Transcriptome Profile Analysis of Intestinal Upper Villus Epithelial Cells and Crypt Epithelial Cells of Suckling Piglets

SIMPLE SUMMARY: To investigate the genetic reprogramming that drives intestinal epithelial cells (IECs) maturation along the crypt-villus axis, entCerocytes were sequentially isolated from the villus tip to the crypts of porcine small intestine. The present study obtained the intestinal upper villus epithelial cells (F1) and crypt epithelial cells (F3) of 21-day suckling piglets using the divalent chelation and precipitation technique. By mRNA high-throughput sequencing analysis of F1 and F3, it was shown that a total of 672 unigenes were differentially expressed between F1 and F3, including 224 highly expressed and 448 minimally expressed unigenes. The greatest number of differentially expressed genes enriched in signal transduction, e.g., Wnt, Hippo, TGF-beta, mTOR, PI3K-Akt, and MAPK signaling pathways, were closely related to the differentiation, proliferation, maturation and apoptosis of IECs. Our results provide important information for identifying new regulators of IECs differentiation and maturation. Moreover, they can also provide insights into the regulatory mechanisms underlying intestinal epithelial cell renewal and the rapid repair of intestinal mucosal oxidative damage. ABSTRACT: It is well known that the small intestinal epithelial cells of mammals rapidly undergo differentiation, maturation, and apoptosis. However, few studies have defined the physiological state and gene expression changes of enterocytes along the crypt-villus axis in suckling piglets. In the present study, we obtained the intestinal upper villus epithelial cells (F1) and crypt epithelial cells (F3) of 21-day suckling piglets using the divalent chelation and precipitation technique. The activities of alkaline phosphatase, sucrase, and lactase of F1 were significantly higher (p < 0.05) than those of F3. To explore the differences at the gene transcription level, we compared the global transcriptional profiles of F1 and F3 using RNA-seq analysis technology. A total of 672 differentially expressed genes (DEGs) were identified between F1 and F3, including 224 highly expressed and 448 minimally expressed unigenes. Functional analyses indicated that some DEGs were involved in the transcriptional regulation of nutrient transportation (SLC15A1, SLC5A1, and SLC3A1), cell differentiation (LGR5, HOXA5 and KLF4), cell proliferation (PLK2 and TGFB3), transcriptional regulation (JUN, FOS and ATF3), and signaling transduction (WNT10B and BMP1), suggesting that these genes were related to intestinal epithelial cell maturation and cell renewal. Gene Ontology (GO) enrichment analysis showed that the DEGs were mainly associated with binding, catalytic activity, enzyme regulator activity, and molecular transducer activity. Furthermore, KEGG pathway analysis revealed that the DGEs were categorized into 284 significantly enriched pathways. The greatest number of DEGs enriched in signal transduction, some of which (Wnt, Hippo, TGF-beta, mTOR, PI3K-Akt, and MAPK signaling pathways) were closely related to the differentiation, proliferation, maturation and apoptosis of intestinal epithelial cells. We validated the expression levels of eight DEGs in F1 and F3 using qRT-PCR. The present study revealed temporal and regional changes in mRNA expression between F1 and F3 of suckling piglets, which provides insights into the regulatory mechanisms underlying intestinal epithelial cell renewal and the rapid repair of intestinal mucosal damage.