Expression Characteristics Of CircRNA In Liver Tissue Of Female Goat Kids And Validation Of Important CeRNA Network

In mammals,liver function is essential for maintaining metabolic homeostasis.The liver is the key hub of many physiological processes,including nutrient metabolism,immune system support,lipid and cholesterol homeostasis,etc.Changes in the transcription level of the liver tissue of the suckling female goat kids will be caused by changes in the intake of nutrients.Therefore,it is of great significance to study the regulation mechanism of the development and function of the liver tissue of suckling female goat kids for improving the molecular biological research of goat liver tissue.However,there is no report on the role of circRNA in goat liver.In this study,RNA-seq technology was used to sequence the liver tissues of five age stages suckling Laiwu black goat: 1 day(D1),2 week(W2),4 week(W4),8 week(W8)and12 week(W12).We analysed the circRNA expression profile of the liver tissue of suckling female goat kids,and performed the differential expression analysis and functional enrichment analysis of its source genes.Joint analysis with the expression profile of differentially expressed mi RNA and differentially expressed m RNA obtained by previous studies,bioinformatics software predicted the binding sites,and constructed a ceRNA network related to goat liver genes.The double luciferase reporter gene assay was used to verify the target relationship of the key ceRNA pairs,and then analyze the biological role of circRNA in the liver tissue of suckling female goat kids.The main results are as follows:1.Twenty-five liver transcriptome libraries of suckling female goat kids were successfully constructed,and a total of 3,023,865,598 clean reads were obtained.The comparison rate in goat genome was above 96.9%,and the sequencing results of each library were good.We identified 29,428 circRNAs,among which 1,087 differentially expressed circRNAs were screened.In D1 vs W12,there are 460 differentially expressed circRNAs,in which 139 were up-regulated and 321 were down-regulated;In W8 vs W12,there are 202 differentially expressed circRNAs,in which 64 were up-regulated and 138 were downregulated;In D1 vs W2,there are 368 differentially expressed circRNAs,in which 149 were up-regulated and 219 were down-regulated;In D1 vs W4,there are 307 differentially expressed circRNAs,in which 145 were up-regulated and 162 were down-regulated;In D1 vs W8,there are 449 differentially expressed circRNAs,in which 175 were up-regulated and 274 were down-regulated;In W2 vs W4,there are 261 differentially expressed circRNAs,in which 160 were up-regulated and 101 were down-regulated;In W2 vs W8,there are 221 differentially expressed circRNAs,in which 116 were up-regulated and 105 were downregulated;In W2 vs W12,there are 334 differentially expressed circRNAs,in which 121 were up-regulated and 213 were down-regulated;In W4 vs W8,there are 282 differentially expressed circRNAs,in which 113 were up-regulated and 169 were down-regulated;In W4 vs W12,there are 279 differentially expressed circRNAs,in which 74 were up-regulated and 205 were down-regulated.Five differentially expressed circRNAs were randomly selected for q RT-PCR verification,and the expression trend was consistent with the results of RNA-seq.2.GO enrichment analysis of the source genes of differentially expressed circRNAs found,they are mainly involved in cell development,growth,metabolism,immunity and other biological processes;KEGG pathway analysis found that they are significantly enriched in Th17 cell differentiation,tryptophan metabolism,propionic acid metabolism,osteoclast differentiation and other pathways closely related to cell development,immunity and metabolism.3.According to |r| > 0.7 and P < 0.05,we screened a ceRNA network composed of 11 DEcircRNAs,13 DEmi RNAs and 44 DEm RNAs.Among them,four genes(ACACB,PCK1,HKDC1,ABCA3)related to amino acid metabolism,lipid metabolism and glucose metabolism are regulated by five circRNA-mi RNA pairs(including five differentially expressed circRNAs and four differentially expressed mi RNAs);One immune-related gene(PRF1)is regulated by three circRNA-mi RNA pairs(including two differentially expressed circRNAs and two differentially expressed mi RNAs);Four development-related genes(HSD17B2,ANGPT2,CRB1,SALL2)are regulated by six circRNA-mi RNA pairs(including six differentially expressed circRNAs and five differentially expressed mi RNAs).4.Cluster the circRNAs and m RNAs in the ceRNA network according to their correlation,the differentially expressed m RNAs are grouped into two categories.The first category of genes: KEGG analysis results showed that differentially expressed m RNAs were significantly enriched in HIF-1 signal pathway;All GO entries are not significantly enriched.The second category of genes: GO enrichment results showed that these differential genes are significantly enriched in 11 GO entries such as glycerol biosynthetic process from pyruvate,monosaccharide metabolic process,organic acid metabolic process,oxaloacetate metabolic process,phosphoenolpyruvate carboxykinase activity;KEGG pathway analysis found that the differentially expressed m RNAs were significantly enriched in PI3K-Akt signal pathway,Pyruvate metabolism,Glycolysis / Gluconeogenesis,Steroid hormone biosynthesis and other pathways related to cell growth and liver metabolism.5.According to the targeting relationship in the ceRNA network,successfully constructed PPP1R14 C,circ＿19＿13892761＿13892862 wild-type and mutant-type pmir GLO recombinant vector which containing chi-mi R-32854 binding site;and CYP8B1 、ENSCHIG00000014983 、 circ＿10＿15045038＿15046738 wild-type and mutant-type pmir GLO recombinant vector which containing chi-mi R-532-3p binding site;Successfully synthesized chi-mi R-32854＿mimics and chi-mi R-532-3p＿mimics.The wild-type and mutanttype pmir GLO recombinant vectors were co-transfected into 293 T cells with corresponding mi RNA-mimics and mi RNA-NC,respectively.Results showed that the expression activity of double luciferase in the experimental group co-transfected with mi RNA-mimics and wild type pmir GLO recombinant vector was significantly lower than that in the other three groups(P <0.01),this indicates that the targeting relationship between chi-mi R-32854 and PPP1R14C、chi-mi R-32854 and circ＿19＿13892761＿13892862、chi-mi R-532-3p and CYP8B1、chi-mi R-532-3p and ENSCHIG00000014983、chi-mi R-532-3p and circ＿10＿15045038＿15046738 is existing.In summary,through bioinformatics analysis of sequencing data,circRNA in the liver of suckling female goat kids may regulate gene expression by competing with m RNA to bind mi RNA,involves in the biological processes of metabolism,immunity and development in liver.The interaction targeting relationship between chi-mi R-32854 and PPP1R14C、chimi R-32854 and circ＿19＿13892761＿13892862、chi-mi R-532-3p and CYP8B1、chi-mi R-532-3p and ENSCHIG00000014983 、 chi-mi R-532-3p and circ＿10＿15045038＿15046738 was verified by double luciferase experiment.