Single-cell RNA sequencing revealed the impact on intestinal epithelial cell homeostasis in mice with intestinal epithelial-specific knockout of Becn2

Abstract:

Objective To establish intestinal epithelial-specific knockout mice of the autophagy protein gene Becn2, and to investigate the regulatory effect of intestinal epithelial-specific Becn2 deletion on intestinal epithelial cell homeostasis. Methods Becn2flox/-/Villin-Cre− mice were mated with Becn2−/−/Villin-Cre + mice. After multiple rounds of screening, control mice (Becn2fl) with the genotype Becn2flox/flox/ Villin-Cre− and knockout mice (Becn2ΔIEC) with the genotype Becn2flox/flox/Villin-Cre + were obtained. Mouse genotypes were identified by PCR, and protein-level knockout efficiency was detected by Western blot. Body weight changes were recorded from 4 to 8 weeks of age. Mouse organs were harvested to compare colon length and spleen weight between Becn2fland Becn2ΔIEC mice. Histological changes in the colon and spleen were examined by hematoxylin-eosin (HE) staining. Single-cell RNA sequencing was performed on mouse colon tissues, and bioinformatic methods were used to analyze alterations in intestinal epithelial cell subsets and key differentially expressed genes in mouse colon tissues. Results PCR and Western blot confirmed the successful establishment of control Becn2fl and knockout Becn2ΔIEC mouse models. Statistical analysis of organ data showed no significant differences in body weight, colon length, or spleen index between knockout and control mice under physiological conditions. HE staining revealed intact structure and no inflammatory lesions in the colon and spleen of both groups. Single-cell RNA sequencing analysis revealed that, compared to control mice, the proportion of goblet cells among the intestinal epithelial cell subsets decreased from 36.0% to 26.5% in Becn2 knockout mice. Furthermore, differential gene expression analysis in colonic epithelial cells revealed a slight downregulation of the Muc2 gene (log2FC =-0.51). Conclusion Under physiological conditions, intestinal epithelial-specific knockout of Becn2 does not alter the colonic tissue architecture , but reduces the proportion of goblet cells and downregulates Muc2 gene expression in colonic epithelial cells, thereby remodeling the intestinal epithelial cell composition.