Expression And Clinical Significance Of CHD1 Gene In Acute Myeloid Leukemia

This thesis will comprise two parts:PartΙ:CHD1 gene expression levels in acute myeloid leukemia:clinical features and prognostic implication;Part II:Research on pathogenesis of CHD1-RUNX1 fusion gene.PartΙ:CHD1 gene expression levels in acute myeloid leukemia:clinical features and prognostic implicationObjective:To reveal CHD1 gene expression in de novo acute myeloid leukemia and its clinical implication,we measured the level of CHD1 gene expression in 188 newly diagnosed AML patients,and retrospectively analyzed its relationship with FAB classification,cytogenetics,gene mutation and prognostic classification.Methods:1.We measured the expression level of CHD1 gene in 188 newly diagnosed AML patients,and 30 non-malignant patients by real-time quantitative RT-PCR.GAPDH was used as the internal control for CHD1 gene expression.For qualitative RT-PCR,the CT method(2^{-（CT 0f gene-CT of internal control）}was applied to quantify relative gene expression.And we divided 188 AML patients into two groups(CHD1^high group and CHD1^low group)by using cut-off vaule 0.038.2.We analysed the relationship between differential expression of CHD1 gene and clinical features,including FAB classification,cytogenetics and gene mutation,overall survival,event-free survival by statistical methods.To reveal its connection with disease progression and clinical significance.Results:1.Low CHD1 gene expression was detected in 134/188（71.3%）cases and occur in all FAB subtypes（M1-M6）.The highest incidence of CHD1 gene low expression was detected in M2 patients with 28.4%,followed by M1（23.1%）,M4（17.2%）,M5（12.7%）,M3（11.2%）,M6（7.5%）respectively.While significant association with FAB subtypes was not observed with respect to CHD1 expression levels.2.Remarkably,lower CHD1 gene expression was evident in 188 de novo AML patients compared to that of 30 patients with non-malignant diseases including newly diagnosed with iron-deficiency anemia and primary immune thrombocytopenia（P<0.0001）.Patients in the CHD1^low group were younger（median,39 vs.44 years,P=0.047）,had lower hemoglobin levels（77 vs.99.5 g/L,P=0.001）and less harbored biallelic CEBPA gene mutations（16.7%vs.31.7%,P=0.043）.No statistical difference were observed between the two groups with respect to gender,white blood cells（WBCs）and peripheral platelet counts,LDH and marrow blasts.Moreover,prognostic classification analysis revealed that the majority of CHD1^low patients had intermediate or poor prognosis（109/119,91.6%）.In the cytogenetic analysis of 188 AML patients,in 164 non-M3 AML patients,CHD1^low group occurred more frequently in patients with complex cytogenetics than those in CHD1^high group（P=0.029）.3.Among de novo non-M3 AML patients undergoing conventional induction chemotherapy,those in the CHD1^low group tended to have a lower complete remission rate than patients in the CHD1^high group,and the difference reached statistical significance（57.1%vs.75.6%,P=0.030）.However,there was no strikingly different rate of relapse between the two groups（14.7%vs.23.5%,P=0.299）.In the cohort comprising 164 AML patients,the CHD1^low group had markedly shorter OS and EFS compared with the CHD1^high group（P=0.024 and P=0.032,respectively）.The subgroup analysis of patients receiving chemotherapy regimens showed that OS and EFS were poorer in the CHD1^low group（P=0.047 and P=0.040,respectively）.In addition,it was shown that low CHD1 gene expression was also associated with poorer EFS（P=0.023）in the normal karyotype group.Multivariate analyses were performed to confirm the prognostic significance of CHD1gene expression level and it was revealed that low CHD1 gene expression had adverse impact on OS.Conclusion:CHD1 gene is a tumor suppressor gene.In our study,lower CHD1 gene expression was detected in de novo AML patients compared to that of patients with non-malignant diseases.And different levels of CHD1 gene expression were closely associated with distinct laboratory characteristics,risk stratification and prognosis.Defferential levels of CHD1 gene expression might be a new marker for risk stratification of de novo AML patients.Part II: Research on pathogenesis of CHD1-RUNX1 fusion geneObjective: To investigate the effect of CHD1-RUNX1 fusion gene expressed by recombinant adenovirus on cell proliferation and apoptosis in mouse fibroblast NIH3T3 cells.Methods: 1.To establish the NIH3T3 cells stably expressing CHD1-RUNX1 fusion protein,we constructed Adenoviral vector（p HBAd-MCMV-REP）containing CHD1-RUNX1 fusion gene and used it to infect NIH3T3 cells.The cells were divided into three groups :Ad-CHD1-RUNX1 group,Ad-RFP group and the untreated cells（NIH3T3）were set as control.2.We used fluorescent microscopy to assess the infection efficiency,optimal infection time and MOI of Ad-CHD1-RUNX1.And expression of CHD1-RUNX1 in NIH3T3 cells was examined by RT-PCR.3.To observe the effect of CHD1-RUNX1 fusion gene on cell proliferation in vitro,CCK8 assay was used.And flow cytometry was used to dectect the cellular apoptosis.Results: 1.The highest infection efficiency of NIH3T3 cells with Ad-CHD1-RUNX1 was about 70% when the infection time was 48 h and with the MOI 150 cfu/ml.The expression of CHD1-RUNX1 m RNA was detected by RT-PCR 48 h after transfection.2.Compared to empty vector group,the growth ability of NIH3T3 cells in experimental group was significantly improved（P<0.05）.Additionally,lower apoptosis rate（4.22%）was detected by flow cytometric analysis in experimental group.Conclusion: In summary,our results indicated that recombinant adenovirus Ad-CHD1-RUNX1 could promote NIH3T3 cells proliferation potential in vitro,and inhibited the cellular apoptosis in NIH3T3 cells.The results of this study initially illustrated the biological behavior of CHD1-RUNX1 fusion gene,and provided experimental basis for the further study on pathogenesis of the fusion gene in leukemia.