2022, Vol. 26 ›› Issue (32): 5119-5125

Characteristic changes of intestinal mucosal morphology and transcriptomics in silent information regulator 6 gene knockout mice

You Yihui1, Song Chunhui1, Chen Xi1, Yao Xiaohong1, Ke Junyu1, Du Qun1, Song Ning2, Li Yanwu1   

Abstract: BACKGROUND: Intestinal mucosal renewal is a key physiological process to maintain body homeostasis, and stem cells at the crypt base are the driving force for intestinal epithelial proliferation and differentiation. Silent information regulator (SIRT) is involved in the regulation of cell gene repair, metabolism, energy balance, and lifespan. Research on its role in intestinal epithelial homeostasis has gradually become a hot spot. Intestinal organoids are formed by 3D culture of single crypt stem cells, which can present the intestinal epithelial renewal and repair process after damage in vitro. It is the latest model for studying intestinal mucosal renewal.
OBJECTIVE: To elucidate the characteristic changes of intestinal mucosal morphology, intestinal organoid formation and its transcriptomics in a SIRT6 gene-specific knockout (SIRT6-i/-i) mouse model.
METHODS: The Cre-loxp method was used to establish the SIRT6-i/-i mouse mode. Paraffin sections of small intestine tissue were prepared to observe the pathological changes using hematoxylin-eosin staining, and the expression of intestinal stem cell marker Lgr5 and Paneth cell marker Lyso was detected by immunohistochemistry. The intestinal organoid SIRT6-i/-i mouse model was constructed in vitro, SIRT6 protein expression in intestinal organoids was detected by western blot assay, and the protein expression of Lgr5, cytokeratin 20 and mucin 2 in intestinal organoids was detected by immunofluorescence staining. The intestinal tissue was used for transcriptome sequencing, and the characteristic changes of the transcriptome in the mouse model were analyzed.
RESULTS AND CONCLUSION: Compared with the control group, SIRT6-i/-i mouse intestinal mucosa showed sparse and shortened villi, shallower crypts, increased number of goblet cells, significantly decreased number of Paneth cells, and significantly reduced expression of Lgr5 and Lyso. The expression of SIRT6 protein in the SIRT6-i/-i mouse organoid was significantly reduced. The budding rate of organoids and the expression of Lgr5 were reduced, while the expression of absorption epithelial cell marker cytokeratin 20 and goblet cell marker mucin 2 increased obviously. Transcriptome results showed that there were 846 differentially expressed genes between the control and model mouse intestine, including 438 up-regulated genes and 408 down-regulated genes. The top five differentially expressed genes enriched in the Kyoto Encyclopedia of Genes and Genomes pathway were chemical carcinogens, drug metabolism, retinoic acid metabolism, linoleic acid metabolism, and steroid biosynthesis. And the differentially expressed genes in the top five pathways focusing on the Cyp450 family included Cyp2c29, Cyp2c65, Cyp3a11, and Cyp3a25. To conclude, SIRT6 gene deletion can lead to the imbalance of intestinal stem cell proliferation and differentiation, and the mechanism may be related to the metabolic regulation pathways of SIRT6. Regulating cytochrome P450 family by SIRT6 is closely related to the homeostasis of intestinal mucosal renewal.
Key words: SIRT6, gene knockout, intestinal organoid, intestinal mucosa, intestinal stem cell, transcriptome, mouse