| Objective: The hypermethylated region of the SOCS3 gene promoter has been found in most malignant tumours,but the correlation between SOCS3 methylation and acute lymphoblastic leukaemia（ALL）has rarely been explored.This study compared and analysed the clinical characteristics of ALL cells with different levels of SOCS3 gene methylation and further verified the effect of SOCS3 gene methylation on ALL cells.Methods:In this study,the included children were divided into rehabilitation control group and study group through the induction and collation of clinical data.BSP technology was used to detect the difference in methylation level of SOCS3 gene CPG island in children with acute lymphoblastic leukemia at different treatment stages.The differences in CPG island methylation of SOCS3 gene between ALL cell lines and PBMC were detected,and the clinical characteristics of children with different methylation levels were collected and discussed.SOCS3 gene expression differences in different cell lines were detected by fluorescence quantitative PCR.Annexin V-FITC/PI combined flow cytometry was used to detect the effects of Annexin V-FITC/PI on cell cycle and apoptosis,and CCK-8 assay was used to detect cell proliferation.Western blot was used to detect the protein expression levels of SOCS3,Caspase-3 and PUMA.SPSS 22.0 software was used for statistical analysis and Graph Pad PRISM 7.0 software was used for graphic production.The count data were expressed as percentages and analyzed by Chi-square test and Fisher’s exact test.Measurement data were expressed as mean ± standard deviation and analyzed by an OVA.T test was used to compare measurement data between the two groups,and one-way an OVA was used for measurement data between multiple groups.P < 0.05 was considered statistically significant.Results: Through statistical analysis,we found that the methylation rate of SOCS3 gene in the newly diagnosed group was 20.7%,and the median methylation density was 19.72%（8.00% ～ 41.21%）.The median methylation density was 30.13%（19.85% ～ 43.98%）in the recurrence group（31.0%）.In the complete remission group,the median methylation density was 4.65%（2.77% ～ 15.25%）.The SOCS3 gene in the control group was almost not methylated,and the difference was statistically significant.The cumulative recurrence rate of the SOCS3 high methylation group was significantly higher than the low methylation group.Fluorescence quantitative PCR was used to verify that SOCS3 gene was significantly overexpressed in control peripheral blood monocytes（P < 0.0001）.The Cp G island of SOCS3 gene was differentially methylated in ALL cells and PBMC by bisulfite sequencing polymerase chain reaction（BSP）.After demethylation treatment,SOCS3 gene expression was significantly up-regulated in ALL cells.Flow analysis showed that the high expression of SOCS3 significantly increased the apoptosis of ALL cells,and Western-blot results showed that the demethylation of SOCS3 might promote cell apoptosis by inducing the expressions of apoptosisrelated proteins such as Caspase3 and PUMA.Further studies showed that SOCS3 demethylation blocked the induced cell cycle in the G0/G1 phase.Conclusion:（1）The CIR of the high level SOCS3 methylation group was significantly higher than that of the low level SOCS3 methylation group.There was no significant difference in overall survival rate between high and low levels of SOCS3 gene methylation.（2）Fluorescence quantitative PCR confirmed that SOCS3 gene was significantly over expressed in control peripheral blood mononuclear cells compared with ALL cells.By BSP analysis of ALL cell lines,we found differential methylation of SOCS3 gene Cp G island in peripheral blood mononuclear cell lines of ALL and healthy controls.（3）We found that the demethylation of SOCS3 significantly inhibited the growth of ALL cells.Further studies showed that SCOS3 demethylation induced cell cycle arrest in G0/G1 phase and simultaneously promoted apoptosis of ALL cells. |
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