Screening For Hypermethylation Abbrrent Genes In Myelodysplastic Syndrome Using Genomic Promoter-CpG Island Microarrays

PartⅠPreliminary screening for hypermethylated genes in SKM-1 using genomic promoter-CpG island microarraysObjective:To analyse and compare the methylation status of CpG islands in promoter between myelodysplastic syndrome(MDS) cell line SKM-1 and control samples using methylated DNA immunoprecipitation(MeDIP) on chip. So we can find hypermethylated genes in SKM-1 compared with control and finally find better target genes in epigenetics contributing to the diagnosis or therapy of MDS.Methods:To collect peripheral blood of twenty healthy persons, extract genomic DNA and mix equally. We consider the mixture as control samples. Extract genomic DNA from SKM-1 cells meanwhile. All kinds of DNA were sonicated to random fragments in size about 500bp. Immunoprecipitation of methylated DNA was performed using mouse monoclonal antibody against 5-methylcytidine and BiomagTM magnetic beads coupled anti-mouse IgG Immunoprecipitated DNA was eluted and purified by phenol chloroform extraction and ethanol precipitation. The Input and IP DNA were labeled with Cy3-and Cy5-labeled random 9-mers, respectively, and hybridized to NimbleGen HG18 CpG Promoter arrays, which is single array design containing all known CpG Islands and all well-characterized promoter regions. Scanning was performed with laser scanner. The intensity of Cy3 and Cy5 was analysed with NimbleScan software. And then we used SignalMap software according to Cy5:Cy3>2(log2 Cy5/Cy3>1) as standard to screen high methylated gene of SKM-1 compared with control in order to find high methylated gene in SKM-1 but no or hypomethylated gene in control, meanwhile located hypermethylated abrrent gene of SKM-1.Results:we initially screened 849 genes which were high methylated in SKM-1 but no or hypomethylated in control. According log2 Cy5/Cy3, we finally chose 6 genes, that were DMRTA2,PAX6,GDNF,POU4F3,LHX1 and PGR. There were 1 to 4 CpG island displaying hypermethylation status in the promoter region of these genes.Conclusion:We found six hypermethylated aberrant genes by methylated gene microarray screening. This will establish the foundation for looking for specific hypermethylated abrrent gene in myelodysplastic syndrome. This study provided a solid foundation for the next experiment and made it easy for screening hypermethylation abrrent genes of myelodysplastic syndyrome.PartⅡBisulfite genomic sequencing PCR for genes initially screened using genomic promoter-CpG island microarraysObjective:Bisulfite genomic sequencing polymerase chain reaction(BSP) were performed in six genes initially screened using methylated microarray in partⅠstudy. To calculate the methylation ratio in CpG island of each gene and theck the accuracy of results in previous study.Methods:To collect bone marrow aspirate of 3 other healthy persons, extract genomic DNA separately. Extract genomic DNA from SKM-1 cells meanwhile. All kinds of DNA were treated with bisulfite, purified and amplified with polymerase chain reaction(PCR). The PCR products were sequenced to identify CpG site methylation situation. Results:The results between two parts were matched well. Among them, GDNF and PAX6 genes were the most obvious. The difference of CpG sites methylation rate between SKM-1 and control in these two genes is very obvious. CpG site locating in these two genes of control DNA has almost no methylation while 100 percent methylation in SKM-1.Conclusion:The method like methylated DNA immunoprecipitation(MeDIP) on chip used for detecting methylation abrrent genes among diffenent cells and tissues is practical. The results from BSP verified the methylation status of these six genes and were considered applicable for future study in function.PartⅢEffects of 5-azacytidine on the growth inhibition of SKM-1 and reversion of hypermethylated genesObjective:To treat SKM-1 cells with 5-aza in different concertrations, observe the varieties in biological characteristics of SKM-1 cells such as growth,apotosis and cell cycle distribution. To explore the mechanics of 5-aza in reversing hypermethylation combined with six genes in previous study. To investigate correlation between DNA methylation alteration and SKM-1 cells as well as to explore mechanisms of 5-aza inhibiting SKM-1 cells and reversing methylation.Methods:SKM-1 cells were treated with 5-aza in concertration among 2μmol/L to lOμmol/L for 24 to 48 hours. Cell growth speed was measured of different concertrations using counting plate under light microscope. And then three groups were chosen for next study, that is low concertration group(2μmol/L),high concretration group(8μmol/L) and SKM-1 with no 5-aza used(control). Cell morphology changes were determined under light microscope and electron microscope, cell cycle distribution and apotosis rate were estimated using flow cytometry, methylation status of these six genes was determined using methylation-specific PCR and mRNA expression of them was determined using RT-PCR. Results:After treatment with 5-aza, significant inhibiting effects were detected in SKM-1 cells. The growth rate was decreased gradually along with the increase of 5-aza. In the treatment group, S phase of SKM-1 cells decreased obviously, and apotosis rate increased apparently. The overt apotosis was detected under electron microscope.5-aza could reverse the methylation status of GDNF and PAX6 genes, increase their mRNA expression. While there was no mRNA expression of DMRTA2,PGR,LHX1 and POU4F3 genes. Conclusion:5-aza inhibits tumor cell growth, decreases cell cycle and increases mRNA expression of GDNF and PAX6 in SKM-1 cells.5-aza inhibits the malignant phenotypes of human SKM-1 cells and reverses hypermethylation of these two genes. Multiple mechanics may be involved in other four genes.Part IVCharacteristic research about methylation status of GDNF and PAX6 genes in primary myelodysplastic syndromeObjective:To study CpG island's methylated character of GDNF and PAX6 genes in primary myelodysplastic syndrome(MDS) using methylation-specific PCR. To establish a stable method about detecting methylation abbrrent genes and build a foundation for MDS's early diagnosis and prognosis judgement.Methods:To collect bone marrow aspirate samples from 5 patients with MDS. The specimen were anticoagulated by heparin. The bone marrow mononuclear cells (BMMC) from specimen were separated by lymphocyte separation medium. CD34+cells were sorted from BMMC using magnetic beads. And then DNA was extracted and modified by bisulfite, finally GDNF and PAX6 genes were amplified by PCR. PCR product were loaded onto 2% agarose gels and visualized by ethidium bromide staining.Results:PAX6 gene displayed positive methylated band but negative unmethylated band in patients with MDS to AML and MDS-RAEB. It also displayed same results in patient with haematological remission of MDS. GDNF gene displayed both positive methylated band and unmethylated band in patients MDS to AML and MDS-RAEB. It displayed only positive unmethylated band in patients with MDS in haematological remission. While there was only positive methylated band in patient with refractory anemia with ringed sideroblasts.Conclusion:The methylated PCR product of GDNF and PAX6 in 5 patients with MDS was partly in accordance with results from MDS cell line SKM-1.It may be due to the individual difference and the heterogeneity of MDS. Additionally MSP itself consist in false negative and false positive results. However, many tasks remains to be done, such as the clinical validation of epigenetic biomarkers to allow the accurate prediction of the outcome of cancer patients, their potential chemosensitivity to current pharmacological treatments and diagnostic value in MDS.