Expression Profile,Mode Of Action And Targeting Relationship Of LncRNA In Female Goat Kids Liver Tissue

In most mammals,the suckling to weaning transition is accompanied by significant nutritional changes.In order to adapt to the changing nutritional environment,the energy metabolism of mammals will change.As an important metabolic organ,the goat liver controls the body’s metabolism and is responsible for nutrient distribution,and plays an important role in growth and development and immunity.Changes in nutrient intake of suckling goat kids can affect the characterization of liver transcriptome,many non-coding RNAs have been shown to participate in known biological development processes by controlling protein-coding genes and non-coding genes.Long non-coding RNA(Lnc RNA)have received widespread attention as potential key regulators of transcription and epigenetic networks.The main purpose of this study was to reveal the lncRNA expression profile in suckling goat kids liver tissue,analyze the mode of action between lncRNA and target genes,and verify the targeting relationship of key competing endogenous RNA(ceRNA)networks.To explore lncRNAs differentially expressed in suckling goat kids liver at different stages,the biological processes or functions involved in target genes,and elucidate the molecular mechanism that is consistent with the physiological changes of the goat kids liver,Those are important reference significance for the study of suckling goat kids liver development,metabolism and immune regulation.In this study,the liver tissue of Laiwu black goat at five age stages(5 replicates for each age group,1 goat for each replicates,25 goats in total)of 1 day old(D1),2 weeks old(W2),4weeks old(W4),8 weeks old(W8)and 12 weeks old(W12)was selected as the research object.Total RNA was extracted,25 libraries were constructed for transcriptomic sequencing data analysis,and screening for differentially expressed lncRNA,lncRNA target gene prediction and GO and KEGG enrichment analysis,key lncRNAs were validated by quantitative real-time PCR and the targeting relationships were verified by dual luciferase assay.The main results are as follows:(1)In this study,a total of 25 transcriptome libraries in five age groups of suckling goat livers were successfully constructed,and a total of 399.52 Gb of Clean Data were obtained by high-throughput sequencing,the percentage of Q20 bases was 97.43% and above,the sequencing error rate of each sample was no higher than 0.0267%.Comparison of quality control data with goat reference genome,and the comparison efficiency was 96.94% and above.A total of 5473 lncRNAs,including 2869 known lncRNAs and 2604 novel lncRNAs,were identified in five age-stage goat liver tissue libraries constructed,a total of 567 differentially expressed lncRNAs were identified by differential expression analysis.(2)LncRNAs had cis-trans mode of action on target genes,with 270 lncRNAs targeting1113 m RNAs.According to the correlation of lncRNA-m RNA expression levels,18 different lncRNAs were finally screened out to have 23 interaction relationships with 22 different m RNAs.According to the targeting relationship between mi RNA-m RNA and mi RNAlncRNA,the ceRNA network was constructed.There were 328 targeting relationships between153 differentially expressed lncRNAs and 111 differentially expressed mi RNAs,and there were2265 targeting relationships between 111 mi RNAs and 862 differentially expressed m RNAs.According to the correlation analysis results of mi RNA-m RNA and mi RNA-lncRNA expression levels,13 interaction relationships were found between 11 different mi RNAs and10 different lncRNAs,and 78 interaction relationships were found between 72 different target genes.8 lncRNAs were selected for q RT-PCR,the results show that the quantitative results were consistent with the transcriptome sequencing results.GO/KEGG enrichment analysis showed that both cis and trans acting target genes of lncRNA were significantly enriched in immune disease,lipid metabolism,cofactor and vitamin metabolism pathways;The trans positive correlation target gene of lncRNA was also related to cell growth and death;In the ceRNA network,the differentially expressed lncRNA coexpressed m RNA was significantly enriched into the pathways of cofactor and vitamin metabolism,lipid metabolism,carbohydrate metabolism,biodegradation and metabolism of allobiotin,Genes related to metabolism,development and immunity were found through enrichment analysis.(3)According to the targeting relationship in ceRNA network,lnc17598.2,HSD11B1,lnc9465.3,FASN wild-type and mut-type dual luciferase reporter vectors were successfully constructed,the corresponding lncRNA,mi RNA and m RNA were analyzed by q RT-PCR,and the results showed that the quantitative results were consistent with the transcriptome sequencing results.Transfect lnc17598.2/HSD11B1 with chi-mi R-433 mimics and mimics NC into 293 T cells,respectively.Transfect lnc9465.3/FASN with chi-mi R-497-5p mimics and mimics NC into 293 T cells,respectively.The results showed that the luciferase expression activity in chi-mi R-433 mimics and lnc17598.2/HSD11B1 wild-type group was significantly lower than other three groups(P < 0.01),the luciferase expression activity of chi-mi R-497-5p mimics and lnc9465.3/FASN wild-type group was significantly lower than other three groups.The results show that chi-mi R-433 could directly bind to lnc17598.2/HSD11B1,chi-mi R-497-5p can directly bind to lnc9465.3/FASN.In conclusion,lncRNA plays an important role in liver metabolism,immunity and development of suckling goat kids,as the age of goat kids increases,the liver undergoes changes in transcript levels due to nutrients and environmental change.There is a targeting relationship between chi-mi R-433 and lnc17598.2/HSD11B1,a targeting relationship between chi-mi R-497-5p and lnc9465.3/FASN.