IL-21 mediates microRNA-423-5p /claudin-5 signal pathway and intestinal barrier function in inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) is a group of chronic and recurrent nonspecific inflammatory disorders, including Crohn's disease (CD) and ulcerative colitis (UC). Due to the persistent inflammation of intestinal mucosa caused by immune disorders, barrier dysfunction may be an essential cause of the pathogenesis of IBD. Therefore, exploring the mechanism is very important to clarify the pathogenesis of IBD. In our research, we provided evidence of IL-21 function in IBD. The junction complex protein claudin-5 may be a downstream gene of the IL-21. Anti-IL-21 administrated prevented DSS-simulative colitis via recovering claudin-5 expression in the human colonic epithelial cells. Meanwhile, we described that miR-423-5p could be involved in IL-21/ claudin-5 pathway by regulating NF-κB/MAPKs/JNK signaling pathway, which may provide a new therapeutic target for IBD.

Keywords: IBD; IL-21; claudin-5; inflammation; microRNA-423-5p.

Figure 1

Increased expression of IL-21 in UC patients. (A) The expression of IL-21 in colonic mucosa from UC patients and healthy volunteers. n=15. (B) The protein level of IL-21in human samples. n=5. (C) Representative images of immunohistochemistry staining with IL-21. (D) Changes in body weight in different groups of mice. n=10. (E) The protein level of IL-21 in different groups. n=10. (F) The mRNA level of IL1β, IL6, and TNFα in plasma. n=5. (G) The length of the colon in different groups of mice. (H) MPO activity detection in different groups of mice.n=5. (I) Serum FITC- dextran was used as a monitoring index of intestinal permeability. n=6. (J) The protein level of NF-κB/MAPKs/JNK signaling pathway components in different groups. n=5 *P < 0.05, **P < 0.01.

Figure 2

IL-21 regulates the intestinal epithelial TJ barrier function by targeting claudin-5. (A) The effect of IL-21 on intestinal epithelial barrier function. n=3. (B) RT-PCR was employed to detect the mRNA level of claudin-1, claudin-4, claudin-5, claudin-8, claudin-11, occludin, and ZO-1. n=7. (C) The effect of claudin-5 on TEER in Caco-2 cells. n=3. (D) The FD4 level was detected after claudin-5/vector transfection in IL-21 treated cells. n=5. (E, F) The protein level of claudin-5 in different groups. n=4. (G) Representative images of immunofluorescence staining with claudin-5. *P < 0.05, **P < 0.01.

Figure 3

MiR-423-5p could bind with the 3'UTR of claudin-5. (A) Binding sequences between miR-423-5p and claudin-5 (CLDN5) (http://www.targetscan.org/cgi-bin/targetscan/vert_72/view_gene.cgi?rs=ENST00000406028.1&taxid=9606&members=miR-423-5p&showcnc=1&shownc=1&shownc_nc=1&showncf1=&showncf2=&subset=1). The luciferase assay report. n=3. (B, C) The expression of claudin-5 in colorectal epithelial cell lines. n=4. (D) The level of miR-423-5p in colonic mucosa from UC patients and healthy volunteers. n=15. (E) Representative images of immunofluorescence staining with claudin-5. *P < 0.05, **P < 0.01

Figure 4

MiR-423-5p regulates the intestinal epithelial barrier function by targeting claudin-5. (A) The effect of miR-423-5p on intestinal epithelial barrier function. n=3. (B) RT-PCR was employed to detect the mRNA level of claudin-1, claudin-4, claudin-5, claudin-8, claudin-11, occludin, and ZO-1. n=6. (C) The effect of miR-423-5p and claudin-5 on TEER in Caco-2 cells. n=4. (D) The FD4 level was detected after claudin-5/vector transfection with miR-423-5p mimic/miR-NC. n=5. *P < 0.05, **P < 0.01.

Figure 5

The effect of miR-423-5p in DSS mice. (A) Changes in body weight in different groups of mice. n=10. (B) The mRNA level of IL1β, IL6, and TNFα in plasma. n=5. (C) The length of the colon in different groups of mice. (D) MPO activity detection in different groups of mice. n=4. (E) Serum FITC- dextran was used as a monitoring index of intestinal permeability. n=5. (F) Representative images of H&E staining. (G) The expression of claudin-5 in different groups. n=4. (H) Representative images of immunofluorescence staining with claudin-5. (I) The protein level of NF-κB/MAPKs/JNK signaling pathway components in different groups. n=5. *P < 0.05, **P < 0.01.

Figure 6

IL-21 regulates miR-423-5p in UC. (A) The expression of miR-423-5p in colorectal epithelial cell lines. n=8. (B) TEER was determined in Caco-2 cells. n=4. (C) The FD4 level was detected after si-miR-423-5p/si-NC transfection in IL-21 treated cells. n=5. (D) The expression of miR-423-5p in different groups. n=7. *P < 0.05, **P < 0.01.