The Prognostic Value Of Methylation Of SOX7and SOX17in Patients With Myelodysplastic Syndrome

Part I The expression of SOX7and SOX17has been downregulated by promoter methylated in THP-1and SKM-1cell line.Objective:Our aim is to investigate the effects of5-aza-2’-deoxycytidine (5-aza-2’-dC) on the expression of SOX7and SOX17genes and cell proliferation in TPH-1and SKM-1cell lines, and further to detect the methylation of SOX7and SOX17in two cell lines and verify the results of methylation-Specific PCR (MSP).Methods:The cell lines were treated with2,4and8μmol/L5-aza-2’-dC. The expression of SOX7and SOX17mRNA were detected by RT-PCT at24h,48h and96h, and cell proliferation was measured by the CCK-8methods. The methylation of SOX7and SOX17were detected by MSP and the results were verified by Bisulfite genomic sequencing (BSP).Results:We found that treatment of two AML cell lines with5-aza-dC increased SOX7and SOX17mRNA levels in a dose-and time-dependent manner (P<0.05), and increasing of SOX7and SOX17genes in THP-1was higher than in SKM-1. The results from the CCK-8assay show that the proliferation of cell lines had been inhibited by treatment with4μmol/L5-aza-2’-dC, and THP-1is more sensitive than SKM-1. The methylation of SOX7and SOX17DNA had been found in THP-1and SKM-1cell lines, and the results of BSP were consistent with MSP.Conclusion:the methylation of SOX7and SOX17genes had been found in THP-1and SKM-1, and the levels of SOX7and SOX17mRNA were increased after treatment with5-aza-2’-dC. MPS methods had been proven by BSP and could be used to detect the clinical samples. Part II Aberrant methylation of SOX7and SOX17CpG island predicts poor prognosis of myelodysplastic syndromeObjective:To investigate the aberrant methylation of SOX7CpG island and to explore its prognostic significance in myelodysplastic syndrome (MDS) patients.Methods:We detected167MDS bone marrow samples which collected by prospective methods form8hospital of Shanghai Leukemia Cooperative Group during June2003and April2009. Methylation-specific PCR (MSP) was used to detect the methylation of SOX7and SOX17. All patients had been followed until death from any cause or until the last follow-up date (4/30/2009). Overall survival (OS) and rate of leukemia transformation were predictive of prognosis. Distributions of OS curves were estimated according to the Kaplan-Meier method. Comparisons of OS between groups were evaluated by the log-rank test. X2test or Fisher’s exact test were carried to rate of leukemia transformation between different groups. The Cox regression model was used for multivariate survival analysis in order to identify the significant independent prognostic factors affecting OS.Results:Among the167MDS patients evaluated, methylation of the gene promoters was observed in SOX7and SOX17genes. The methylation frequencies were as follows:58.1%for SOX7,58.1%for SOX17. A total of69(41.3%) patients had methylation of both SOX7and SOX17genes. Aberrant SOX7and SOX17methylation was significant difference in each staged of WHO subtypes (P<0.001, P=0.007); in addition, the frequency of SOX7and SOX17methylation was significantly correlated with the IPSS risk score (P<0.001, P=0.009). The median survival of patients with SOX7and SOX17methylation was16.5months and18.5months, respectively, compared to31.8months and26.93months for patient without methylation (P=0.012, P=0.021). For the leukemia transformation, we did not find difference between methylated and unmethylated patients (P=0.131for SOX7, P=0.550for SOX17). We further classified patients into two groups according to the number of the two genes that were involved in aberrant methylation: patients with two of genes methylation were included in the high risk group, the other patients were included in the low risk group. The median survival of high risk group patients was13.23months, compared to28.37months for low risk patients (P=0.002). The classification based on the number of genes was useful predictor for leukemia evolution in patients with MDS (P=0.012).Conclusion:The methylation of SOX7and SOX17genes were presented in patients with MDS, and was a useful prognosis marker and predicts poor prognosis:methylation status of SOX7and SOX17was associated with poor OS in MDS (P=0.012, P=0.021). There were shorter OS and higher rate of leukemia transformation in the patients with both SOX7and SOX17gene methylation. Our findings suggest that the hypermethylation of SOX7and SOX17promoter may play important role in the Pathogenesis and development of MDS.