| MicroRNAs (miRNAs), a class of small (about 22nt) endogenous small noncoding RNAs, mediate posttranscriptional regulation of protein-coding genes by binding chiefly to the 3 untranslated region of target mRNAs, miRNAs play an integral role in many different biological processes like cell proliferation, differentiation and apoptosis, neurodevelopment and fat metabolism. Recent studies have also revaled that miRNAs play an important roles in the regulation of the host response to bacterial and virus infection. Salmonella enterica serovar Enteritidis (SE) is recognized as food-borne pathogenic bacteria which has important public health significance. It caused severe inflammation of the gut mainly by invading the intestinal mucosa. Moreover, several previous studies have shown that the host genetic background plays an important role in response to SE infection, significantly different susceptibility has been observed between different chicken lines. Recent years, Solexa high-throuthput sequencing technology has become a powerful tool for microRNA analysis and identification of each species, some small RNAs have been demonstrated to have important functions in the regulation of the host response to bacterial and virus infection. However, the regulatory mechanism of miRNAs in SE infection of chicken has not been known.SE was used to infecte twenty-week old SE negative white leghorn layers in the current study. Cecum tissues was collected from the control and treat group 7 days post SE infection and used for sRNA libraries construction, in total, six sRNA libraries were constructed, Cp1,Cp2,Cp3 in the control group and Tp1,Tp2,Tp3 in the treatment group. Then the Illumina/Solexa high-throughput-sequencing technology was used to identify and analyze the differentially expressed miRNAs post SE infection. The differentially expressed miRNAs were further verified by quantitative real-time PCR. Meanwhile, quantitative real-time PCR used to analyze the expression of some different miRNAs and its target genes related to immune response at 14 days post SE infection in white leghorn layers and 7 days post SE infection in Shouguang chicken. The results showed that:(1) A total of 1972566,3017995,2244711,10673073,1852677 and 2708904 filtered clean data were obtained from six libraries, respectively, accounted for 17.724%,33.542%, 17.995%,58.832%,17.570% and 30,987% of total reads.(2) Through bioinformatics analysis,404 known chicken miRNA and 194 new predicted miRNAs were identified. The results of differentially expressed analysis showed that a total of 37 expressed microRNAs were significantly different expressed between treatment group and control group, in which there were 19 known miRNAs including 15 up-regulated and 4 down-regulated and 18 novel predicted miRNAs including 7 up-regulated and 11 down-regulated.(3) The targets of significantly differential expressed miRNAs were predicted using miRanda algorithm with the software Vienna package. In total,2,897 unique targets regulated by differentially expressed miRNAs were predicted including 636 target genes only regulated by down-regulated miRNAs(G1),841 target genes only regulated by up-regulated miRNAs(G2)and 1,420 target genes both regulated by up and down-regulated miRNAs(G3). The blast with the immune related genes in the Biomart indicated that 176 target genes were immune related genes.(4) DAVID was used to perform the functional analysis for the target genes. The results showed that 118 GO-BP terms were significantly enriched in the group 1,73 GO-BP terms were enriched in group 2 and 178 GO-BP terms were enriched in group3. Several immune related GO-BP terms were enriched in the three groups. The enriched pathways including ECM-receptor interaction, Glycolysis/Gluconeogenesis, Focal adhesion, Melanogenesis and Proteasome.(5) The quantitative analysis of twelve differentially expressed miRNA that randomly selected, the result shown that among 12 miRNAs, in the white leghorn layers at 14 days post infection 11 miRNAs verified were consistent with the results of the high-throughput sequencing. Four miRNAs were differentially expressed in the white leghorn layers at 14 days post infection and the regulation direct were all different with the sequencing result; 11 miRNAs were significantly down-regulated in the Shouguang chickens at 7 days post infection.(6) The analysis of the interaction of miRNA with the immune-regulated targets indicated that in the white leghorn layers at 7 days post infection, in the relationship of the gga-miR-1416 with TLR21?gga-miR-1416 with BCL10?gga-miR-1662 with TLR1LA? gga-miR-34a-5p with NOTCH and gga-miR-34a-5p with THBS1, the regulated direction of the miRNA was different with its targets; in the Shouguang chickens at 7 days post infection, in the relationship of gga-miR-1416 with IGJ?gga-miR-1416 with TLR21?gga-miR-1662 with TLR1LA and gga-miR-34a-5p with CCL4, the regulated direction of the miRNA was different with its targets, which showed the interaction.The results show that 37 miRNAs were differentially expressed in chickens 7 days post SE infection. The miRNAs related to the biological processes, such as inflammatory response, the immune system development, lymphocyte activity and NF-kB cascade and the pathways including ECM-receptor interaction, Glycolysis/Gluconeogenesis, Focal adhesion, Melanogenesis and Proteasome may play an important role in SE infection. Genetic background influence the interaction of microRNAs with target genes; Gga-miR-1416, gga-miR-34a-5p and gga-miR-1662 plays an important regulatory role in SE infection.This result herein will lay the foundation for the further study of regulatory mechanism of miRNAs in the response to SE infection. At the same time provide a new theoretical basis for the chicken disease molecular genetics and breeding. |
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