| Enteritis is the most serious digestive and metabolic diseases in rabbitry. Classified by the origin of enteritis etiology, there were known pathogen enteritis enteritis (enteritis) and of unknown etiology enteritis (nonspecific enteritis). At present, genetic, immunologic, environmental factors generally sufferred from enteritis in rabbit, however the molecular mechanism of enteritis is still not well known or needed to further study. A few studies have presented those JAKs-STATs signaling pathway widely participated in cell proliferation, differentiation, survival, apoptosis, and mediated immunoregulation and tumorigenesis. Reportedly, IL-23R-JAKs-STAT3 signaling pathway played a critical role in IBD. In this study, we expect to detect candidate genes involved in the resistant to enteritis from JAKs and STATs gene families and to investigate the J AKs-STAT3 signaling pathway in the pathogenesis of rabbit enteritis. Mainly, we screen cSNP of JAK1, JAK2, TYK2, STAT1, STAT3 and STAT5a genes through 480 New Zealand rabbits (case 253 and control 227), and the association analysis between JAK1/STAT3 genes and the susceptibility to enteritis.And then we detect the expression level of JAK1/STAT3 gene in ileum and colon tissues from 60 low fiber induced rabbit enteritis group. The primary intestinal epithelial cells (IEC) system was indentified and the inflammatory model of rabbit IEC also was successfully constructed. Under the inflammatory cell mode, effective siRNA down-regulation of JAK1, JAK2 and TYK2 genes were detected and STAT3 protein expression levels were also examined by Western blot. Finally, the inflammatory model of IEC cells transfected with si-JAK1 and the control group were investigated with RAN-Seq. The molecular of mechanism of JAKs-STAT3 signaling pathway in rabbit enteritis was studied. The main results were as follows:1. Using purification of PCR products by direct sequencing, total 22 cSNP were detected from six members of JAKs and STATs two families, involved in JAK1 (3), JAK2 (1), TYK2 (5), STAT1 (5), STAT3 (4) and STAT5a (4). Among cSNP, JAK1 c.1421 C>T and TYK2 c.1477 C>T were non synonymous mutations, which caused amino acid change in p.Thr 474 Met (T>M) and p.Leu 404 Phe (L>F), respectively. The rest of the cSNP are synonymous mutation. According to nonsynonymous and synonymous mutation analysis, JAKl (c.1421, c.3036) and STAT3 (c.399, c.831), a total of four cSNP were selected to association analysis. The case-control association analysis revealed that in JAK1 allele C (c.1421) and G (c.3036) were the risk allele and protective allele, respectively, but in STAT3 allele G (c.399) and C (c.831) were protective allele and the risk allele, respectivley. The multifactor dimensionality reduction (MDR) method was used to evaluate genetic interactions between JAK1 and STAT3. Data on the genetic interactions indicated that the JAK1 and STAT3 risk alleles described above contribute to enteritis susceptibility in combination with each other, and the model (c1421, c3036, c831) was the best model (OR:2.7262; 95% CI:4.7408-5.1986, P<0.0001).2. Through test induced by low fiber diet, fluorescence quantitative PCR (qPCR) experiment indicated that mRNA exression levele of JAK1 (c.1421 C>T) in severe group of ileum and colon were 0.2398 ± 0.0145 and 0.1835 ± 0.0175, respectively. mRNA expression level of JAK1 gene in severe group of the ileum was extremely signifiecanted with that of the healthy group (P=0.0056) and significanted with the mild inflammation group (P=0.039). mRNA expression level of CC genotype in the ileum reached significant level with CT and TT genotype, respectively (P=0.031, P=0.0082). However mRNA expression level of CC genotype in the colon was significanted with the TT genotype (P=0.026). mRNA expression level of STAT3 (c.399 G>A) in severe inflammation group of the ileum and colon were 0.3598 ± 0.0249 and 0.2881 ± 0.0671, respectively. mRNA expression level of STAT3 gene in severe inflammation group of the ileum was significanted with that of the healthy group and the mild inflammation group, respectively (P=0.0089, P=0.013), and the difference between health group and mild inflammation group also reached significant level (P=0.045). mRNA expression level of AA genotype was significanted with GG and AG genotype (P=0.036, P=0.029, respectively), but the difference of mRNA expression level between AG and GG genotype was not significantly difference (P=0.219).3. Rabbit primary IEC culture system was construced with collagenase (0.2 mg/ml) and purified according to different digestion and adherent times. And then the purity more than 95% was identified by CK-18 monoclonal antibody. The purified IEC culture system added to a final concentration of 1000 ng/ml LPS 24 h, and then using the ABC-ELISA method to detect two classic early acute inflammatory cytokines IL-1β and TNF-α. The results elucidated that expression levels of these two cytokines were higher than those in the control group (P<0.0I) and IEC inflammatory models of rabbits with LPS was successfully constructed.4. The optimal Lip2000 transfection system was determined through positive control si-Pcontrol-GAPDH and negative control si-Ncontrol 05815 marked with Cy3. Using the optimal Lip2000 transtection system, we obtained three siRNAs which could down-regulate JAK1, JAK2 and TYK2 gene expression levles, respectively (si-JAK1-001, si-JAK2-001 and si-TYK2-003). In order to obtain similar transfection efficiency (70%), we adjust the final concentration of siRNA. And then the siRNAs were transfected into IEC inflammatory models. qPCR and Westeren blot were applied to analysis the mRNA expression levels and p-STAT3 (STAT3) protein expression levels. The results showed JAK1, JAK2 and TYK2 downregulated 76.34%,23.89% and 42.14% at STAT3 gene mRNA expression level, respectively, and downregulated 0.71%,0.24% and 0.35% at p-STAT3 expression level, respectively, suggesting JAK1 palyed a key role in STAT3 Tyr705 phosphorylation.5. IEC inflammation cell model transfected with si-JAKl-001 and the control group were sequenced by RNA-Seq technology. The sequencing data revealed ideal sequencing randomness assessment and high quality data were confirmed using RNA fragmentation strategy to contruct sequencing library, which approximately 40.05% genes could match to the reference gene and approximately 65% gene of coverage reach to 90-100%. Gene expression difference analysis found that 491 up-regulated genes and 780 down-regulated genes in two repeat sequencing, involved in response to stress, response to stimulation of steroid hormone and the development of the immune system and other biological processes. Furthermore, RNA-Seq proved again that JAK1 gene could effectively silence by si-JAKl-001. Pathway enrichment analysis of DEGs revealed some genes in the JAKs-STATs signaling pathway were significanted with immune response from IEC inflammatory models of rabbits, and selectively down-regulate expression levels of STATI, STAT3, and STAT5b genes,In this study, the significant relationship between JAKs/STATs genes and susceptibility to enteritis in rabbit was revealed. Moreover, JAK1 played a key role in STAT3 Tyr705 phosphorylation, which aggravates the enteritis of rabbit. This parer provides the reference basis for the molecular breeding of rabbit and development of JAKs and STATs kinase inhibitors. |
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