The FⅧ Gene’s Expression Does Not Depend On The Methylation Of CpG Island In Its 5’ End

Objective:To establish stable bisulfite sequencing PCR(BSP) testing platform,explore the methylation status of coagulation factor Ⅷ gene upstream CpG island in human immortalized hepatocyte LO2. detecting the methylation status of CpG island in normal liver cells and stimulated LO2 cells,explore the regulation function of methylated CpG island in FⅦ gene’s expression.Methods:We predicted the CpG island using Methprimer software, The first CpG island was divided into three separate pieces:CpG is1-1, CpG is 1-2 and CpG is 1-3; The second CpG island as a whole fragment: CpG is2. and designed BSP primers, BSP combine TA cloning and blue white spot selection, and then plasmid PCR tested ten positive monoclone and sent it to a sequencing facility, BiQ Analyzer software analysed the methylation site, and calculated the methylation rate of every CpG site using (methylated CpG sites)/(methylated CpG sites+unmethylated CpG sites). Rt-pcr detected the F VⅧ expression in LO2 cells, normal liver cells and L-Arg stimulated LO2 cells, BSP detected the methylation status of the CpG is2 in normal liver cells and L-Arg stimulated LO2 cells, compared the whole methylation status among them.Result:the 5’end of F Ⅷ gene existed two CpG islands, the CpG island 1 located in-4879 --4071,809 bp; CpG island2 located in-3600-3323,278 bp.in LO2 cells, CpG isl-1, a total of 12 CpG loci, the methylation rate was 0%; CpG isl-2 a total of 40 CpG loci, the methylation rate was 0-20%; CpG is 1-3 a total of 27 CpG loci, the methylation rate was 0-20%; CpG is2, a total of 23 CpG loci, the whole methylation rate was 80-90%. Rt-PCR showed that there is FⅧ express in normal liver cells and stimulated LO2 cells but not in LO2 cells. The methylation status of CpG is2 was 70% -100%in normal liver cells, 60%- 100%in stimulated LO2 cells. And there was no statistical difference among the three of them.Conclusion:There is a highly methylated CpG island in the 5’end of FⅧ gene in human immortalized hepatocyte LO2.and the methylation status of CpG is2 has no significant changes In LO2 cells, normal liver cells and stimulated LO2 cells, which shows that F Ⅷ gene expression does not depend on its 5’CpG island methylation.