1 and occludin was not changed (n=5). *P<0.05, # P<0.01, ns,no significance."

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