Study Of The Association Between Regulatory Regions Of The WWOX Gene Methylation And Its Expression In Leukemia Cell Lines Of HL-60and K562

Part I Study of the regulatory regions of the WWOX Gene methylation in leukemia cell lines of HL-60and K562Objective:The aim of the study was to to detect and analyze the level of the regulatory regions(promoter and exon1) of the WW domain-containing oxidoreductase (WWOX) Gene methylation in leukemia cell lines of HL-6and K562;To investigate the relationship between the regulatory regions of the WWOX Gene methylation and its mRNA expression.Methods:The leukemia cell lines of HL-60and K562were chosen as the object of the study. DNA was extracted from each of the two cell lines and then it was bisulfite-modified using the EZ DNA Methylation-GoldTM Kit. Methylation of the WWOX promoter and exon1were determined by methylation-specific PCR(MSP) and the same time the promoter was determined by bisulfite sequencing (BSP). Total RNA was extracted and WWOX mRNA expression was determined by realtime fluorescence quantitative reverse transcription PCR (FQRT-PCR).By this way, we can learn the relationship between the regulatory regions of the WWOX Gene methylation and its mRNA expression. Results:1. There were unmethylated bands of the WWOX promoter and exon1,and methylated bands is negative of the leukemia cell lines of HL-60by the method of MSP; The bisulfite sequencing showed that WWOX promoter have no methylated CpG sites,2. The positive bands were amplified by the methylated primer and the amplification of unmethylated primer were negative in the WWOX promoter of the leukemia cell lines of K562, and all above were detected by the method of MSP; The bisulfite sequencing showed that WWOX promoter have28(82.4%) CpG sites.3. The mRNA expression of WWOX in the leukemia cell lines of K562(0.53±0.12) is lower than in HL-60(1.25±0.14), and the difference was significant(P<0.01).Conclusion:1.There were different level of methylation in leukemia cell lines K562and HL-60. There was no methylated CpG site in the regulatory regions of the WWOX Gene in HL-60,but were methylated CpG site inK562.2.WWOX mRNA expression was significantly reduced in the region of methylated WWOX promoter of leukemia cell line K562when compared with that in HL-60with no methylated CpG sites. It points out methylation of WWOX promoter maybe is an important reason led to low expression of mRNA. Part Ⅱ The influence of Zebularine on the methylation of WWOX Gene and the mRNA expression in leukemia cell lines of HL-60and K562Objective:To investigate the change of methylation and expression of mRNA of the WWOX after using different concentrations of Zebularine (Zeb) to treat the leukemia cell lines of HL-60and K562; To analyze the meaning of using Zeb as a kind of treatment of leukemia.Methods:Treated the leukemia cell lines of HL-60and K562with Zeb, and took5-Aza-CdR as control group. Methylation of the WWOX promoter and exon1were determined by MSP and at the same time the promoter was determined by bisulfite sequencing; the WWOX mRNA expression was determined by FQRT-PCR and the protein was determined by Westen-Blot. Then analyze the level of methylation both in the experimental groups which were treated with different medicine and the control groups and their relationship with the expression of WWOX mRNA or protein.Results:1. There were methylated bands, and the unmethylated bands were negative in the region of WWOX promoter,and there were unmethylated bands, while the methylated bands were negative of the exon1of the leukemia cell lines of K562, and the all above were detected by the method of MSP before treated; There were unmethylated bands, however the methylated bands were negative of the WWOX promoter and exon1of the leukemia cell lines of K562by the method of MSP after treated by drugs; There was only one(2.9%) methylated CpG site in the WWOX promoter of K562after treated by drugs, and when compared this result with the control group, the difference is significant(P<0.01).2. There were unmethylated bands, and the methylated bands were negative of the WWOX promoter and exon1of the leukemia cell lines of HL-60by the method of MSP after the treatment of Zeb and5-Aza-CdR3. The WWOX mRNA and protein expression in the leukemia cell lines of K562was increased after Zeb and5-Aza-CdR treated, and the difference is significant (P<0.05). There is a significant differences among the different experimental groups treated with different concentration of Zeb(P<0.05); However, there is no significant differences among the different experimental groups treated with different concentration of5-Aza-CdR(P>0.05).4. There is no significant differences between drug(Zeb and5-Aza-CdR) treated and untreated group in WWOX mRNA and protein expression in the leukemia cell lines of HL-60.Conclusion:1. Zeb can play a part in the effect of indemethylation in a way of upregulating the WWOX mRNA and protein expression in the leukemia cell lines of K562, nor does HL-60.2.5-Aza-CdR can upregulate the WWOX mRNA and protein expression in the leukemia cell lines of K562.