RESUMO
OBJECTIVE: To investigate the effect of SARI overexpression on the proliferation and apoptosis of core binding factor leukemia (CBFL) cells and explore the potential molecular mechanisms. METHODS: C-KIT N822K mutation status in Kasumi-1 cell line was detected by exon 17 sequencing. Then the SARI lentiviral vector (pGC-FU-SARI) was constructed, meanwhile Kasumi-1 cells were transfected with the SARI lentiviral vector. Quantitative PCR and Western blot were employed to identify efficacy of SARI overexpression after the transfection of cells. Cells were divided into three groups, including the cells infected with pGC-FU-SARI (OE group), the cells infected with pGC-FU-GFP (NC group) and the untreated cells (blank control group). Cell proliferative activity was tested by MTT assay, cell apoptosis was measured by flow cytometry (FCM) and the expression of apoptosis-related proteins: BCL-2,BAX,Cyto C,Caspase 9,Caspase 3,cleaved-Caspase 3,PARP and cleaved-PARP as well as PI3K/Akt pathway proteins: PI3Kï¼p85ï¼,p-PI3Kï¼p85ï¼,Akt and p-Akt were detected by Western blot. RESULTS: The Kasumi-1 cells were detected to bear c-KIT N822K (T>A) mutation. The Kasumi-1 cells with SARI was overexpression were construeted successfully. Compared with NC group, the cell proliferation was decreased and cell apoptosis was increased; BCL-2 expression was reduced, BAX expression was enharued; cyto C expression appeared; the expression of Caspase 9 and Caspase 3 was down-regulated, the expression of cleaved Caspase 3 was up-regulated; the PARP expression was decreased, cleaved PARP expression was increased; the phosphorylation level of PI3K/Akt pathway proteins: p-PI3K/PI3K, p-Akt/Akt was down-regulated in OE group (P<0.05). CONCLUSION: SARI gene may suppress the proliferation of CBFL cells, and induce their apoptosis through the mitochondrial pathway, which may be related with the inhibition of PI3K/Akt pathway.
