| Circular RNA is a kind of non-coding RNAs with a closed loop structure without a 5’ end cap and a 3’poly A tail.It has a wide range of regulatory functions,not only as an endogenous competing RNA(ceRNA)but also as a regulator of parent gene expression.Studies have found that lipid metabolism in liver is regulated by non-coding RNAs mediated before or after transcription.However,there is relatively little research on the expression and regulation of circular RNA in the liver currently.In this study,70-day old uncastrated Landrace pigs and Jinhua pigs were selected as test subjects.The serum biochemical levels of the two breeds were compared and high-throughput sequencing technology was employed to explore mRNA,miRNA and circRNA expression in the liver of Landrace and Jinhua pigs.Differentially expressed genes,miRNAs and cire RNAs were screened and GO enrichment and KEGG pathway analysis were performed.Quantitative real-time PCR was performed to validate the sequencing results.In addition,integrated analysis of differentially expressed miRNAs and genes,and interaction analysis between circRNA and miRNA were performed.The results are as follow:(1)The body weight and liver weight of Landrace pigs were significantly higher than those of Jinhua pigs(P<0.01)5 but there was no difference in the ratio of liver/body weight.The serum insulin,TT3 and TT4 of Landrace pigs were significantly higher than those of Jinhua pigs(P<0.05).The serum TCH and LDL-C of Jinhua pigs were significantly higher than those of Landrace pigs(P<0.01).Serum glucose and HDL-C were higher in Landrace pig,but the difference is not significant.(2)Approximately 78 M(million)of clean reads(1×50 bp)obtained by digital gene expression sequencing and more than 86.64%of them were aligned to the reference genome.A total of 467 differential genes were filtered for differential expression analysis(|log2(fold change)|>1 and p-adjust<0.05),of which 172 genes were up-regulated in Jinhua pigs and 295 genes were up-regulated in Landrace pigs.The GO and KEGG analysis revealed that they are mainly involved in biological processes such as oxidation-reduction,lipid biosynthetic and cellular lipid metabolic process,and interact with ECM-receptor interaction,and protein digestion and absorption,fatty acid metabolism,PPAR signaling pathways,and fat digestion and absorption.(3)About 75.5 M of clean reads were obtained by small RNA sequencing and more than 98.96%were able to be aligned to the reference genome.A total of 266 known miRNAs were identified in the livers of the two breeds,14 of which were specifically expressed in Landrace pigs,and 7 miRNAs were specifically expressed in Jinhua pigs.What’s more,all of the specific miRNAs were low expression.A total of 35 differentially expressed miRNAs were identified from the two breeds,of which 8 miRNAs were up-regulated in Jinhua pigs and 27 miRNAs were up-regulated in Landrace pigs.Bioinformatics analysis demonstrated that differential miRNAs are involved in lipid metabolism-related pathways such as endocytosis,fatty acid biosynthesis,fatty acid metabolism pathway,PI3K-Akt signaling pathway.TGF-βsignaling pathway,and cAMP signaling pathway.(4)A total of 61858 circRNAs were identified by circular RNA library sequencing.Most of the circRNAs were derived from exons,and about 93%of circRNAs showed low expression levels(RPM value<100).A total of 376 differential circRNAs were screened by differential analysis,of which 116 circRNAs were up-regulated in Jinhua pigs and 250 circRNAs were up-regulated in Landrace pigs.Host gene enrichment analysis of differentially expressed circRNAs revealed that they were mainly involved in fat metabolism and oxidation-reduction processes.Comparing with gene expression profiles by DGE,these host genes were found to be highly expressed in both breeds.(5)To validate reliability of sequencing results,six differentially expressed genes,six differentially expressed miRNAs and eight differentially expressed circRNAs were randomly selected and subjected to qRT-PCR.The expression results were found to be consistent with high-throughput sequencing results.(6)Combined analysis of differential miRNA,mRNA and circRNA,the resulting PPAR pathway-related regulatory network map includes 10 mRNAs,11 miRNAs and 32 circRNAs. |
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