Study On The Regulation Of CBX7 In Proliferation And Differentiation Of Sheep Preadipocytes

The fat content of mutton directly affects its meat quality.The proliferation and differentiation of preadipocytes are important biological processes for fat development and function maintenance.Studies have shown that CBX7 and SP1 play an important role in lipid metabolism.The mice which knockout CBX7 showed an obese phenotype.Overexpression of CBX7 in vitro inhibited the differentiation and lipid deposition of 3T3-L1 cells.The addition of SP1-specific inhibitors to human mesenchymal stem cells inhibited adipocyte differentiation and lipid vesicle formation.However,the effects and regulatory mechanisms of CBX7 and SP1 on the proliferation and differentiation of sheep preadipocytes are not clear.In this experiment,3 different adipose tissues and tail pre-adipocytes of sheep were the research objects.Immunocytochemistry is used to determine where the CBX7 protein functions in sheep preadipocytes.Real-time Quantitative PCR(RT-q PCR)was used to detect the expression pattern of CBX7 in different adipose tissues of sheep.PCR was used to clone full-length CDS(coding sequence)of sheep CBX7 and SP1.Bioinformatics software was used to analyze nucleic acid and amino acid sequences in the CBX7 and SP1 coding regions.The expression of CBX7 and SP1 in sheep preadipocytes were overexpressed or inhibited by lentivirus.The regulation effects of CBX7 and SP1 in sheep preadipocytes were studied by RT-q PCR,Western blotting,CCK-8 and Oil Red O staining.Five vectors of CBX7 promoter fragments with different lengths were constructed and transfected into C3H10T1/2 and sheep preadipocytes.The luciferase activity of each deletion fragment was detected by dual luciferase reporter assay to determine the key regions affecting the transcriptional activity.In order to study the effect of SP1 on the differentiation of sheep preadipocytes by regulating CBX7,bioinformatics methods and dual luciferase reporter system experiments predicted and verified the binding site of SP1 on the CBX7 promoter;RT-q PCR was used to detect the expression of CBX7 after SP1 was overexpressed or inhibited in sheep preadipocytes.The results of this study are as follows:1.Cloning,expression analysis and subcellular localization of CBX7 in sheepThe length of sheep CBX7 CDS was 756 bp,encoding 251 amino acids.CBX7 protein had 109 potential phosphorylation sites,and the conserved domain analysis showed that CBX7 had one Chromodomain and one Polycomb repressor box.Sequence similarity analysis showed that sheep CBX7 CDS and its coding sequence had the highest similarity with Capra hircas,followed by Bos taurus,Sus scrofa,Equus caballus,Capra hircas and Homo sapiens,and the lowest similarity with Mus musculus and Rattus norvegicus,which was consistent with the phylogenetic analysis results.The expression level of CBX7 m RNA was the highest in adipose tissue of sheep tail,followed by perirenal adipose tissue and the lowest in subcutaneous adipose tissue.CBX7 was expressed in both C3H10T1/2 cells and sheep preadipocytes,and was localized in the nucleus.2.CBX7 promotes the proliferation of sheep preadipocytesCompared with the control group,the overexpression of CBX7 in sheep preadipocytes extremely significant up-regulated the m RNA expression of CBX7,Cyclin B and PCNA(P<0.01),and significantly up-regulated the m RNA expression of Cyclin D(P<0.05).);Cell viability is significantly enhanced.Compared with the control group,the inhibition of CBX7 in sheep preadipocytes extremely significant down-regulated the m RNA expression of Cyclin D(P<0.01),and significantly down-regulated the m RNA expression of CBX7,Cyclin B and PCNA(P<0.05);Cell viability is significantly reduced.These results showed that CBX7 promotes the proliferation of sheep preadipocytes.3.CBX7 inhibits the differentiation of sheep preadipocytesThe expression of CBX7 decreased with during the differentiation of sheep preadipocytes.When overexpressing CBX7 in sheep preadipocytes,the m RNA expression levels of adipogenic marker genes PPARγ,C/EBPα,Adiponection and FABP4 were were significantly down-regulated detected(P<0.01).After inhibiting CBX7,the m RNA expressions of PPARγ,C/EBPα and FABP4 were significantly up-regulated(P<0.05),Adiponection was extremely up-regulated(P<0.01).These results showed that CBX7 inhibites the differentiation of sheep preadipocytes.4.Transcription factor SP1 promotes CBX7 expressionAfter transfecting five promoter-deficient vectors into C3H10T1/2 and sheep preadipocytes,the CBX7promoter-554~+302 region showed the strongest fluorescence activity.SP1 binding site was found in the-311~-298 bp region of CBX7 promoter.After overexpressing SP1,the m RNA expression of CBX7 was significantly increased(P<0.05),and extremely significantly decreased when inhibitiing SP1(P<0.01).These results showed that transcription factor SP1 promotes CBX7 expression.5.Transcription factor SP1 promotes differentiation of sheep preadipocytesThe total length of sheep SP1 CDS was 2 340 bp,encoding 779 amino acids.SP1 was localized in the nucleus and has 109 potential phosphorylation sites,and three zinc finger domains were found in both secondary and tertiary structures.Sequence similarity analysis showed that sheep SP1 CDS and its coding sequence had the highest similarity with goat,followed by cow,horse,pig,human,mouse and rat,and the lowest similarity with chicken.Phylogenetic analysis results were consistent with these results.When overexpressing SP1 in sheep preadipocytes,the m RNA expression levels of SP1 and adipogenic marker genes was significantly up-regulated(P<0.01).Inhibition of SP1 resulted in the opposite result.These results indicates that SP1 promotes the differentiation of sheep preadipocytes.In conclusion,SP1 has a positive regulatory effect on the differentiation of sheep preadposytes;CBX7can promote the proliferation of sheep preadipocytes;CBX7 is regulated by SP1 and has an inhibitory effect on the differentiation of sheep preadipocytes.