Selection And Validation Of MiRNA Related To Shedding In Sheep

In this study,the extreme phenotypes of shedding sheep(group S,5 animals)and non-shedding sheep(group N,4 animals)were studied in Dorper sheep ewes,and skin tissue samples were collected from the body side of their skin at three stages of hair follicle development during the eSeptember 2019,January 2020,and March 2020 for paraffin sectioning and miRNA sequencing to observe and analyze the hair follicle morphology,and to differential miRNAs for target gene prediction,and analyzed the function and signaling pathways of target genes by GO and KEGG enrichment,and finally performed qRT-PCR validation tests,with the following results:1.The hair follicle developmental stages at three time points in this study,group S were:anagen,telogen,and anagen,and group N were:anagen,pre-catagen,and mid-catagen.Statistics on the hair loss of the sheep in May showed that 81.53%had different degrees of hair loss,and 18.47%had non-shedding hair.2.By comparing and analyzing the difference of miRNA expression levels between S group and N group by transcriptome sequencing,a total of 489 differential miRNAs were detected in S group,of which 281 were up-regulated and 208 were down-regulated;a total of 305 differential miRNAs were detected in N group,of which 146 were up-regulated,down 159.A total of 215 differential miRNAs were detected by S-vs-N,of which 138 were up-regulated and 77 were down-regulated.Repeated and abnormally expressed miRNAs were removed,and a total of 244 differential miRNAs were obtained after screening.3.The 244 differential miRNAs were screened according to A trend(high expression in growth phase,low expression in regression and telogen phase)and T trend(high expression in regression and telogen phase,low expression in growth phase),and finally 50 T trends in A trend were obtained.14,and then perform target gene prediction,and screen according to the correlation coefficient between miRNA and target gene mRNA expression(less than-0.7),and finally get 26 differential miRNAs with A trend and 186 target genes;7 differential miRNAs with T trend,53 target genes.These target genes were intersected with the differential genes that conformed to the A and T trends obtained by mRNA analysis,and finally 36 target genes affecting hair follicle growth and development were screened,corresponding to 15 Atrend miRNAs.4.The above 36 target genes were subjected to GO enrichment analysis,and the results showed that more target genes were enriched in cellular process,biological process regulation;organism process,cell,cell part;binding,catalytic activity and other functional items;KEGG analysis was performed.,more target genes are enriched in the signaling pathways related to hair follicle growth and development,such as MAPK,Hedgehog,TGF-beta,RAP1,TNF,NF-kappa B,Jak-STAT,Ras,PI3K-Akt,etc.5.The four signaling pathways of NF-kappa B,Jak-STAT,Ras,and PI3K-Akt are important pathways in the resting stage of hair follicle growth and development.The differential miRNAs enriched in the four pathways and their corresponding target genes were selected,and 6 pairs of miRNA-mRNA were screened out.Yes,respectively:miR-184-3p-CARD11,miR-146-3P-PDGFRB,miR-210-3p-STAT6,miR-429-3p-NTRK2,miR-17-3p-NTRK2,miR-146-5p-TRAF1-NTRK2,these differential miRNAs and target genes may play an important regulatory role in the periodic growth and development of sheep follicles.6.Real-time quantitative PCR technology was used to quantitatively detect the expression levels of 13 differential miRNAs and 2 target genes.The results showed that the trends of real-time quantitative PCR and transcriptome sequencing results were basically the same.This study provides new information for the miRNA expression profile of Dorper wool follicle tissue through miRNA sequencing analysis of three periods of periodic changes in Dorper wool follicles,and provides a basis for continuing to explore the targeting of these miRNAs and their target genes.The relationship provides a preliminary basis for the analysis of the molecular regulatory mechanisms related to hair removal sheep traits in sheep.