Differential Expression Analysis And Functional Verification Of MiRNA In The Small Intestine Of Laying Ducks Induced By Caged Stress

Cage rearing mode has improved the efficiency of duck production,biosecurity prevention and control and manure treatment,breaking the bottleneck of the healthy development of duck farming,and is gradually being used in the industry.Micro RNAs（miRNAs）are 20-24 nt,non-coding,highly conserved endogenous RNA molecules that are widely found in eukaryotic cells.miRNAs play an important role in the growth and development of the animal intestine,the proliferation and differentiation of enterocytes and the repair of intestinal barrier damage.They play an important role in the growth and development of the animal intestine,the proliferation and differentiation of intestinal cells and the repair of intestinal barrier damage.There are few reports on the mechanism of miRNAs on the repair of intestinal damage in duck cage culture,and few reports on the function of target genes of differentially expressed miRNAs during intestinal damage in ducks.Therefore,in this study,we analyzed the role of differentially expressed miRNAs on the post-transcriptional regulation of target genes by mapping the multi-omics expression profile of duck cage stress intestinal tissues,and analyzed the response mechanism of differentially expressed miRNAs involved in intestinal mucosal injury in duck cage stress.The study consisted of two main parts as follows.Experiment 1:The aim of this experiment was to investigate the effects of cage stress on growth performance and intestinal morphology in ducks,and to map the expression profiles of proteins and miRNAs in intestinal mucosal damage using TMT protein sequencing and high-throughput transcriptome sequencing analysis.Sixty-four 70-day-old Nonghu No.2ducks were randomly assigned to the flat-rearing group（TB）and the cage-rearing group（CR）.Tissue paraffin sections were used to observe intestinal villus structure,multi-omics sequencing to detect intestinal tissue protein and miRNA expression abundance,and q PCR and WB to detect target miRNA and protein expression.The results showed that the weight gain and feed intake of ducks in the CR group were significantly lower compared with those in the TB group（P<0.05）.Paraffin sections of small intestine tissues showed that compared with the TB group,the CR group showed damage to the villus structure,with a break in the upper and middle part of the villus,and a significant decrease in the ratio of villus height and villus height to crypt depth（P<0.05）and a significant increase in crypt depth（P<0.05）.miRNA high-throughput sequencing yielded 21 differentially expressed miRNAs,of which 6were up-regulated and 15 were down-regulated.TMT protein sequencing yielded 45differentially expressed proteins,of which 23 were up-regulated and 22 were down-regulated.KEGG analysis of differentially expressed proteins showed that differentially expressed proteins between TB and CR groups were mainly enriched in MAPK signaling pathway,PPAR signaling pathway,tight junctions and other cellular signaling pathways related to oxidative stress and intestinal barrier,in which MAP3K2 protein expression was upregulated related to MAPK signaling pathway.miRNA sequencing results and differentially expressed protein association analysis revealed that MAP3K2 was a candidate target gene for apla-miR-106a-5p.The results of previous studies showed that MAP3K2 was not significantly differentially expressed in transcriptome and q PCR results.We further performed WB and q PCR on the expression of MAP3K2 protein,MAP3K2 and apla-miR-106a-5p in the intestinal tissues of laying ducks,and the results showed that the expression of MAP3K2 protein was significantly up-regulated（P<0.05）and apla-miR-106a-5p was significantly down-regulated in the CR group compared to the TB group（P<0.05）,while there was no significant difference in MAP3K2 gene expression between the two groups,validating the results consistent with high-throughput sequencing and proteomic analysis.In summary,we predict that the apla-miR-106a-5p/MAP3K2 targeting relationship in the MAPK signaling pathway may influence the repair process of intestinal barrier damage in upper caged ducks through post-transcriptional regulation.Experiment 2:The aim of this experiment was to verify the relationship between apla-miR-106a-5p and MAP3K2 targeting and its effect on the proliferation apoptosis and barrier function of oxidatively stressed intestinal epithelial cells.The experiment used primary duck intestinal epithelial cells（d IEC）as test subjects and was divided into 6 groups:blank group I（Blank）,blank group II（Blank OS）,control group I（NC mimics）,control group II（NC inhibitors）,test group I（miR-106a-5p mimics）and test group II（miR-106a-5p inhibitors）.Blank group I was not treated,and the rest of the groups were treated with 50μmol/LH₂O₂ for4h to induce oxidative stress in the cells,and three replicates were set up for each group.The expression of apla-miR-106a-5p,MAP3K2 and MAP3K2 proteins in the cells was measured by q PCR and WB,dual luciferase reporter assay for target verification,CCK-8 assay for cell viability,scratch test for cell migration rate,cell transmembrane resistance and FITC-dextrose for intestinal barrier integrity and permeability.The results showed that MAP3K2 protein expression was significantly decreased in test group I（P<0.05）and significantly increased in test group II（P<0.05）compared to control groups I and II and blank group II.apla-miR-106a-5p had a negative regulatory relationship with MAP3K2 protein expression;while MAP3K2 gene expression was not significantly different.The results of the dual luciferase reporter assay indicated that MAP3K2 was the target gene of apla-miR-106a-5p.The results of the CCK-8 assay and the scratch assay showed that transfection with apla-miR-106a-5p inhibitors contributed to the effect of enhancing cell activity and cell migration rate of d IEC after oxidative stress;similarly,the cell transmembrane resistance assay and the FITC-dextran permeability assay revealed that transfection with apla-miR-106a-5p inhibitors helped to improve monolayer cell barrier function and reduce cell permeability（P<0.05）.In conclusion,apla-miR-106a-5p regulates MAP3K2 gene translation and low expression of apla-miR-106a-5p improves cell viability and cell migration rate due to oxidative stress,reduces cell permeability and promotes repair of intestinal barrier damage.Conclusion:cage stress in laying ducks leads to intestinal barrier damage.apla-miR-106a-5p can participate in the repair of barrier damage caused by oxidative stress by targeting and regulating MAPK protein expression.