| Fat deposition is an important economic trait in livestock,which has an important impact on animal carcass quality,production efficiency,meat quality and even reproductive performance.Excessive deposition of adipose also plays an important role in the development of human-related diseases,including obesity,type 2 diabetes mellitus(T2DM)and other diseases.Therefore,to further explore the molecular mechanism of adipogenesis is great significance for livestock and poultry production and the treatment of human diseases.The molecular mechanism of adipocyte proliferation and differentiation has been deeply studied.Among them,CCAAT enhancer binding proteins α(C/EBPα),as one of the most important adipogenic regulators,regulated adipogenic-related genes through transcriptional regulation.It played an important regulatory role in the process of cell differentiation.Recent studies have shown that epigenetics played an important regulatory role in numerous biological processes including adipogenesis.Among them,the most representative is noncoding ncRNA(non-codingRNA,ncRNA)regulation,including long non-codingRNA(long non-codingRNA,lncRNA)and smallRNA(microRNA,miRNA)and so on.Therefore,to explore the epigenetic mechanism of adipogenesis is great significance for animal production and human health.At present,the epigenetic regulatiory mechanism of C/EBPα on adipogenesis remains to be further explored.In this study,we explored the epigenetic mechanism of C/EBPα regulating porcine adipocytes from two aspects of miRNA-mediated competitive endogenousRNA(ceRNA)effect and lncRNA regulatory pathway,and further enriched the mechanism of porcine adipocyte differentiation.The specific results of this study are as follows:Study 1 C/EBPα influences the formation of C/EBPα-FOXO1 protein complex through ceRNA effect to regulate porcine lipogenesis1.CeRNA effect of C/EBPα and FOXO1 regulates adipogenesis in porcine adipocytesBy bioinformatics analysis of miRNAs targeting C/EBPα,5 miRNAs that have been shown to target C/EBPα were selected as candidate miRNAs for predicting ceRNAs of C/EBPα,meanwhile,the relative expression levels of these miRNAs were detected in pig adipose tissue.The qPCR results showed that the relative expression level of miR-144 was the highest among them(P<0.001).Therefore,miR-144 was used to predict the ceRNA of C/EBPa.By the analysis,a forkhead box O1(FOXO1),which has been shown to be involved in adipogenesis regulation,was identified for subsequent analysis.To verify whether C/EBPα and FOXO1 are ceRNAs,adipocytes were transfected with pcDNA3.1-C/EBPα-3’UTR plasmids or C/EBPα siRNAs,and the expression pattern of FOXO1 gene was quantitatively detected.On the other hand,pcDNA3.1-FOXO1-3’UTR plasmids or siRNAs were transfected into adipocytes,then to detect the expression pattern of C/EBPα as well.The data showed that,compared with the control group,pcDNA3.1-3’UTR of C/EBPα or FOXO1 could significantly increase the expression levels of C/EBPα and FOXO1(P<0.05);on the contrary,the expression levels of C/EBPα and FOXO1 in the siRNAs groups were significantly decreased(P<0.05).According to the rule of ceRNA effect,C/EBPα and FOXO1 were ceRNAs of each other.In order to verify that miR-144 was a key regulator of the ceRNA effect,after interfering with miR-144 in adipocytes,the pcDNA3.1-3’UTR plasmids of C/EBPα and FOXO1,or their specificity siRNAs were transfected into adipocyte,respectively,and detected the ceRNA effect again.The results showed that the expression levels of C/EBPα and FOXO1 did not change after interfering with miR-144(P>0.05).Therefore,which indicated that miR-144 regulated the ceRNA effect of C/EBPα and FOXO1.Adipocyte differentiation was induced after transfecting pcDNA3.1-3’UTR plasmids of C/EBPα and FOXO1 into porcine adipocytes,respectively.The results showed that the adipogenic differentiation level of C/EBPα and FOXO1 pcDNA3.1-3’UTR groups was significantly lower than that of the control group(P<0.05).These results indicated that the ceRNA of C/EBPα and FOXO1 inhibited adipogenesis in adipocytes.2.CeRNA effect regulates the formation of C/EBPα-FOXO1 protein complex to regulate adipogenesisBy report,C/EBPα and FOXO1 forming a protein complex to regulate Adiponectin gene(Adipo Q)transcription by binding to its promoter.Therefore,this study used CoImmunoprecipitation(Co-IP)assay to detect whether the formation of C/EBPα-FOXO1 protein complex was regulated by ceRNA effect.After transfecting the pcDNA3.1-3’UTR plasmids of C/EBPα and FOXO1 in adipocytes,the expression level of C/EBPα-FOXO1 protein complex was detected by IP method using C/EBPα and FOXO1 specific antibodies.Results showed that transfection of C/EBPα and FOXO1 pcDNA3.1-3 UTR in adipocytes promoted the formation of C/EBPα-FOXO1 protein complex(P<0.05).Above results indicated that the ceRNA effect regulated the formation of the C/EBPα-FOXO1 protein complex.Moreover,chromatin immunoprecipitation PCR(ChIP-PCR)was used to detect whether the ceRNA effect of them influenced the binding transcription of C/EBPα-FOXO1 complex to