The Role And The Dysregulation Mechanism Of The SARI Expression In CML

ObjectiveSARI (suppressor of activator protein (AP)-1, regulated by (interferon) IFN) is a novel tumor suppressor gene which has been found recently. The constitutive expression of SARI mRNA was detected in multiple lineage-specific normal cells, but not detected in their tumorigenic counterparts. However, the suppression mechanisms of SARI expression are still obscure. This study was purposed to investigate the SARI expression level in patients with chronic myeloid leukemia (CML) and healthy volunteers. This study also aimed to explore the role and the regulation molecular mechanism of SARI in CML-derived cell line K562. The study will provide a new clue for the treatment of CML.Methods(1) SARI expression in patients with CML and healthy volunteers:46 patients with CML as the experimental group,40 healthy volunteers as the normal control group. Peripheral blood mononuclear cells (PBMCs) of patients with CML and healthy volunteers were collected. SARI expression in two groups was detected using real-time quantitative PCR. (2) The molecular mechanisms of down-regulation of SARI in CML:in vitro, K562 cells were treated by Bcr-Abl inhibitor STI571. Set the concentration gradient:0,0.5,1.5, 2.5μM and time gradient:6,12,24 hours. Then K562 cells were collected to detect SARI expression using real-time quantitative PCR. In respective experiment, K562 cells were treated by the PI3K inhibitor LY294002 (20μM), MEK inhibitor PD98059 (50μM), JAK inhibitor Ag490 (50μM) for 24 hours. Then the cells were collected to detect SARI expression using real-time quantitative PCR. (3) The role of SARI in CML:K562 cells were transfected with transfection reagent as the Mock control group, non-transfected K562 cells as the control group. K562 cells were transfected with SARI specific siRNA for 72 hours following detection of SARI expression using real-time quantitative PCR. Further, transfected K562 cells were treated by STI571 (2.5μM) for 24 hours. Then K562 cells were collected to detect the apoptosis rate by using flow cytometry.Results1. SARI expression in patients with CML and healthy volunteersCompared with the normal control group (n=40), SARI mRNA expression in the experimental group (n=46) was significantly lower. The data were obtained after three experiments. Student's unpaired t-test was employed to analysis the data. The difference between two groups has statistically significant with p<0.001.2. The molecular mechanisms of down-regulation of SARI in CML2.1 The influence of STI571 on SARI expression in K562 cellsIn time-course and dose-course study, SARI mRNA expression is enhanced in K562 cells as early as 12 hours after treated with STI571 (1.5μM). And the results show that SARI mRNA expression was up-regulated by STI571 in a time-dependent manner, but not dose-dependent manner at a different time period.2.2 The influence of signaling pathway inhibitors on SARI expression in K562cellsTreatment with the MEK inhibitor PD98059 for 24 hours, SARI mRNA expression in K562 cells was higher (p<0.001). Similarly, treatment with JAK inhibitor Ag490 for 24 hours, SARI mRNA expression increased in K562 cells (p<0.05). While the PI3K inhibitor LY294002 down-regulated SARI mRNA expression in K562 cells.3. The role of SARI in CML3.1 Interference effect of SARI-special siRNACompared with the Mock group, SARI mRNA expression in transfected K562 cells was decreased by 72%(p<0.001); while SARI mRNA expression in the control group and the Mock group was no significant difference (p>0.05).3.2 The influence of SARI-special siRNA on apoptosis of K562 cellsUsing flow cytometry to detect the apoptosis rate. Compared with the Mock control group, the apoptosis rate of the transfected K562 cells was increased by (1.74±0.30)% (p=0.05).3.3 The influence of SARI-special siRNA on STI571-induced apoptosis in K562 cells Treated with STI571 (2.5μM) for 24 hours, then the apoptosis rate of the transfected group and Mock group were detected by flow cytometry. Compared with the Mock group, the apoptosis rate of the transfected group was reduced by (7.61±0.16)%(p<0.001).ConclusionThe suppression of SARI expression may be associated with the pathology of CML. Bcr-Abl mediates the down-regulation of SARI mRNA expression in K562 cells. Further, Bcl-Abl down-stream signal pathways, including JAK signal pathway and MEK signal pathway are involved in the down-regulation of SARI mRNA expression in K562 cells. In addition, SARI silencing may decrease the sensitivity of K562 cells against STI571-induced apoptosis.