Mechanism Of Caudal Type Homeobox Transcription Factor 2 Promoting Pig Intestinal Epithelial Cell Proliferation

CDX2(caudal type homeobox transcription factor 2),an intestinal specific transcription factor,regulates intestinal epithelium cells proliferation and differentiation by modulatingintestine genes expression.The objective of this study was to investigate the mechanism of CDX2 on the proliferation of porcine jejunal epithelium cells(IPEC-J2).Firstly,IP EC-J2 cells,transfected with CDX2-pcDNA3.1+ vector or pcDNA3.1+ vector,were used as models.The effect of CDX2 overexpression on cell proliferation weredetermined by cell counting assay and MTT.Then,CDX2 interference fragment were designed and synthesized to investigate the effect of CDX2 knockdown on cell proliferation.In the end,the specific antagonists of mTOR and Wnt signaling pathways,Rapamycin and XAV939,were used to explorethe role of mTOR and Wnt signaling pathways on the CDX2-mediated cell proliferation.Western blot was used to analyze the expression of related proteins under each treatment.Thus,it is expected to laid foundation for the comprehensive understanding of the funcation of CDX2in piglet intestine.The results were as follows:(1)The results showed that the proliferation ability of EPEC-J2 cells were significantly increased(P<0.05)when tranfected with CDX2-pcDNA3.1+ vector and significantly decreased(P<0.05)after CDX2 knockdown.(2)Compared with the null vectorgroup,the overexpression group markedly increased(P<0.05)the protein levels of p-mTOR(Ser2448),p-S6K1(Thr389)/p-S6(Ser235),p-4EBP1(Thr70)/eIF4E,c-Myc,Cyclin D1 and ?-catenin.Interestingly,the phosphorylation of mTOR(Ser2448),followed with its downstream key proteins,c-Myc,Cyclin D1 and?-cateninwere significantly down-regulated(P<0.05)in the knockdown group compared with negativecontrol group.(3)The mTOR and Wnt pathway specific antagonists,Rapamycinand XAV939,both suppressed the proliferation of CDX2-overexpression IPEC-J2 cells(F<0.05),with the combination of Rapamycinand XAV939 group leading an additive effect(P<0.05).(4)Compared with the DMSOgroup,Rapamyicn significantlydecreased(P<0.05)the protein levels of p-mTOR(Ser2448),p-S6K1(Thr389)/p-S6(Ser235),p-4EBP1(Thr70)/eIF4E,c-Myc,Cyclin D1,?-catenin and CDX2 so did XAV939(P<0.05)and the combination of Rapamycinand XAV939(P<0.05).Conclusion:(1)CDX2 promotes the proliferation of pig intestinal epithelium cells via activating the mTOR and Wnt signaling pathway.(2)Both mTOR and Wnt pathways participate in thesynthesis of CDX2.