The Genetic Effects Of IGFBP-6on The Pig Body Size And Its Roles On The Cell Proliferation

Animal growth and development are a series of complex and sophisticatedbiological metabolic processe, the regulating mechanism of this process has alwaysbeen a hot topic. The Bama Xiang pig and the Xizang Tibetan pig are our preciousgermplasm resources of miniature pigs in China, but neither their dwarf traits nor themechanism of growth or development are unclear. In the animal growth anddevelopment systems, the regulation of the growth mainly through the neuroendocrineaxis of growth hormone (GH-IGF axis), in which insulin-like growth factor bindingprotein-6(IGFBP-6) is concerned because of its structure and function, as well as itsstrong affinity with insulin-like growth factor2(IGF-2).In this study, real-time fluorescence quantitative PCR was used to test theexpression levels of IGFBP-6gene mRNA in different tissues of The Bama Xiang pig,the Xizang Tibetan pig and Junmu No.1White pig at different growth stages.Meanwhile, in order to analyze the IGFBP-6action in the drawf mechanisms ofminiature pigs, the promoter and part of the IGFBP-6gene sequences were sequencedin The Bama Xiang pig, the Xizang Tibetan pig, Junmu No.1White pig, Large Whitepig and northeast wild boar to screened mutations and SNPs, and promoter oftranscription factor binding sites were predicted and analyzed. To study the functionsof IGFBP-6, mouse IGFBP-6shRNA vectors which would reduce IGFBP-6expression were reconstructed, the density and growth rate of the cells were detectedby MTT after transfection. The results in vitro will lay the foundation for theconstruction of IGFBP-6transgenic mouse model.The results of the expression levels of IGFBP-6gene mRNA suggests thatduring the same stages, expression patterns of IGFBP-6in the Bama Xiang pig andthe Xizang Tibetan pig are consistent among of different organizations, but differentfrom the Junmu No.1White pig. The mRNA expression levels of IGFBP-6gene in the liver of Junmu No.1White pigs at adult stage were higher than Xizang Tibetanpigs and Bama Xiang pigs (p<0.05) respectively; and in spleen, Junmu No.1Whitepigs at born stage were higher than Xizang Tibetan pigs and Bama Xiang pigs(p<0.01) respectively; this results indicated the different expression pattern ofIGFBP-6genes mRNA after birth were shown in different pig breeds with large orsmall body sizes.A cluster Sp1binding sites and GATA-1, GATA-2, Ik-2, NF-kap and SREBP-transcription factor binding sites were predicted in the5′-end of IGFBP-6geneindicating that the IGFBP-6may be associated with the occurrence and inhibition ofcancer and lipid metabolism. Two (A>G at-403bp and T>C at-488bp) of the sevenmutations in the5′-end of IGFBP-6gene may affect the expression of IGFBP-6. TheA>G at-403bp leads to a new a c-Myb binding site added, and T>C at-488bp ofnortheast wild boar leads to a MZF1binding site added, but a AP-1binding sites loss.The SNPs of promoter genotype frequencies analysis showed that at-726bp,-649bp,-403bp,-378bp may be related to body size of the Bama Xiang pig and the XizangTibetan pig. Linkage analysis of IGFBP-6promoter in Junmu No.1White pig, LargeWhite pig and northeast wild boar found that the two SNPs at-403bp and-378bpwere in linkage and formed three kinds of haplotypes, of which AT was as the mainhaplotype. In IGFBP-6gene, six mutations were detected, in which the A>G at67bpin exon4may related to body size of the Bama Xiang pig and the Xizang Tibetan pig.Linkage analysis found that the three SNPs at636bp and745bp in intron3and at67bp in exon4were in linkage and formed four kinds of haplotypes, of which CCTwas as the main haplotype.Cell proliferation studies of IGFBP-6showed that down regulation of IGFBP-6promotes hepa1-6cells proliferation to53.31%at24h post transfection. Theproliferation to20.22%of3T3cells were inhibited by the IGFBP-6reduction at48hpost transfection.Based on the above results, we concluded that IGFBP-6gene may be afunctional gene in the regulation of the miniature pig growth and development, and acandidate gene affecting on the drawfism of pigs, but its specific mechanism of actions in further studies are still necessary. This study provided molecular basis forthe drawfism study of pigs, and laid foundation for depth studies on the actions ofIGFBP-6on growth and development mechanism of miniature pigs, and provided atheoretical basis for exploring and application of miniature pigs’ resources.