The Effect Of Two Haplotypes In Bic Combined With Foxp3 On The Expression Of MiR-155 In Pig

Disease resistant breeding of the swine has been the key research content in the field of pig breeding,especially the immune differences between native and foreign pig breeds.From the perspective of genetics to study the disease resistant of native breeds is very important to breed new varieties of pig disease resistant.miR-155 plays a key role in immune balance and the immune system,and is widely involved in the physiological processes,including T cell activation,proliferation and differentiation.This study explores the different binding ability between SNP of the porcine miR155 host gene Bic intron and the transcription factor Foxp3 by Electrophoretic Mobility Shift Assay,At the individual level,we sorted Treg cells of two haplotypes by flow cytometry and extracted the total RNA for sequencing,and we did the functional clustering of gene expression profiles of two haplotypes.The effects of Poly I: C stimulation on miR155 expression and the regulation of the transcription factor Foxp3 were investigated.The specific results are as follows:(1)According to these two A/C mutations SNP loci of 305 bp and 168 bp of Pre-miR155 upstream of host gene Bic intron,two haplotypes of AA and CC individual were identified by PCR-RFLP in cultivated breeds-Hubei white pig.(2)Regulatory T cells(Treg)were isolated from two haplotypes of AA and CC with incubating CD3+CD4+CD25+ by flow cytometry,and the total RNA of Treg cells were extracted by microscale kit for sequencing.By analyzing the RNA-Seq data,we find that there were 126 genes related to immune system,122 genes were up-regulated in CC haplotype Treg cells,and 4 genes were down-regulated.Differences in gene cluster analysis showed that there were 23 genes associated with the immune response.Ten target genes of miR155 were found,among which three target genes were significantly different,and the other seven were not significantly different.The relative expression of SOCS1 and SLA-6 in CC haplotype Treg cells were higher than the AA haplotype Treg cells,while MYLK was not expressed in CC haplotype Treg cells.The proportion of ribosomal RNA obtained by sequencing was high and the effective data was not enough.(3)Two haplotypes individual were identified in Duroc × Erhualian F2 population.The Affymetrix expression profile was carried out to screen the different gene between genotype AA and genotype CC.By analyzing expression profile of Duroc × Erhualian F2 population in 33 d and 35 d,554 and 232 differentially genes of two haplotypes were detected respectively(P <0.05),and then we did the enrichment analysis of the differentially expressed genes,the important miR-155 functional regulation related genes including ETS2,ATF3,TGF?-1,TGFB1,IL15,SLA-DRB1,SOCS1 and CASP8 were screened.(4)The different binding ability of Foxp3 protein and the A/C fragment of the Bic gene was studied by Electrophoretic Mobility Shift Assay,the results showed that the binding ability of Foxp3 and genotype AA was stronger than that of genotype CC.(5)In the case of Poly I: C stimulation,we verified the expression of miR155 and the transcription factor Foxp3 on regulating the expression of miR155 on macrophage.The results showed that the expression of miR155-5p was up-regulated within 12 h,but had not significant difference;and after Poly I: C stimulation and Foxp3 overexpression we found a significant increase in the expression of miR155-5p.The above results of this research show that the SNP mutation on the host gene Bic intron is functional mutation,and related to the binding ability of transcription factor Foxp3.This research provides some theoretical basis for revealing the difference of immune ability between native pig breeds and foreign pig breeds,and provides molecular marker for disease resistant breeding.