Transcriptome Analysis Of MRNA And MiRNA And Study On The Regulation Mechanism Of IGF1 Expression In Prenatal And Postnatal Livers Of Large White

The liver is the mainorganizationwhich produces IGF1.A large number of studies have found that the system of insulin-like growth factor 1(IGF1),its receptor(IGF1R)and related binding proteins(IGFBPs 1-6)play a key role in regulating the growth,development and metabolism of animals.Our previous studies showed that IGF1 expression was significantly different in porcine livers before and after birth:the prenatal expression level was relatively low,and its expression level was significantly increased after birth.But the reasons for the expression pattern have not been clarified.As one of the largest internal organs in mammals,the liver exhibits endocrine and exocrine properties,and can secretes many hormones,such as the secretion of IGF1.Meanwhile the liver has a series of metabolic functions,such as glycogen storage,drug detoxification,metabolic control and cholesterol synthesis and transport regulation and so on.The expression and function of miRNAs and mRNAs in liver tissues of different animals have been reported in some studies.However,there are fewer reports on transcriptomics of prenatal and postnatal porcine liver tissues,including differentially expressed miRNAs and mRNAs and the biological processes and pathways which they are involved in are still to be addressed.MiRNA is a non-coding RNA of about 22bp,and has attracted lots of scientists because of its important roles in the regulation of cell growth,cell proliferation,cell differentiation,apoptosis,glucose metabolism,body fat formation,protein synthesis and decomposition and so on.Sequencing of miRNA and mRNA can obtain all genes and miRNAs of different tissues or cells at a given time.The integrated analysis of miRNA and mRNA can accurately and in-depth identify the key genes and miRNAs related to pig liver tissue development,which can help us better understand the molecular mechanisms on liver tissue development.In this study,miRNAs and transcription factors which regulated the expression of IGF1 and the biological processes and pathways those affected the development of liver tissues and lipid metabolism in pigs were obtained by miRNAs and mRNAs sequencing in liver tissues of different developmental stages.In this study,a total of 8 liver tissue samples of 70-day-old fetal pigs and 70-day-old adult pigs were studied(4 samples of each period respectively).The results were as follows:(1)Using RNA-Seq technique to explore miRNA and mRNA expression profile of porcine livers in different developmental stages(prenatal and postnatal).The differentially expressed miRNAs,mRNA and a joint analysis between them were carried out,which provided us a solid theoretical basis on the miRNA regulation of the liver tissue development.(2)to further provide more reliable evidence that C/EBPβ is a transcription factor for IGF1 in porcine liver;(3)to study the expression pattern of C/EBPβ in prenatal and postnatal porcine livers,and to explore the mechanism of significant changes in the expression level before and after birth;(4)to investigate the effect of methylation differences in IGF1 promoter region on the IGF1 expression(P<0.05);(5)to investigate the miRNAs which potentially regulates IGF1 expression.According to the above research content,the following results were obtained:1.MiRNA sequencing analysis on liver tissue of 70-day-old fetal pigs(P70)and and 70-day-old adult pigs(D70)A total of 8 miRNA libraries were constructed in this study(4 samples of each period respectively).Average 28M(million)raw reads were obtained respectively.After removal of invalid data(tRNA,rRNA,snRNA,snoRNA and adaptor contamination sequences),the average 20 M(million)clean reads were obtained for subsequent analysis.The length of the miRNAs were mainly distributed in the range of 21-23nt,which was consistent with the length distribution characteristics of miRNAs.The differential expression analysis showed that there were 116 differentially expressed miRNAs.Compared with the 70-day-old adult pig(D70),61wasup-regulated and 55 was down-regulated.Hierarchical clustering analysis of differentially expressed miRNAs revealed that the four samples of the same period could be well together,indicating that the samples were highly homogeneous and that subsequent bioinformatics analyses were more reliable.The GO functional annotation and KEGG pathway enrichment analysis found that the main enrichment GO terms were:cell