Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 7, 2019; 25(5): 584-599
Published online Feb 7, 2019. doi: 10.3748/wjg.v25.i5.584
Claudin-7 gene knockout causes destruction of intestinal structure and animal death in mice
Chang Xu, Kun Wang, Yu-Han Ding, Wen-Jing Li, Lei Ding
Chang Xu, Kun Wang, Yu-Han Ding, Wen-Jing Li, Lei Ding, Department of Oncology, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China
Author contributions: Ding L designed the study; Xu C performed the research and wrote the paper; Wang K and Ding YH fed the mice; Li WJ analysed the data.
Supported by the National Natural Science Foundation of China, No. 81372585 and No. 81772557; and Beijing Health System High Level Training Plan of Health Technical Personnel, No. 2014-3-048.
Institutional review board statement: The study was reviewed and approved by the Medical Ethics Committee of the Capital Medical University Affiliated Beijing Shijitan Hospital Institutional Review Board.
Institutional animal care and use committee statement: All protocols were carried out in accordance with relevant guidelines and regulations.
Conflict-of-interest statement: The authors declare no conflict of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Lei Ding, MD, PhD, Associate Professor, Chief Doctor, Surgeon, Surgical Oncologist, Department of Oncology, Beijing Shijitan Hospital, Capital Medical University, No. 10, Tieyi Road, Haidian District, Beijing 100038, China. dinglei1005@126.com
Telephone: +86-10-63926296 Fax: +86-10-63926296
Received: October 24, 2018
Peer-review started: October 25, 2018
First decision: November 29, 2018
Revised: January 10, 2019
Accepted: January 18, 2019
Article in press: January 18, 2019
Published online: February 7, 2019
Processing time: 99 Days and 1.3 Hours
Abstract
BACKGROUND

Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanism. Therefore, we constructed three lines of Claudin-7 knockout mice using the Cre/LoxP system.

AIM

To determine the function of the tumor suppressor gene Claudin-7 by generating three lines of Claudin-7 gene knockout mice.

METHODS

We crossed Claudin-7-floxed mice with CMV-Cre, vil1-Cre, and villin-CreERT2 transgenic mice, and the offspring were self-crossed to obtain conventional Claudin-7 knockout mice, conditional (intestinal specific) Claudin-7 knockout mice, and inducible conditional Claudin-7 knockout mice. Intraperitoneal injection of tamoxifen into the inducible conditional Claudin-7 knockout mice can induce the knockout of Claudin-7. PCR and agarose gel electrophoresis were used to identify mouse genotypes, and Western blot was used to confirm the knockout of Claudin-7. The mental state, body length, and survival time of these mice were observed. The dying mice were sacrificed, and hematoxylin-eosin (HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and proliferation markers.

RESULTS

We generated Claudin-7-floxed mice and three lines of Claudin-7 gene knockout mice using the Cre/LoxP system successfully. Conventional and intestinal specific Claudin-7 knockout mice were stunted and died during the perinatal period, and intestinal HE staining in these mice revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. The inducible conditional Claudin-7 knockout mice had a normal phenotype at birth, but after the induction with tamoxifen, they exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were also observed. Intestinal immunohistochemistry analysis showed abnormal expression and distribution of Ki67, and the normal intestinal proliferation balance was disrupted. The intestinal crypt size in inducible conditional Claudin-7 knockout mice was increased compared with control mice (small intestine: 54.1 ± 2.96 vs 38.4 ± 1.63; large intestine: 44.7 ± 1.93 vs 27.4 ± 0.60; P < 0.001).

CONCLUSION

The knockout of Claudin-7 in vivo causes extensive inflammation, atypical hyperplasia, and adenoma in intestinal tissue as well as animal death in mice. Claudin-7 may act as a tumor suppressor gene in the development of colorectal cancer.

Keywords: Claudin-7; Gene knockout; Inflammation; Adenomas; Colorectal carcinoma

Core tip: The intestinal tract of conventional and intestinal specific Claudin-7 knockout mice was characterized by extensive and severe inflammation. The development of inducible conditional knockout mice can control the knockout of Claudin-7 in a temporal and compartment specific manner and prolong the survival time of mice, which exhibited atypical hyperplasia and adenoma in the intestine. This study revealed the inhibitory role that Claudin-7 plays in colorectal inflammation and colorectal cancer.