The Experimental Research On The CpG Island Methylation Of Progesterone Receptor's Promoters In Leukemia

Objective:The research of promoter methylation before was mainly focused on mono-promoter gene. But the gene regulation of multiple promoters is more complex than that of mono-promoter. Research of methylation of progesterone receptor (PR) with two promoters was mainly focused on steroid-related neoplasms. This experiment was designed to disclose the methylation status of PR double promoters and clarify the significance of PR promoter methylation in silencing PR expression in leukemia. The correlations of PR methylation and PR expression together with the methyltransferase (DNMT1) were further studied which provided theory basis for the relation between the epigentic and the formation of neoplasm and new direction for the therapy of neoplasm. Methods:1. Treatment with 5-Aza CdR on and transfection with antisense oligonucleotide MG88 into leukemia cell lines HL-6CK K562 and Jurkat2. Using methylation specific PCR (MSP) to detect the CpG island methylation changes of PRA & PRB promoters in the leukemia cell lines before and after 5-Aza CdR treatments and the MG88 transfection.3. Using RT-PCR to detect the expressions of PRAB and PRB before and after5-Aza CdR treatment; and using semi-quantitative RT-PCR to detect the expressions of DNMT1, PRB mRNA in the leukemia cell lines before and after 5-Aza CdR treatment and the MG88 transfection.4. Using MSP to detect the methylation status of PRA and PRB in 44 cases of patients with leukemia and 10 normal samplesResults:1. MSP analysis showed CpG islands of PRA & PRB promoters were hypermethylated without unmethylation bands and PRAB & PRB mRNA expressions were inactivated in the three leukemia lines HL-60, K562, Jurkat.2. CpG islands of PRA and PRB promoters were demethylated and PRAB and PRBmRNA were re-expressed after 5-Aza CdR treatment.3. DNMT1 mRNA was decreased notably compared with that before 5-Aza CdR treatment(p<0.001)4. After two days transfection in MG88 groups, the DNMT1 mRNA expression was significantly downregulated in the three leukemia cell lines(p<0.001).Along with the elongation of the transfection time, the DNMT1 mRNA was also decreased gradually. Whereas there wasn't such result in control MG208 groups.5. After transfection in MG88 groups, the DNMT1 mRNA was decreased notably in a dose-dependent manner(p<0.01). The most suitable concentration of MG88 was 40nM.6. After transfection in MG88 groups, PRB mRNA was significantly upregulated(p<0.01)from the second day. PRB expression had negative coefficient correlation with DNMT1 mRNA (Spearman, r=-0,900, p = 0.037) . Whereas there wasn't such result in control groups.7. After transfection in MG88 groups, the CpG islands of PRA and PAB promoters were demethylated from the second day. Whereas there wasn't such result in control groups.8. The positive rates of PRA & PRB methylation in 44 cases of leukemia were 65.9% and 61.4%, respectively. But the PRA and PRB in normal samples were unmethylated. Three cases of patient had PRA hypermethylation but PRB unmethylation. Another case had PRB hypermethylation but PRA unmethylation. Five cases of patient had PRA/PRB methylation partly.Conclusions:1. Two isoforms of progesterone receptor, the CpG islands of PRA & PRB promoters were hypermethylated in leukemia cell lines. Furthermore, both mRNA of the two isoforms had no expressions.2. After 5-Aza CdR treatment, the CpG islands of PRA & PRB promoters were demethylated. Furthermore, PRB mRNA was re-expressed, suggesting that inactivation of PRB was caused by methylation in leukemia.3. Analyzing DNMT1 mRNA before and after 5-Aza CdR treatments, we can conclude that 5-Aza CdR may induce PR demethylation through decreasing DNMT1 mRNA expression.4. Antisense oligonucleotide MG88 can downregulate the DNMT1 mRNA expressions in leukemia cell lines. The DNMT1 mRNA expressions were decreased gradually in doseand time dependent manners.5. MG88 can have the CpG islands of PRA & PRB prom...