Alpha-fetoprotein Gene Expression And Methylation In F9 Cells During Retinoic Acid-induced Differentiation

[Background and objectives]Alpha-Fetoprotein (AFP) is a large serum glycoprotein belonging to theintriguing class of onco-developmental proteins. AFP has attracted considerableattention since it was shown that the change in its serum level during pregnancy is ahallmark of the development of numerous embryonic disorders, while the increase inits content in the plasma of adults correlates with the appearance of severalpathological conditions. The AFP gene can also be induced in other oncogenic states,such as in hepatoid adenocarcinomas occurring in tissues other than the liver, and interatoblastomas. Therefore, the AFP gene provides an excellent model to studytranscriptional regulation during mammalian development and oncogenictransformation. DNA methylation is an important mechanism in transcription, so thestudy of methylation in AFP gene expression is of great significance. Studies on thecorrelation of the methylation level of the AFP gene or its regulatory region and thegene expression level have given contradictory results. No clear correlation betweenthe expression level and the level of the structural gene demethylation in embryonicand adult hepatocytes has been observed. A few critical sites, the methylation level ofwhich correlates with possible or real activity of the AFP gene were mapped at the 5'-end region and the first intron.F9 cells are one of the most extensively studied embryonal carcinoma cells thatoriginate from a teratocarcinoma. When treated with RA and allowed to grow insuspension, F9 cells form aggregates. Most of the cells on the outer surface of theaggregates differentiate into visceral endoderm and produce AFP as a marker ofdifferentiation.This study use RA to induce F9 cells into visceral endoderm, then testthe expression and methylation status of AFP gene, in order to find out therelationship between them and identify some critical sites, the methylation level ofwhich correlates with AFP gene expression. [Materials and methods]1.RA was used to induce F9 cells differentiation. The morphological changes wereobserved using phase contrast microscope and F9 cell differentiation model wasestablished.2.Semi-quantitative reverse transcription-PCR was used to test the variation of AFPmRNA in different time points. Relative expression level was analysed with quantityone v4.4.0 after electrophoresis.3.MSP was used to test the methylation status in various time points of different sites, including AFP gene upstream promoter area, non-promoter area and the intron ofAFP gene.[Results]1. F9 cell differentiation model was successfully established. Simple embryoid bodieswere formed: cell mass was divided into three layers. Those endoderm-like cells withclear boundary were in the outside layer; base membrane was in the middle;undifferentiated cells with unclear boundary were in the inside layer.2. AFP expression varied during F9 cells differentiation. During day 0 to day 3, AFPexpression was undetectable; From day 4 to day 10, AFP expression was detectedincreasingly and at its maximum in day10; In day 11, the expression level droppeddown significantly; In day 12, little AFP mRNA was detected.3. The methylation status of different methylation site changed differently duringdifferentiation. The methylation status in primer 1 methylation site and primer 5methylation site was unchanged during differentiation. Primer 1 methylation sitestayed unmethylated and primer 5 methylation site stayed methylated. Themethylation status in primer 2 methylation site, primer 3 methylation site primer 4methylation site changed during differentiation. The methylation status of primer 2methylation site and primer 3 methylation site was linked to the expression of AFP.[Conclusions]1. F9 cell differentiation model was successfully established. Simple embryoid bodieswere formed: cell mass was divided into three layers. Further more, AFP gene expression was induced.2.The methyaltion status of both primer 1 methylation site and primer 5 methylationsite is incorrelate with AFP expression. Primer 1 methylation site which is in the AFPgene upstream CpG island stayed unmethylated and primer 5 methylation site whichis in the AFP gene upstream out of CpG island stayed methylated during AFPexpression.3.The methyaltion status of primer 2 methylation site which is in the AFP geneupstream CpG island is correlated with AFP gene expression. From day 0 to day 3when the AFP mRNA was undetectable the methylation status of this site was high; inthe following days when AFP expression was induced and increased the methylationstatus was lower obviously.4. The methyaltion status of primer 3 methylation site which is in the first intron ofAFP gene is correlated with AFP gene expression. Changing from methylation tounmethylation of this site could promote the expression of AFP gene which wasinitially silent. However, as the AFP gene expressed, methylation of this site couldnot silent AFP gene expression.5. The relationship between the methyaltion status of primer 4 methylation site whichis in the AFP gene upstream CpG island and AFP gene expression is unclear.