Screening Of Genes Associated With The Transformation Of Myelodysplastic Syndrome To Acute Leukemia And Preliminary Study Of TUBB Gene

PartⅠFoundation of MDS nested case-control study cohort and study of risk factors associated with MDS evolution to leukemiaObjective To establish a nested case-control study cohort in myelodysplastic syndrome (MDS) patients and investigate the clinical characteristics, WHO subtype and risk factors associated with MDS evolution to leukemia of this cohort.Methods All patients,≥18 years of age, presenting at 24 Shanghai hospitals with initial clinical findings consistent with a hematopoietic abnormality between June, 2003 and April,2007 were candidates for inclusion in this study. Every patient provided blood and bone marrow samples at baseline. Diagnosis was made by incorporating morphologic, immunophenotypic, cytogenetic, and molecular features according to WHO classification criteria. Cytogenetic analysis was performed using conventional G-banding karyotyping and fluorescence in situ hybridization (FISH) techniques. Cumulative risk of evolution was estimated by Kaplan-Meier method. Prognostic factors were evaluated by univariate Log-rank method and multivariate Cox proportional hazard models.Results A total of 435 patients were diagnosed as MDS. The median age of onset of MDS was 58(18-90) years, with 248 male patients and 187 female patients (male: female 1.33:1). The percentage of cases with refractory cytopenia with multilineage dysplasia (RCMD) was the highest (65.5%), while that of RA (2.3%), RARS (1.1%) and 5q-syndrome (0.5%) was lower. Trisomy 8 (+8) was the most common chromosome abnormalities (71cases,12.7%). The mean follow-up period was 20.3 (4.2～57.1) months. Cases were patients with evolution by the end of follow-up, while controls were patients without evolution by that time. Case group included 41 patients and control group included 342 patients. Univariate analysis showed age, gender, WHO subtype, WBC count, absolute neutrophil count (ANC), IPSS cytogenetic subgroup, IPSS group and bone marrow blast percentage were significant factors for Leukemia free survival (LFS). Multivariate analysis of COX model showed age, gender, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast were independent factors for LFS.Conclusion We established a nested case-control study cohort of MDS patients. The clinical characteristics and WHO subtype of MDS patients in Shanghai were different from that in western countries. The independent risk factors for MDS evolution were age, gender, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast percentage.PartⅡScreening of genes associated with evolution of myelodysplastic syndrome to acute leukemiaObjective To investigate the genes related to progression of MDS to acute leukemia so as to identify specific molecular marker predicting evolution of MDS to acute leukemia.Methods Based on our nested case-control study cohort of MDS patients, we chose paired patients for gene expression microarray test, in 1:1 ratio, according to age, gender, WHO subtype and IPSS cytogenetic subgroup. We analyzed the gene expression profile of patients in case group (with evolution) at diagnosis and after evolution (self control study). Also, we compared the gene expression profile of paired patients (case-control study) at diagnosis. By incorporating the results of two parts, we identified genes related to transformation of MDS to acute leukemia.Results A total of 958 deregulated genes were identified according to bioinformatics analysis by incorporating the results of self control study and case-control study. These genes are involved in many biological processes, including cell growth, cell differentiation, cell adhesion, signal transduction, stress and protein metabolism. We further analyzed the 958 genes and identified a subset of 6 genes that distinguishes between case and control group, including TUBB, PSMD1, SLC7A5, ATG3, TUBB2C and TIMM10.Conclusion Combing gene expression microarray and nested case-control study method, we identified a subset of 6 genes that distinguishes between case and control group. The 6 genes may play critical role in evolution of MDS to acute leukemia.PartⅢTUBB gene expression in MDS patients with and without evolution:a nested case-control studyObjective To study the differential expression of TUBB gene in MDS patients with evolution and that without evolution in our MDS nested case-control study cohort.Methods Based on our nested case-control study cohort of MDS patients, we chose paired patients in 1:1 ratio, according to age, gender, WHO subtype, IPSS cytogenetic subgroup and follow-up period (≥1 year). We examined TUBB gene expression changes in paired patients from case group (with evolution) and control group (without evolution), using quantitative real time PCR.Results We chose 11 patients in each group of our MDS nested case-control study cohort excluding patients underwent gene expression microarray test. TUBB gene expression of MDS patients in case group were significantly higher than that in control group (P<0.01).Conclusion TUBB gene expression was significantly higher in case group than that in control group of our MDS nested case-control study cohort, indicating that TUBB gene may play a role in evolution of MDS into acute leukemia. Part IV The study of effect of small interfering RNA against TUBB gene on SKM-1 cell lineObjective To investigate the effect of small interfering RNA (siRNA) against TUBB gene on SKM-1 cell line.Methods TUBB gene independent siRNA (TUBB-siRNA) were synthesized chemically. Transient transfection with TUBB-siRNA was implemented by LipofectamineTM2000 according to the manufacturer's instructions. Cells were typically seeded in 24-well plates before transfection. Transfection complexes were prepared in sereum-free medium. Immediately before transfection, complete growth medium was removed from all wells, and wells were rinsed with PBC. Cells were typically exposed to transfection complexes for 6 hours before replacement of complete growth medium. Cells were divided into 6 groups:no transfecion group (UT), negative control group (NS), positive control group (PS), TUBB-siRNA1 group (S1), TUBB-siRNA2 group (S2), and TUBB-siRNA3 group (S3). The effect of TUBB-siRNA on the growth rate of SKM-1 was tested by CCk-8 kit and flow cytometry. The effect of TUBB-siRNA on the colony formation percentage of SKM-1 was tested on soft agar. The effect of TUBB-siRNA on the ultrastructure of SKM-1 was observed by transmission electron microscope.Results Satisfactory transfection efficiency (>80%) could be obtained with lipofectamine. Real time PCR showed the down-regulated expression of TUBB after transfection (P<0.05). Growth inhibition of SKM-1 cell was obvious 24 hours after TUBB-siRNA transfection (P<0.05). The cell cycle analysis by flow cytometer showed the percentage of SKM-1 before transfection in S phase was higher than that after transfection. Colony formation percentage of SKM-1 was inhibited obviously with TUBB-siRNA transfection (P<0.01). The ultrastructure of SKM-1 cell after transfection exhibit several features, such as vacuole in cytoplasm and nuclear fragmentation.Conclusion Growth and colony formation inhibition are the result of siRNA against TUBB. This indicates that the activation of TUBB may play a role in the evolution of MDS.