Methylation Of CpG Island Regulates The Expression Of C/EBPβ Gene And The Differentiation Of Preadipocyte To Adipocyte

Obesity, Ⅱ type diabetes, cardiovascular disease and tumor have a close relationship, R&D drugs prevented obesity and its related diseases are important researchs.C/EBP p plays a key role during the differentiation from preadipocytes to adipocytes, C/EBP β activated C/EBP a, then several genes involved in synthesis of fat acid would be activated (such as422/ap2, SCD1, GLU4, etc.), thereby promoting the differentiation of preadipocytes into adipocytes.We have found that C/EBP β promoter sequences have been rich in CpG islands through software analysis, we suspect that methylation of CpG island controls the expression of C/EBP β gene. When preadipocytes were added no inducer, the methylation of CpG islands in the promoter was in high degree, and C/EBP β gene expression was silenced; after added with the inducer MDI, the methylation of CpG islands in the promoter of C/EBP β will be demthylated, and the expression of C/EBP β gene begins, then lead the differentiation of preadipocytes. We analyzed the association between the methylation status of CpG island in the C/EBP β gene promoter region and the expression level of C/EBP β gene. We found that the methylation status of CpG islands in the C/EBP β gene promoter regulated C/EBP β gene expression and played a important role in the differentiation of preadipocyte. In front of the first demethylation site there was a potential GATA-2protein binding site. It seemed that the expression curve GATA-2and C/EBP β expression have a negative correlation, and it was indicating GATA-2protein regulated the C/EBP β expression in a certain extent. Molecular docking with Discovery Studio software had been shown that GATA-2protein can be bound a binding site in C/EBP β gene promoter.Our previous study showed that Rehmannia extract inhibited preadipocyte differentiation and decreased the concentrations of blood glucose and lipid levels of dietary obesity. We suspected that Rehmannia extract and its active components inhibit the methylation of CpG islands in the C/EBP β gene promoter and preadipocyte differentiation. Rehmannia as one of the four Huai medicines has a high medicinal value. Studies had shown Rehmannia Extraction could inhibit preadipocyte differentiation and reduce high-fat diet-induced hyperglycemia and hyperlipidemia. Rehmannia Extraction could also inhibit the expression of C/EBP β protein in six days, but within24hours no significant inhibition of its expression was observed, it was indicating that Rehmannia extraction decreased C/EBP p gene expression not through the inhibition of CpG demethylation within C/EBP p gene promoter. RE could significantly increase the expression of GATA-2protein in1-4days of MDI induction. It was shown that the mechanism of RE inhibition of pre-adpocyte differetiation may be similar to berberine, which inhibited the expression of GATA-2protein.1、Western Blot analyzed the expression level of C/EBP β geneIn order to detect C/EBP β expression by Western Blot, we preparated the antiserum against C/EBP β protein, We constructed the cDNA clone encoding C/EBP β gene. The expression product was formed inclusion body, after preliminary purification we immunize rabbits with the product, and get antiserum against C/EBP β.Using the antiserum we analyzed the expression of C/EBP β protein during differetiation of preadipocyte. The expression of C/EBP β protein reached the highest level in1days, then decreased slowly.2、The relationship between the differentiation of preadipocyte and the methylation status of CpG islands in the C/EBP β gene promoter regionWe analyzed the promoter sequence of C/EBP β gene by software, which showed that approximately-3000bp prior to the ATG start site of C/EBP p gene had been rich in GC, existing multiple CpG islands. The methylation status of C in CpG island would affect the expression level of downstream gene, the higher methylation, the lower expression levels.We detected the methylation status of the CpG island during the differentiation of preadipocytes by BSP-PCR, Bisulfite sequencing PCR (referred to BSP) converse the cytosine (C) into uracil (T), and to make sequencing of PCR products, which would show the methylation status of CpG island. The results showed that the expression of C/EBP β protein reached the highest level in24hours, then gradually reduced,7C bases in the C/EBP β gene promoter region were methylated before induction of preadipocyte,2of the7methylated C sites are demethylated after adding the inducer, and the express of C/EBP β gene from silence to a high level. It was indicated that the demethylation of CpG island in the C/EBP p gene promoter region regulated the expression C/EBP β gene during the differentiation of preadipocyte.AZA was azacitidine deoxyribose analogue, the methyltransferase enzyme inhibitors. Methyltransferase enzyme catalyzed the methylation of DNA C bases. The expression of C/EBP β gene was observed when AZA was added during the differetiation of3T3-L1. The results showed that AZA did not increase C/EBP β gene expression, but reduced its expression. It seems that the methylation site of C/EBP β gene promoter may be conservative, not modified by methylation transferase. The expression of GATA-2and C/EBP P protein during the preadipocyte differentiation was detected and showed a negative correlation between the expression of these two proteins. When inducer was added, GATA-2protein expression was rapidly decreased, the C/EBP β protein abundantly expressed immediately. And Computer Molecular Docking proved this further:GATA-2protein could bind to C/EBP β gene promoter methylation site, indicating that when the GATA-2protein binds to the methylated site in the promoter and prevent the occurrence of demethylation, after adding inducer, the expression of GATA-2protein had been rapidly decreased, the methylation site losed the protection of GATA-2protein and became to demethylate. The mechanism of DNA demethylation has a variety of ways to go, further research and analysis are needed.3、Rehmannia glutinosa extract (RE) inhibites the expression of C/EBP β, decreases differentiation of preadipocyte and dietary obesity of young rats, but RE does not inhibit the demethylation of CpG island in the C/EBP β gene promoterWe had established a preparation method for RE, using HPLC for the quality control of each batch of herbs. It has been shown that RE inhibits preadipocyte differentiation and dietary obesity of young rats.With high performance liquid chromatography-mass spectrometry (HPLC-MS) technique, we separated RE into18components called Hx-H17, only Hx showed the strongest inhibition of preadipocyte differentiation, half of the activity was remained in Hx.With3T3-L1preadipocytes differentiation model, Western Blot showed that RE inhibited the expression of C/EBP β gene during preadipocyte differentiation. Through the establishment of high fat diet induced young rat obesity model, we found that the RE was able to inhibit obesity of young rat, as weight, body mass index, adipose tissue weight, and lower the levels of blood glucose and lipids.The experiment of RE on the methylation of CpG island in the C/EBP β promoter region during preadipocyte differentiation were showed that when RE and inducer added at the same time, in24h the expression level of C/EBP β gene did not change, but in4days, at the concentration of2mg/ml the expression level was severly inhibited. These results showed that Rehmannia extract inhibited the expression of C/EBP β gene but it did not act on CpG methylation status, it may act on other biological pathways.Observation of RE effection on the expression of GATA-2gene, we found: RE improved the expression level of GATA-2protein significantly in1-4days, inhibited of C/EBP β gene expression. It was shown that the action of RE may be similar with berberine, reducing C/EBP β gene expression through the GATA-2pathway during the preadipocyte differentiation.