Cloning Of Pig Lgr5 And Its Effect On Intestinal Epithelial Cell Proliferation

Lgr5(Leucine-rich repeat-containing G-protein coupled receptor 5)is the biomarker of crypt-base columnar cells(CBCs),one of intestinal epithelial stem cells(IESCs).The objective of this study was to investigate the roles of Lgr5 in the proliferation of pig intestinal epithelial cells.In this study,the Lgr5 c DNA of pig was cloned and the structure and function of Lgr5 protein were predicted by bioinformatics analysis.The IPEC-J2 cells transfected with Lgr5-pc DNA3.1+ vector and overexpressed the Lgr5 protein.The cell count assay and MTT assay were performed to test the proliferation of Lgr5 overexpression IPEC-J2 cells.Western blot analysis was used to detect the regulatory effect of Wnt/β-catenin signaling pathway on cell proliferation.The IPEC-J2 cell proliferation and the key proteins of Wnt/β-catenin signaling pathway were detected after Wnt/β-catenin signaling pathway was activated by Wnt3 a or inhibited by XAV939.It laid the foundation for the study of pig IESCs marked by Lgr5.The results are as follows:(1)Cloning of pig Lgr5 cDNAPig Lgr5 c DNA sequence(2832 bp)containing a coding sequence(CDS,2724 bp)and a 3’ untranslated region(UTR,108 bp)was cloned by 3’ RACE,RT-PCR and overlapping PCR.(2)Bioinformatics analysis of pig Lgr5Pig Lgr5 protein was predicted to be a membrane protein containing a signal peptide and seven alpha helix transmembrane domains.The homology of the pig Lgr5 protein with the human Lgr5 protein is 90.30%.Phylogenetic analysis showed that Lgr5 is a conserved protein.(3)Identification of Lgr5 overexpression in IPEC-J2 cellsThe Lgr5-pc DNA3.1+ vector was constructed and the IPEC-J2 cell lines stably transfected with Lgr5-pc DNA3.1+ were obtained.The real-time PCR assay and Western blot analysis showed that Lgr5 m RNA abundance and protein level in the overexpression group were significantly higher than the control group at 72 h after seeding(P<0.05).(4)The effect of pig Lgr5 overexpression on IPEC-J2 cell proliferationThe cell count assay and MTT assay showed that the cell proliferation ability of the overexpression group significantly increased at 48,72 and 96 h after seeding(P<0.05).(5)Lgr5 overexpression activated Wnt/β-catenin signaling pathwayThe Western blot analysis showed that compared with the control group,levels of Axin2 and GSK-3β proteins were significantly increased(P<0.05)and β-catenin,c-Myc and cyclin D1 proteins were significantly decreased(P<0.05)in the overexpression group at 72 h after seeding.(6)Lgr5 promoted the proliferation of IPEC-J2 cells by activating the Wnt/β-catenin signaling pathwayThe cell proliferation activity of the Wnt3 a group significantly higher(P<0.05)than the control group at 48 h after treatment.The Western blot analysis showed that compared with the control group,levels of Axin2 and GSK-3β proteins in the Wnt3 a group were significantly decreased(P<0.05)and levels of β-catenin,c-Myc,cyclin D1 and Lgr5 proteins were markedly increased(P<0.05)at 48 h after treatment.The cell proliferation activity of the XAV939 group significantly lower(P<0.05)than the overexpression group at 24 h and 48 h after treatment.Compared with the overexpression group,levels of Axin2 and GSK-3β proteins were significantly increased(P<0.05)and levels of β-catenin,c-Myc,cyclin D1 and Lgr5 proteins were significantly decreased(P<0.05)in the XAV939 group at 48 h after treatment.Conclusion:(1)Pig Lgr5 c DNA containing CDS was first cloned.(2)Lgr5promotes the proliferation of pig intestinal epithelium cells by activating the Wnt/β-catenin signaling pathway.