differentiation,developmental process,metabolic process,signal transduction process and biological regulation process and so on;KEGG terms were:cAMP signaling pathway,Wnt signaling pathway,Notch signaling pathway,PI3K-Akt,MAPK signaling pathway,FoxO signaling pathway,adipocytokine signaling pathway,thyroid hormone signaling pathway and so on.These pathways played important roles in the development of animal livers.Ten differential expressed(5 up-regulated,5 down-regulated)miRNA were chosen to verify the reliability of the sequencing results by qRT-PCR.The results showed that the expression trends of those selected genes were consistent with the sequencing results,indicating that the sequencing results were reliable.2.MRNA sequencing analysis on liver tissues of 70-day-old fetal pigs(P70)and and 70-day-old adult pigs(D70)Totally,we obtained 25323 known genes and 9986 new transcripts from 8 mRNA libraries.4916 transcripts were differentially expressed between P70 and D70 samples.Among them,126 genes were expressed only in P70 samples,75 genes were expressed only in D70 samples,and 4715 genes were expressed in all samples.Compared with D70 samples,2502 genes were up-regulated and 2414 genes were down-regulated.Hierarchical clustering analysis of differentially expressed miRNAs revealed that the four samples of the same period could be well together,indicating that the samples were highly homogeneous and that subsequent bioinformatics analyses were more reliable.The GO function annotation and the KEGG pathway enrichment analysis showed that the main GO items were:metabolic,response to stress,immune system process and so on;KEGG items were:DNA replication,cell cycle,drug metabolism,amino acid degradation and so on.Ten differential expressed(5 up-regulated,5 down-regulated)mRNAs were chosen to verify the reliability of the sequencing results by qRT-PCR.The results showed that the expression trends of those selected genes were consistent with the sequencing results,indicating that the sequencing results were reliable.3.The integration analysis of miRNA and mRNA dataThe integrated analysis of differential expressed miRNrAs and mRNAs totally found 60 pairs of "up regulated miRNA-down regulated mRNA ",55 pairs of "down regulated miRNA-up regulated mRNA ".The GO function annotation and the KEGG pathway enrichment analysis showed that the main GO items were:thrombopoietin receptor activity,antigen binding and so on;KEGG items were:TNF,immune system signaling pathways and so on.4.Verification of C/EBPβ regulation on IGF1 by dual-luciferase reporter systemC/EBPβ could significantly increase the luciferase activity of wild type IGF1 promoter,and the luciferase activity of site2 mutation was significantly reduced than that of the wild type.It only had a slight reduction in luciferase activity when site 1 was mutated.The luciferase activity reached the lowest when both sitel and site 2 were mutated.The above results indicated that C/EBPβ regulated the IGF1 mRNA expression by acting at site2 of IGF 1 promoter.5.Study on methylation pattern of IGF1 promoters in different developmental porcine liversMethylation sequencing results showed that there were no significant changes in methylation level of IGF 1 two potential promoters.There were noly minor but not significant differences in individual loci.This suggested that the rapid IGF1 mRNA changes in the porcine livers before and after birth were not determined partially by the methylation differences in its promoters.6.miR-18 b and miR-130b can directly target IGF1 3’-UTR,and thus regulates its expressionMiRNA and mRNA sequencing results suggested that miR-18b and miR-130b could regulate IGF1 expression.The results of qRT-PCR and sequencing showed that the above two miRNAs had the opposite expression pattern compared with IGF1 mRNA in different development stages of porcine liver tissue.Meanwhile,the dual luciferase reporter system showed that miR-18b and miR-130b could regulate the expression of IGF 1 by directly targeting to IGF 1 3,-UTR.7.Study on expression pattern of C/EBPβ potential transcription factors in different developmental porcine liversSTAT3,CALR,CUGBP1 and Spl mRNA had the similar expression patterns as C/EBPβ mRNA in different developmental stages of porcine liver:Expression levels were significantly higher in postnatal stages than in prenatalstages.Myb,KARA and SREBP mRNA had the opposite expression pattern compared with C/EBPβ mRNA:Expression levels were significantly higher in prenatalstages than in postnatal stages.