Methylation Analysis Of OY-TES-1 Gene Promoter In Hepatoma Cells

Objective:To predict the OY-TES-1 gene promoter region, understand the methylation status of OY-TES-1 gene promoter and the influence of methyltransferase inhibitoy (5-aza-2'deoxycytidine,5-Aza-CdR) on hepatoma cell.Methods:(1) Online bioinformatics software was used to obtain OY-TES-1 gene 5' end upstream sequence, which was applied to predict promoter region and its transcription factor binding sites, locate CpG islands.(2) Genomic DNA of 6 hepatoma cell lines was extracted. Bisulfite sequencing PCR (BSP) was performed to detect methylation status of OY-TES-1 gene promoter in 6 types of hepatoma cells, QGY-7703, BEL-7404, BEL-7402, QGY-7701, SMMC-7721 and HepG-2, respectively.(3) Hepatoma cell BEL-7404 was treated by 2μmol/L DNA methyltransferase inhibitor (5-Aza-CdR) for 72h. Then Genomic DNA was exacted from those cells and methylation status of OY-TES-1 promoter was detected. The changes of methylation status before and after treatment of 5-Aza-CdR on cells were compared with that before.Results:(1) Bioinformatics analysis showed that OY-TES-1 promoter was located in-184bp-67bp and contained several potential transcription factor binding sites including 9 SP1,4 GCF and 1 CREB. There are 2 CpG islands, the 1st located in-200bp-+126bp, the 2nd located in+137bp-+587bp.(2) With BSP results methylation frequency of each hepatoma cell were calculated. The methylation frequency of these 6 type of hepatoma cells was as followed,51.25%(QGY-7701),62.08%(HepG-2),76.67%(BEL-7402), 79.58%(SMMC-7721),87.08%(BEL-7404) and 87.92%(QGY-7703), respectively.(3) Methylation frequency of BEL-7404 was greatly decreased from 87.08% to 21.25% after 5-Aza-CdR treatment.Conclusion:OY-TES-1 promoter contains CpG islands and potential transcription factor binding sites. The hepatoma cell lines tested showed higher methylation status of OY-TES-1 promoter. The methyltransferase inhibitor can greatly decline the methylation frequency of OY-TES-1 promoter in hepatoma cell.