circRNA_Atp8a1 Promotes Glycolytic Reprogramming in Damage of Intestinal Mucosal Barrier by Upregulating IGF2 through miR-200b-3p.

Abstract

Background: This study investigated a circRNA (Circ_Atp8a1) in regulating intestinal epithelial repair in intestinal mucosal barrier damage.

Methods: A mouse model of intestinal mucosal barrier damage caused by burn injury was constructed. Skin and intestinal histopathologic changes in injured and control mice were compared. Glycolytic enzyme protein expression, lactate production, and glucose consumption in intestinal tissues were detected. Microarray analysis was used to screen differentially expressed circRNAs in mucosal tissues, and RT-qPCR, Sanger sequencing, RNAse R test, nucleoplasmic isolation experiments, and fluorescence in situ hybridization (FISH) were used to characterize the circular structure and localization of Circ_Atp8a1. In Caco-2 cells, adenoviral overexpression vector and small interfering RNA (siRNA) were constructed to regulate Circ_Atp8a1 expression. Cell proliferation and migration were detected by combining with the experiments of CCK-8, EdU, wound healing, and Transwell. The interaction between Circ_Atp8a1 and miR-200b-3p was investigated by dual luciferase reporter assay, RNA pull-down assay, and FISH assay. The target gene of miR-200b-3p was predicted and validated. Finally, the effects of intraperitoneal injection of KD-Circ_Atp8a1 and OE-Circ_Atp8a1 on intestinal mucosal damage in burned mice were observed by in vivo experiments.

Results: Mice with burn-induced intestinal mucosal damage had higher CMDI scores, increased expression of glycolytic enzymes in intestinal tissues, and altered glycolytic processes. A total of 308 aberrantly expressed circRNAs were screened, among which Circ_Atp8a1 was significantly down-regulated and mainly distributed in cytoplasm and jejunal crypts. In Caco-2 cells, overexpression of Circ_Atp8a1 inhibited cell proliferation, migration, and glycolysis, and knockdown of Circ_Atp8a1 did the opposite. Circ_Atp8a1 acted as a sponge for miR-200b-3p, which targeted and inhibited IGF2, which affected glycolysis-related metrics. Circ_Atp8a1 regulated IGF2 indirectly through miR-200b-3p, which in turn regulated intestinal mucosal damage. in vivo experiments showed that overexpression of Circ_Atp8a1 could inhibit miR-200b-3p expression, promote IGF2 expression, reduce intestinal mucosal damage and decrease mucosal permeability.

Conclusion: Circ_Atp8a1 plays a key regulatory role in the process of intestinal mucosal damage and affects the process of glycolysis through adsorption of miR-200b-3p to regulate IGF2. It is expected to be a new target for the treatment of intestinal mucosal damage.