Molecular Characterization And Functional Study Of Porcine APC1 And APC2 Genes

The adenomatous polyposis coli protein is a tumor suppressor gene of family adenomatous polyposis and colon cancer. APC mutations have a close relationship with the development of cancer. The APC protein is a multi-functional tumor suppressor protein family that regulation intra-cellular functions by binding with numerous proteins, including Axin-2, JUP, EB1, microtubules, etc. APC gene play a crucial role in the process of tumor suppression, cell proliferation and differentiation, cell apoptosis and cell migration, etc. APC plays a central role suppressing the Wnt signaling pathway by regulation the levels ofβ-catenin that controls cell proliferation and differentiation in the intestines, skin, immune system, bone, brain, etc.Adding linseed-source polyunsaturated fatty acids of ration to feed growth fattening swine, the result display that it increase muscle weight and internal fat content. We detected a series of differential expressed of gene, and the variability of APC gene is distinguished, so we choose the APC gene as research object. In this study, we identified and clone APC1 and APC2 genes of porcine APC gene family, comparative analyses to elucidate the structural and functional feature, promoter region, evolutionary relation and tissue expression profile of APC1 and APC2 genes, and mapped the APC1 and APC2 genes onto relevant chromosomes in pigs. We isolated porcine adipose tissue-derived stem cells, and studied the effect ofω-3 polyunsaturated fatty acids andω-6 polyunsaturated fatty acids on the relative expression levels of APC1 and APC2 genes. We also studied the effect of APC gene family in adipocyte differentiation. The detailed results are as follows:1. We performed the method of in silico cloning to get APC1 and APC2 genes cDNA sequences of porcine APC1 and APC2 genes, and proofed the results by using RT-PCR. The result of research shows that the cDNA sequence of porcine APC1 gene with a length of 8829 bp, it contained an ORF with the longest length of 8367 bp and encodes 2788 piece amine acid. The structure of APC 1 protein contained the conservative structures, such as ARM repetitive sequence, APC basic structures, EB1 binding domain, etc. The cDNA sequence of porcine APC2 gene with a length of 7067 bp, it contained an ORF with the longest length of 6798 bp and encodes 2265 piece amine acid. The structure of APC2 protein contained the conservative structures, such as ARM repetitive sequence, APC tumor suppressor protein, APC basic structures, etc.2. According to pig genome physics map and using electron mapping, we located the porcine APC1 and APC2 genes on pig 2q24 and 2q11, respectively. According to available APC1 and APC2 protein sequence of species in GenBank database and using MEGA 4.1 software, we analysised the phylogenetic analysis tree of APC gene family, the result revealed a high level of conservation at the protein structure level.3. Using bioinformatics, we predicted the CpG island and transcriptional factor binding site of promoter region of porcine APC1 and APC2 genes. The result revealed that there is no CpG island in the promoter region of porcine APC1, there are two CpG island that located on 235-448 and 589-821 region of the promoter of porcine APC2 genes. The promoter region of porcine APC1 and APC2 genes contain a lot of transcriptional factor binding site relate to adipogenesis.4. The tissue expression profile of the porcine APC gene family was detected by semi-quantitative RT-PCR. Porcine APC1 and APC2 genes was detected in all ten tissues, the mRNA expression is widespread.5. We successfully isolated porcine adipose tissue-derived stem cells by enzyme digestion method, and detected the cell mark protein CD44 by immunohistochemistry and molecular biology. The result revealed that cell is porcine adipose tissue-derived stem cell.6. Porcine adipose tissue-derived stem cells was treatment withω-3 polyunsaturated fatty acids(a-LA) andω-6 polyunsaturated fatty acids(LA), we detected the effect ofω-3 polyunsaturated fatty acids and co-6 polyunsaturated fatty acids on the relative expression levels of APCl and APC2 genes. The result revealed that porcine APC genes family was up-regulated dependent on the concentrations ofω-3 polyunsaturated fatty acids andω-6 polyunsaturated fatty acids.7. Porcine adipose tissue-derived stem cells are induced to adipocyte by treatment with inducer. We detected the expression of APC1 and APC2 genes in the differentiation process of adipocyte, the result revealed that the high expression of APC genes in the prophase of adipocyte differentiation. And we detected the expression ofβ-catenin, GSK3β, Axin gene that consisted in Wnt signaling pathway, the result revealed that the mRNA and protein level ofβ-catenin is down-regulation in the differentiation process of adipocyte. The expression tendency of GSK3βis coincidental with APC gene, and the expression tendency of Axin and APC is contrary. APC plays a regulation role in adipogenesis by regulation the levels ofβ-catenin.