The Wnt/β-catenin signaling pathway is aberrantly activated in most colorectal cancers. High-dose 1,25(OH)2D3 has anticancer effect by regulating Wnt signal pathway. However, it is not clear whether high-dose of 1,25(OH)2D3 have an effect on normal cells. The aim of the present study was to investigate the mechanism of high-dose 1,25(OH)2D3 on the Wnt signaling pathway in bovine intestinal epithelial cells. The potential mechanism of action was investigated after knockdown and overexpression of the Wnt pathway inhibitor, DKK2, in intestinal epithelial cells by observing the effects of 1,25(OH)2D3 on proliferation, apoptosis, pluripotency and the expression of genes related to the Wnt/β-catenin signaling pathway. In the present study, we introduced the method of isolation and culture of primary bovine intestinal epithelial cells. After cells were treated with 50 ng/mL 1,25(OH)2D3 or DMSO for 48 h, total RNA was extracted, and six differentially expressed genes, including SERPINF1, SFRP2, SFRP4, FZD2, WISP1 and DKK2 were identified by transcriptome sequencing, which were related to Wnt signaling pathway. To further explore the mechanism of 1,25(OH)2D3 on the Wnt/β-catenin signaling pathway, we constructed knockdown and overexpression plasmids of DKK2. After transfecting these plasmids into bovine intestinal epithelial cells, we measured the expression of DKK2 mRNA and protein through GFP expression, qRT-PCR and western blot analyses to verify the transfection efficiency. In addition, the CCK-8 assay was used to detect the cell proliferation rate after transfection. Subsequently, the transfected cells were treated with 1,25(OH)2D3 for 48 h, and the proliferation- (Ki67 and PCNA), apoptosis- (Bcl-2, p53, casp3 and casp8), pluripotency- (Bmi-1, Lrig1, KRT19 and TUFT1) and Wnt/ β-catenin signaling pathway- related genes (LGR5, DKK2, VDR, β- Catenin, SFRP2, WISP1 and FZD2) were detected by qRT-PCR and western blot analyses. Our results showed that the expression trend of some genes in bovine intestinal epithelial cells under high-dose 1,25(OH)2D3 was consistent with the sequencing results, including SFRP2 (P < 0.001), SFRP4 (P < 0.05), FZD2 (P < 0.01), WISP1 (P < 0.001) and DKK2 (P < 0.001). In addition, knockdown of DKK2 inhibited cell proliferation (P < 0.01), but DKK2 overexpression promoted cell proliferation (P < 0.01). Compared to the control group, 1,25(OH)2D3 promoted the expression of Wnt/β-catenin signaling pathway-related proteins in bovine intestinal epithelium, thus maintaining intestinal homeostasis in normal intestinal epithelium. In addition, knockdown and overexpression of DKK2 indicated that 1,25(OH)2D3 weakened the inhibitory effect of DKK2 on the Wnt/β-catenin signaling pathway. Together, these results suggest that high-dose 1,25(OH)2D3 has no killing effect on normal intestinal epithelial cells and regulates Wnt/β-catenin signaling pathway through DKK2.

BackgroundIntestinal health is an important guarantee for the healthy development of mammals. Therefore, the research on intestinal development and function continues to be a hot spot. And intestinal organoids are a hopeful alternative for the intestine, especially in large animals. In this study, we selected intestine of fetus calves as a target to study the differences in intestinal development and the development and functions of organoids in vitro, which were less reported in the past. The fetus intestine tissue is considered a preferred source for obtaining organoid because of its sterility and excellent developmental potential. MethodsWe compared the morphology of crypts and epithelial cells from duodenum, jejunum, ileum and colon at 4-6 month age fetus calves by HE staining, immunohistochemistry and transmission electron microscopy. And we cultured organoids from jejunum tissue and analyzed the structure by optical and electronic microscopy, immunological methods. Subsequently, through RNA sequencing, we confirmed the characteristics of gene expression between organoids and crypts. ResultsThe results showed the villus of jejunum and ileum was significantly higher than others, but the depth of the duodenum crypt was significantly deeper than the other two (P<0.001). And a large number of fissions were observed in duodenum crypts. In addition, intestinal epithelial stem cell markers LGR⁵ and CTNNB1 were highly expressed in the epithelium, but there was significant difference in villus and crypt of different intestinal segments. And By electron microscopy, we noticed that crypt stem cells were typically long and arranged in single and multiple layers closely, with a big nucleus and a large number of microvilli at the top of the cells. Each cell contained a large number of organelles, such as mitochondria and ribosomes. There are more free ribosomes in jejunum, ileum and colon crypt. Some Paneth cells and goblet cells were interspersed among crypt cells. There are typical tight junctions and adherent junctions (fingers cross pattern) between crypt cells. In addition, we obtained jejunum organoids by tissue culture possessing similar traits, such as microvillus and cell tight junctions. CTNNB1, PCNA and Lyso were highly expressed in the crypt cells around the surface of the organoids which hinted at the prominent capacity of cell proliferation. Furthermore, according to RNA sequencing, we found metabolism-related genes were significantly higher expression in organoids than in crypt stem cells (Q<0.001). On the contrary, immune and disease related genes were significant higher in crypt stem cells (Q<0.001). Finally, we found metabolic genes existed significantly differential (Q<0.001): crypt stem cells were biased towards energy metabolism (fructose and mannose) and vitamin metabolism(retinol), and organoids were biased towards amino acid process(glutathione metabolic process) and cholesterol process. This also implied that enteral nutrition absorption needed to be further processed in myofibroblasts except the crypt epithelium. ConclusionsIn short, different intestinal epithelial morphology, structure and development were different and related to function and organoids from jejunum. The organoids from jejunum were recommended as an ideal model for studying nutrient metabolism in cattle.