Adipo Q promoter.The results showed that the transfection of C/EBPα and FOXO1 pcDNA3.1-3’UTR plasmids or siRNAs,the transcription level of Adipo Q was promoted and inhibited(P<0.05),the protein level of Adipo Q was incerased and descreased as well(P<0.05).Above results showed that the ceRNA effect of them regulated the transcription level of Adipo Q,which in turn to influence the expression of Adipo Q protein.At the same time,in order to verify the regulatory effect of miR-144 on C/EBPα-FOXO1 protein complex formation and adipocytes adipogenesis.After interfering with miR-144,results showed that the formation of C/EBPα-FOXO1 protein complex was not changed after transfection of C/EBPα and FOXO1 pcDNA3.1-3’UTR plasmids or siRNAs(P>0.05),meanwhile,the transcriptional expression level and protein level of Adipo Q did not change as well(P>0.05).These results indicated that miR-144 influenced the formation of C/EBPα-FOXO1 protein complex and regulated adipogenesis.Study 2 C/EBPα regulates porcine adipocyte proliferation and differentiation via MSTRG.12568.2/FOXO3/STYX pathway3.C/EBPα transcriptional regulates MSTRG.12568.2 to promote the proliferation and differentiation of porcine adipocytessThree healthy 7-day-old Erhualian piglets were selected for adipocyte isolation,C/EBPα-specific siRNAs were used to interfere with adipocytes,andRNA samples were collected for sequencing.By analysis of theRNA-seq data(n=3),it found 4 differentially expressed lncRNAs.After coding ability and longest open reading frame(ORF)analysis,2real new differentially expressed lncRNAs were identified,namely MSTRG.10955.2 and MSTRG.12568.2,respectively.Due to the transctipts of MSTRG.10955.2 inRNA-seq data was rare,thus,MSTRG.12568.2 was selected as a candidate lncRNA.The full length of MSTRG.12568.2 was amplified by rapid amplification of cDNA ends method(RACE),and the sequencing and splicing results showed that the full length of MSTRG.12568.2 was 430 bp,including 2 exons.The dual-luciferase assary and quantitative PCR detection results showed that C/EBPα bound to the promoter of MSTRG.12568.2 and regulated its transcription.To identify the adipogenic role of MSTRG.12568.2 to adipocytes,transfecting pcDNA3.1-MSTRG.12568.2 or MSTRG.12568.2 siRNAs into adipocytes,respectively,then detected its adipogenic role.The results showed that MSTRG.12568.2promoted adipocyte differentiation and proliferation(P<0.05).4.MSTRG.12568.2/FOXO3 regulates STYX to promote the proliferation and differentiation of porcine adipocytesBy analysis and screening of differentially expressed mRNAs inRNA-seq data(n=3),97 differentially expressed mRNAs were found.Used the GO enrichment analysis to enrich97 differentially expressed genes,results found "regulation of cell proliferation" and "cell proliferation" GO enrichments in the top 20 ones,which contained 9 differentially expressed genes.Besides,the correlated regulatory network between MSTRG.12568.2 and mRNA was studied by correlation analysis,and it found that MSTRG.12568.2 was trans-correlated with serine/threonine/tyrosine interacting protein gene(STYX),meanwhile,STYX was one of the differentially expressed mRNAs enriched in "cell proliferation".By detecting the regulation effect of STYX on the proliferation and differentiation of adipocytes,it found that STYX promoted the proliferation and differentiation of adipocytes(P<0.05).Due to the trans-correlation between MSTRG.12568.2 and STYX,the key regulatory factors between MSTRG.12568.2 and the promoter of STYX were predicted by bioinformatics analysis,the binding ability of regulatory factors to MSTRG.12568.2 was verified byRNA pull-down assay,used dual-luciferase assary and quantitative PCR detection to identify regulatory role of the factors to STYX.The results showed that MSTRG.12568.2 cooperated with Forkhead box O3(FOXO3)to regulate STYX transcription in the nucleus,and FOXO3 promoted the proliferation and differentiation of porcine adipocytes as well(P<0.05).In order to further determine the regulatory effect of MSTRG.12568.2/FOXO3/STYX pathway on the proliferation and differentiation of porcine adipocytes,the expression level of STYX and proliferation and differentiation of adipocytes were detected after dualinterfering with MSTRG.12568.2 and FOXO3.Results indicated that MSTRG.12568.2cooperated with FOXO3 to regulate STYX to promote the proliferation and differentiation of porcine adipocytes(P<0.05).In summary,the results of the study 1 found that C/EBPα and FOXO1 were ceRNAs of each other,and miR-144 regulated the ceRNA effect of C/EBPα and FOXO1.Their ceRNA influenced formation of the C/EBPα-FOXO1 protein complex,thus regulated the transcriptional regulation of Adipo Q,thereby influencing adipogenesis in porcine adipocytes.Meanwhile,the results of study 2 showed C/EBPα transcriptionally regulated the expression of MSTRG.12568.2,and then,MSTRG.12568.2 cooperated with FOXO3 to transcriptionally regulate STYX,thereby promoting the proliferation and differentiation of porcine adipocytes. |
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