Molecular cloning, sequence analysis, and function of the intestinal epithelial stem cell marker Bmi1 in pig...

In the present work, we cloned the full-length cDNA of the pig Bmi1 gene (BMI1 polycomb ring finger oncogene), which has been indicated as an intestinal epithelial stem cell (IESC) marker in other mammals. This paper provides the first report of the function of Bmi1 in pig intestinal epithelial cells and a brief description of its underlying mechanism. Rapid amplification of cDNA ends technology was used to clone the complete pig Bmi1 sequence, and a Bmi1-pcDNA3.1 vector was constructed for transfection into an intestinal porcine epithelial cell line (IPEC-1). The proliferation ability of the cells was estimated using the MTT assay and the EdU incorporation method at different time points after seeding. Cell cycle information was detected by flow cytometry. The mRNA abundances of cell cycle-related genes were also measured. The results indicated that the pig Bmi1 cDNA is 3,193 bp in length and consists of a 981 bp open reading frame, a 256 bp 5´ untranslated region (UTR), and a 1,956 bp 3' UTR. The transcript contains no signal peptides, and there are no transmembrane regions in the pig Bmi1 coded protein, which has a total of 326 AA. The overexpression of the pig Bmi1 in the IPEC-1 cells led to increased cell proliferation and a lower percentage of cells in the G1 and S phases (P < 0.05), along with a higher percentage of cells in the G2 phase (P < 0.05). Furthermore, the gene expression levels of PCNA, Cyclin D1, Cyclin D2, Cyclin B, CDK1, and CDK2 were all elevated (P < 0.05) by Bmi1 overexpression, while the gene expression levels of Cyclin A2 and p21 showed little difference (P > 0.05). Our data suggested that pig Bmi1 can increase the proliferation of IPEC-1 cells by promoting the G1/S transition and the overall cell cycle process.