Establishment Of PER2 Gene Promoter Methylation Detection Method And Preliminary Application In Myeloid Leukemia

Objective: A real-time fluorescence quantitative detection method based on Taq Man probe was established to detect the methylation level of PER2(Period 2)gene promoter.This detection method was used to study the level of PER2 methylation in patients with myeloid leukemia and its relationship with related gene expression and cell proliferation and apoptosis,in order to explore the role of PER2 gene methylation in the occurrence and development of myeloid leukemia.Methods:(1)According to the sequence of PER2 searched by Gen Bank,a Cp G sequence enrichment region of the PER2 gene promoter was selected,and the methylated and unmethylated target sequences were designed and synthesized according to the law of bisulfite conversion of DNA.The positive and negative reference materials of PER2 methylation were constructed,and simulated samples with a 10-fold dilution concentration were prepared.The primers and probes with the best specificity were designed and synthesized,and the method for detecting PER2 promoter methylation by Taq Man real-time fluorescence quantitative MSP(Taq Man real-time FQ-MSP)was established.The analytical sensitivity,specificity,accuracy and reproducibility of the established detection method were evaluated by using the simulated samples,and the analytical sensitivity was compared with that of conventional MSP method and q PCR based on dye method(SYBR GREEN).Moreover,the new-established Taq Man real-time FQ-MSP assay and the conventional MSP assay were used to detect the PER2 methylation level of 81 patients with leukemia,and the samples with inconsistent detection results of the two assays were sent to pyromethylation sequencing to evaluate the clinical detection performance.(2)AML cell lines HL-60 and CML cell lines K562 were cultured and treated with different concentrations of demethylation drug 5-aza-2d C.Taq Man real-time FQ-MSP was used to detect the level of PER2 methylation in cell lines.QPCR method was used to detect the expression of PER2,p21,p53,DNMT3 A and mi R-217 in cell lines.Western blot method was used to detect the expression of PER2 protein.CCK-8 method was used to detect the level of cell viability.Flow cytometry was used to detect the rate of apoptosis.Results: In this study,we established a method based on Taq Man real-time FQ-MSP to detect the methylation level of PER2 gene promoter.The minimum detection limit of Taq Man real-time FQ-MSP assay for detecting PER2 methylation level was 6 copies/μL,which is more sensitive than the conventional MSP assay,and the coefficient of variation(CV)of intra-assay and inter-assay was less than 3%.The results of detection of 81 samples of leukemia patients showed that the method established in this study had a higher detection rate and was more consistent with the "gold standard" pyrophosphate sequencing method for methylation detection.After treatment of K562 and HL-60 with 5-aza-2d C,with the increase of drug concentration,the methylation level of PER2 decreased,the expression of PER2 m RNA and protein increased,the relative expression of DNMT3 A decreased,the relative expression of mi R-217 and p21 increased,the cell viability decreased and the apoptosis rate increased.Conclusion: 1.Taq Man real-time FQ-MSP assay of PER2 methylation established in this study has high sensitivity,specificity and reproducibility and can be used for the detection of clinical samples.2.Demethylation can decrease the methylation level of PER2 promoter in HL-60 and K562 cells,which can increase the expression of PER2,inhibit cell growth and promote apoptosis.