Expression Patterns Of MRna And MiRNA In The Liver Of Sucking Doe Kids And Their Regulatory Mechanisms

The liver is the largest digestive gland in mammals and plays an important role in substance metabolism and immunity.The liver of goat kids undergoes characteristic changes in transcript levels due to the digestion and absorption of substances and stress response.The main objective of this study was to reveal the changes in the m RNA transcriptome and miRNA transcriptome expression profiles in the liver of goat kids and to elucidate the characteristics of the transcriptome expression and possible regulatory mechanisms associated with the liver of goat kids.In this study,liver tissues from five age groups(5 replicates per age group,1 goat per replicate,25 goats in total)of Laiwu black goats at 1 day of age(D1),2weeks of age(W2),4 weeks of age(W4),8 weeks of age(W8)and 12 weeks of age(W12)were used to construct liver m RNA and miRNA expression profiles of goat kids by transcriptome sequencing,and the differential expression m RNA and miRNA expression patterns with age,and demonstrated the molecular mechanisms consistent with physiological changes in the liver of goat kids,and combined analysis of m RNA and miRNA.The main findings are as follows.1 Construction of m RNA library and data analysis in the liver of goat kidsIn this study,a total of 25 transcriptomic libraries were successfully constructed in five age groups of goat kids liver,and 3,023,865,598 m RNA clean reads were detected by highthroughput sequencing of the libraries.A total of 37,497 m RNAs were detected as expressed in the liver of goat kids from the 25 libraries,and differential expression analysis identified1,271 differentially expressed genes,which were clustered into four categories based on expression patterns.The expression level of the first category of DEGs decreased with increasing age of goat kids,and they were significantly enriched in the PPAR signaling pathway(FDR < 0.05).In the second category,W2 and W8 showed high expression of DEGs and low expression in the other groups,with significant enrichment in pathways related to amino acid biosynthesis and metabolism and lipid metabolism(FDR < 0.05).In the third category,D1 and W4 groups showed high expression of DEGs and low expression in the other groups were significantly enriched in pathways mainly associated with cell growth and development(FDR < 0.05).The expression level of the fourth group of DEGs increased with the age of the goat kids and the significantly enriched pathways were mainly related to immunity(FDR < 0.05).The regulatory network analysis screened 14 key genes,including CD4,MYC,CDK1,IL7 R,and PPARG,which were mainly associated with cell growth and development,endocrine,and immunity.In addition,DEGs between adjacent and non-adjacent groups were analyzed for functional enrichment,and the largest number of DEGs were found between D1 and W12,and upregulated genes were significantly enriched in most enriched in substance metabolism and immune-related pathways,and metabolism-related pathways included steroid hormone biosynthesis,retinol metabolism,cytochrome P450 metabolism of xenobiotics,drug metabolism-cytochrome P450,linoleic acid metabolism,arachidonic acid metabolism.Immune-related signaling pathways include Ig A production in the intestinal immune network,rheumatoid arthritis,Staphylococcus aureus infection,autoimmune thyroid disease et al;down-regulated genes are significantly enriched in immune-related signaling pathways such as alcoholism,cancer,malaria.,fatty acid metabolism,galactose metabolism,amino acid metabolism,PPAR and other metabolism-related pathways.2 Construction of miRNA library and data analysis in the liver of goat kidsIn this study,a total of 25 small RNA libraries were successfully constructed in five age groups of goat kids liver,and 231,497,118 pure reads were detected by high-throughput sequencing of the libraries.Among the top 20 miRNAs expressed in each group,the seven most highly expressed miRNAs were all known miRNAs,namely chi-miR-122,chi-miR-143-3p,chi-miR-21-5p,chi-miR-30a-5p,chi-miR-194,chi-miR-26a-5p,and chi-let-7f-5p.In this study,198 DE miRNAs were identified and clustered into three categories according to their expression patterns,the expression level of the first category of DE miRNAs decreased with the age of goat kids,and their target genes were significantly enriched in pathways mainly related to immunity(FDR < 0.05);the expression level of the second category of DE miRNAs increased with the age of goat kids,and the expression level of these DE miRNAs increased with the age of goat kids.The expression level of the second category of DE miRNAs increased with the age of goat kids,and these DE miRNAs were involved in the regulation of transcriptional levels related to the digestion and absorption of substances,and their target genes were significantly enriched in pathways mainly related to substance metabolism(FDR< 0.05).The third group of DE miRNAs was highly expressed in D1,W2 and W4 groups and lowly expressed in W8 and W12 groups.These DE miRNAs were involved in transcriptional level regulation related to dietary changes and weaning stress,and their target genes were significantly enriched in pathways mainly related to in substance metabolism and immunity(FDR < 0.05).Nine miRNAs,including chi-let-7b-5p,chi-miR-342-5p,chi-miR-432-5p,and chi-miR-412-3p,were screened according to the interactions between miRNAs and genes;five genes,including IQGAP2,LBP,NOTCH2,C3,and FBN2,were mainly enriched in substance metabolism,disease genesis and immune-related pathways.3 Combined analysis of m RNA and miRNA in the liver of goat kids and Validation of the targeting relationship between key miRNAs and target genesBased on the results of KEGG enrichment analysis of DE miRNA target genes in the liver of goat kids,the signaling pathways related to cell proliferation and apoptosis were selected to construct a target relationship network.There were 10 key miRNAs and 10 key genes related to liver growth in goat kids,such as chi-miR-342-5p,chi-miR-27a-5p,chi-miR-432-5p,MAPK13,IGF1,NFATC4.The signaling pathways related to liver metabolism in goat kids were selected to construct a target relationship network.There are 15 key miRNAs and 15 key genes related to liver metabolism in goat kids were screened,such as chi-miR-342-5p,chi-miR-665,chi-miR-27a-5p,chi-miR-432-5p,CYP1A2,GALT,GSTA4.In this study,the targeting relationships of four pairs of miRNAs related to liver growth,metabolism and immunity with genes were selected and validated at the cellular level using 293 T cells as the study subjects.The results of dual luciferase activity assay showed that chi-miR-532-3p significantly inhibited the activity of reporter gene luciferase containing 3′UTR of NFATC4(P < 0.01),chi-miR-222-3p did not bind to the 3′UTR of GPX7 and did not affect the activity of reporter gene luciferase(P > 0.05),chi-miR-432-5p significantly inhibited the reporter gene luciferase activity of the 3′UTR containing GPX2(P < 0.01),and chi-miR-92a-5p significantly inhibited the reporter gene luciferase activity of the 3′ UTR containing IL7R(P <0.01).q RT-PCR and Western blot results showed that,compared with the control,chi-miR-532-3p significantly reduced the m RNA transcript level and protein expression of NFATC4(P< 0.01),and thus inhibited NFATC4 expression,possibly by targeting binding to the 3′UTR region of NFATC4.chi-miR-432-5p significantly reduced GPX2 at the m RNA transcript level and protein chi-miR-432-5p may target the 3′UTR region of GPX2 and significantly reduce the m RNA transcript level and protein expression of GPX2(P < 0.01),thereby inhibiting GPX2 expression;chi-miR-92a-5p may target the 3′UTR region of IL7 R and significantly reduce the m RNA transcript level and protein expression of IL7 R with highly significant differences(P < 0.01),thereby inhibiting IL7 R expression.In conclusion,the liver of goat kids plays an important role in metabolism and immunity,and as the age of goat kids increases,the liver undergoes characteristic changes in transcript levels due to substance digestion and absorption and stress,and there are targeted regulatory relationships with key miRNAs and genes related to growth,substance metabolism and immunity.