Study On The Function Of C/EBPZ In Fat Cells And Its Expression Regulation Mechanism

Objective:Adipocytes are important energy-storage and endocrine regulatory cells in human being and animals.GATA2/3 played important regulatory roles in adipocyte formation.Our previous studies showed that C/EBPZ might bind to the promoter of GATA2/3 to regulate their transcription.At present,the function of C/EBPZ in adipose tissue and its transcriptional regulation mechanism were not clear.The purpose of this study is to explore the function and transcriptional regulation mechanism of C/EBPZ expression in adipose tissue using bioinformatics and cellular and molecular biology methods.Methods:1.Bioinformatics analysis: The expression pattern of C/EBPZ in human adipose tissue was studied using combining human protein atlas(HPA)database information with statistical methods.2.Cell biology experiment: p CMV-HA and p CMV-HA-C/EBPZ plasmids were transfected into chicken proadipocytes to realize C/EBPZ overexpression.The experiment was divided into EV group(transfected p CMV-HA plasmid)and C/EBPZ overexpression group(transfected p CMV-HA-C/EBPZ plasmid).The differentiation of preadipocytes induced by 100 μM oleate for 48 h was observed by oil red O staining.Preadipocyte proliferation was tested using the Cell Tite Glo(?) Luminescent Cell Viability Assay kit.The cell cycle of preadipocytes was detected by flow cytometry.The effects of overexpression of C/EBPZ on the promoter activities of PPARγ,GATA2,GATA3,LPL,FASN,C/EBPα and FABP4 in preadipocytes,the promoter activity of C/EBPZ gene 5’ flanking region,and the regulation of C/EBPZ promoter activity by KLF2 overexpression were investigated using luciferase reporter assay.3.Molecular Biology experiments: RNA-seq and bioinformatics techniques were used to study the changes of overexpressed C/EBPZ on the transcriptomic expression patterns of preadipocytes.The expression of C/EBPZ,PPARγ and KLF2 in preadipocytes was analyzed by Western blot.Real-time PCR was used to study the effect of overexpressed C/EBPZ on the transcription of some functional genes in chicken preadipocytes.The 2031 bp sequence upstream of chicken C/EBPZ transcription start site was obtained by molecular cloning,and its promoter sequence was analyzed by truncation mutation technique and luciferase reporter assay.The 179 bp long chicken C/EBPZ promoter sequence was studied by DNA pull down,and the regulation mechanism of KLF2 on C/EBPZ expression was studied by luciferase reporter assay,expression analysis,site-directed mutation and Ch IP-PCR.Results:1.Expression pattern of C/EBPZ in adipose tissueC/EBPZ was expressed in human adipose tissue and down-regulated with the increase of adipose cells(P<0.05).The expression level of C/EBPZ in high metabolism abdominal adipose tissue was significantly lower than that in subcutaneous adipose tissue(P<0.05).With the increase of age,the expression level of C/EBPZ in adult adipose tissue was significantly up-regulated(P<0.05).2.C/EBPZ regulates the proliferation and differentiation of chicken preadipocytesRNA-seq showed that compared with the control group,448 genes and 354 genes in overexpressed C/EBPZ cells were up-regulated and down-regulated,respectively.Gene enrichment analysis(GSEA)showed that C/EBPZ may be involved in the pathological process of infectious diseases(GO:0019048/GO:0044003)and the regulation of adipose tissue development(GO:0060612).Real-time PCR analysis showed that C/EBPZ was expressed in chicken proadipocytes,and the expression of C/EBPZ was down-regulated with differentiation of chicken proadipocytes induced by oleate-(P<0.05).Cell proliferation experiments showed that overexpression of C/EBPZ promoted the proliferation of chicken proadipocytes.Flow cytometry analysis showed that compared with the control group,there was no significant difference in the proportion of preadipocytes in G2 phase and S phase in C/EBPZ overexpressing