The Regulation Mechanism Of HOXC8 On Differentiation Of Ovine Preadipocytes

Sheep is one of the most important domestic animals,which has important economic value in producing meat,skin,wool and milk.In the aspect of meat performance,the content of adipose tissue has great influence on the quality of mutton.To date,many transcription factors have been found to play important roles in adipocyte development.It is of great significance to study the molecular mechanism of fat deposition in sheep for further understanding the adipose metabolism of sheep and improving the quality of mutton.In this study,the Cp G islands and their methylation levels in the promoter and the first exon sequences of HOXC8 were analyzed by bioinformatics software and bisulfite sequencing PCR(BSP),to investigate the effect of HOXC8 DNA methylation during the differentiation of ovine preadipocytes.In order to elucidate the mechanism of HOXC8 on the differentiation of ovine preadipocytes,the overexpression and interference of HOXC8 in ovine preadipocytes were mediated by lentivirus.The target relationship between miR-301 a and HOXC8 was predicted and verified by bioinformatics software analysis and dual luciferase reporting assay.In order to elucidate the regulatory mechanism of miR-301 a on HOXC8 and the differentiation of ovine preadipocytes,miR-301 a was overexpressed and interfered within ovine preadipocytes.The main results of this study are shown as follows:1.The methylation of the first exon of HOXC8 positively regulates its expression at transcriptional level.Bioinformatics analysis showed that there was a Cp G island in the promoter region and the first exon of HOXC8,respectively.The expression level of HOXC8 before differentiation was extremely significantly higher than that after differentiation in ovine preadipocyte.Between day 0 and day 2 of ovine preadipocyte differentiation,no significant difference of methylation level was found in the promoter,but significant difference was found at the methylation level of the first exon(P<0.05).The methylation level of HOXC8 first exon was significantly higher than that of the promoter region(P<0.001).These results indicate that the methylation of the first exon of HOXC8 positively regulates its expression at transcriptional level during the differentiation of ovine preadipocytes.2.MiR-301 a inhibits the differentiation of ovine preadipocytes by targeting HOXC8.In ovine preadipocytes,overexpression of miR-301 a significantly inhibited the expression of HOXC8(P<0.001)and adipogenic marker genes that including Adiponectin(P<0.01),CEBP/α(P<0.001),FABP4(P<0.01)and PPARγ(P<0.01),and lipid droplet accumulation.While interference with miR-301 a significantly promoted the expression of HOXC8 and adipogenic marker genes like FABP4(P<0.05)and PPARγ(P<0.05)and lipid droplet accumulation.MiR-301 a significantly reduced the luciferase activity of report vectors containing the 3′-UTR of HOXC8(P<0.001).These results indicate that miR-301 a inhibits the differentiation of ovine preadipocytes and lipid droplet deposition by targeting HOXC8.3.HOXC8 promotes the differentiation of ovine preadipocytes.After overexpressing HOXC8 in ovine preadipocytes,the m RNA expression of adipogenic markers that including Adiponectin(P<0.01),CEBP/α(P<0.01),FABP4(P<0.001)and PPARγ(P<0.01)was significantly up-regulated,and the lipid droplet deposition in adipocytes was increased.In addition,interfering HOXC8 showed the opposite result.These results indicate that HOXC8 promote the differentiation of ovine preadipocytes and lipid droplet deposition.In summary,HOXC8 expression is positively regulated by the methylation of its exon 1 at the transcriptional level,and negatively regulated by miR-301 a at the post-transcriptional level.HOXC8 plays an important role in the differentiation of ovine preadipocytes.The results of these studies provide a theoretical basis for further research on the adipose metabolic mechanism of sheep.