cells(P>0.05),however,the proportion of G0 cells was significantly lower than that of EV group(P<0.05).In addition,oil red O staining showed that overexpression of C/EBPZ inhibited the oleateinduced differentiation of chicken preadipocytes(P<0.05).Luciferase reporter assay showed that overexpression of C/EBPZ significantly inhibited the promoter activities of PPARγ,C/EBPα,LPL and FASN in chickens(P<0.05),and promoted FABP4 and GATA2 promoter activities(P<0.05),it may also promote GATA3 promoter activity(P=0.07).Real-time PCR and Western blot showed that overexpression of C/EBPZ significantly inhibited the expression of PPARγ in chicken preadipocytes(P<0.05).3.Transcriptional regulation of C/EBPZ expression in chickensThe 5’ flanking region of chicken C/EBPZ(-2025 bp/+6 bp)showed obvious promoter activity(P<0.01).The promoter plasmid p GL4.10-C/EBPZ(-2025/+6)was co-transfected with overexpressed plasmids GATA2,GATA3,KLF2 and KLF3.Luciferase reporter assay showed that compared with EV group,overexpression of KLF2 significantly promoted promoter expression of chicken C/EBPZ(-2025/+6)(P<0.01),overexpression of KLF3 inhibited the expression of chicken C/EBPZ(-2025/+6)promoter(P<0.05),GATA2 and GATA3 had no significant effect on chicken C/EBPZ(-2025/+6)promoter(P>0.05),overexpression of C/EBPZ it self had no significant effect on chicken C/EBPZ promoter(P>0.05).For the six of 5’ flanking region of chicken C/EBPZ(-94 bp/+6 bp),(-173 bp/+6 bp),(-485 bp/+6 bp),(-793 bp/+6 bp),(-1409 bp/+6 bp)and(-2025 bp/+6 bp),luciferase reporter assay showed: all these sequences showed promoter activities(P<0.01).and(-94 bp/+6 bp)may be a basic promoter region for C/EBPZ transcription.The C/EBPZ promoter reporter plasmid was co-transfected with KLF2 overexpression plasmid,and the results showed that: KLF2 greatly promoted luciferase activity of C/EBPZ(-94/+6)promoter(P<0.01).in addition,with the mass increase of p CMV-myc-KLF2 plasmid,C/EBPZ luciferase activity gradually increased,and there was a significant dose effect(P<0.001).Real-time PCR and semi-quantitative PCR showed that KLF2 up-regulated C/EBPZ m RNA expression(P<0.01).Western blot results showed that overexpression of KLF2 promoted the protein expression of C/EBPZ.The results of Ch IP-PCR showed that there were multiple nucleotide sequence binding sites between KLF2 and C/EBPZ promoter sequence.Point mutation of the C/EBPZ(-94/+6)plasmid and co-transfection with overexpressed KLF2 showed that the luciferase activity of mutant p GL4.10-C/EBPZ(-94/+6)was lower than that of wild type p GL4.10-C/EBPZ(-94/+6)(P<0.01).A total of 189 proteins binding to chicken C/EBPZ promoter(-173 bp/+6 bp)were screened by DNA pull down.In COG analysis,12 factors related to transcription regulation were found.GO and KEGG enrichment analysis showed that C/EBPZ was correlated with ribosome structure formation,cytoplasmic large ribosome subunit,nucleoli and ribosome.Enrichment analysis of Reactome showed that proteins were mainly related to RNA metabolism,ubiquitination,ubiquitin E3 ligase ubiquitination target protein,exon junction complex enhancement and meaningless mediated decay.Panther database enrichment analysis revealed that the protein was associated with P53 and Wnt signaling pathways.Conclusion:1.C/EBPZ is an important regulator for the formation of adipose tissue.Overexpression of C/EBPZ could inhibit adipocyte differentiation and promote adipocyte proliferation by inhibiting the expression of PPARγ and C/EBPα.2.The(-2025 bp/+6 bp)sequence upstream translation start site of chicken C/EBPZ gene showed obvious promoter activity.The basic promoter may be located in the(-94 bp/+6 bp)sequence upstream of the translation initiation site.KLF2 might act on chicken C/EBPZ promoter sequence to up-regulate C/EBPZ expression.The upstream(-173 bp/+6 bp)sequence of the chicken C/EBPZ translation start site could bind to ribosomal proteins and was associated with protein translation.