| Background and Objective: Acute lymphoblastic leukemia(ALL)is one of the most common malignant tumors in children and adults.With the continuous improvement of medical technology,the 5-year survival rate of childhood ALL has been greatly improved(up to 80%);but the situation of adult ALL cannot be optimistic(the survival rate is still less than 50%).Thus,nowadays the first duty of improving survival is to elucidate further the pathogenesis of adult ALL,to find new molecular targets and to better evaluation system for prognosis.KHARAS et al found that Musashi-2(Msi2)gene was highly expressed in primitive cells of hematopoietic system and decreased rapidly in the course of with the differentiation and maturation of these primary cells ones.Increasing evidences have revealed that Musashi-2(Msi2)gene is closely related to leukemia,but its molecular mechanism is still unclear.It is proved that the expression of Msi2 is closely related to the NOTCH signaling pathway in hematopoietic stem cells and myeloid leukemia.However,our previous results showed that unlike normal human bone marrow CD34 + cells sorting and peripheral blood mononuclear cells(mainly mature B lymphocytes),the Msi2 in adult B-ALL had no significant correlation to expression levels of cmyc and Hes1 expression.Our study also confirmed that the c-myc and Hes1 expression is no obvious difference between Msi2 high expression group and low expression group.In addition,some studies had shown that over expression of Msi1 can activate the Wnt signal.Because of highly homology between Msi1 and Msi2,we hypothesized that Msi2 might activate Wnt signaling in B-ALL leukemia cells.In this paper,we observe the cell growth,apoptosis and the expression of potential target genes(LEF1)by Msi2 gene silencing on B-ALL cell to explore the molecular mechanism of Msi2 gene in B-ALL.Methods:(1)Cell culture:NALM6 cells were culturedin saturated humidity incubator culture with 37 C,5% CO2 and 1640 medium containing 10% FBS.(2)Construction of Msi2 siRNA:Synthesised by Shanghai Han Heng Biotechnology Company.(3)Transfection of virus and screen the cells: The small interfering fragment was transfected by the slow virus(MOI=100)transfection method and used the 5μg/mL of puromycin to screen the cells.The transfection efficiency was observed by the fluorescence microscope,and the cells were collected when the transfection rate reached more than 90%(4)Total RNA extraction: The cells were collected from each group using Trizol-centrifugal column method to extract RNA,and the purity and content were determined.The cDNA were synthesized by reverse transcription experiments.(5)Verification of the down-regulaiton of Msi2 Gene: The RT-PCR and western-blot were used to detect the expression levels of Msi2 mRNA and protein in each group.(6)Detection of cell growth: Using cell counting method,MTT method and V/PI Annexin method,the effects of silenced the expression of Msi2 gene on cell growth rate,cell proliferation inhibition rate,apoptosis and cycle were observed.Hochest staining was used to observe the apoptosis of cells,and the levels of apoptosis protein capase-3,PARP and D1,Cyclin,P21 were measured by the western-blot method(7)Detection of cell differentiation: The positive rates of CD38 and CD10 were measured by flow cytometry.(8)LEF1 expression levels in each group were detected by RT-PCR and western blot methods.Results:(1)The fluorescence rates were detected after lentivirus infection:The fluorescence rate of each group reached 90%.In siRNA1,siRNA2,siRNA3,the Msi2 mRNA was decreased by24.48±3.65%,71.73±3.20% and 33.03±3.81%.The Msi2 gene in the Msi2 siRNA2 group was most significantly decreased,and the corresponding protein level was significantly reduced too.Therefore,the Msi2 siRNA2 group was chosen to do the further experiment.(2)The CCK-8 results showed that 24 hours after infection,cell proliferation activity of siRNA2 group was1.093±0.081,and the unrelated sequence group was 1.106±0.074,there were no obvious differences between the two groups;after 72 hours,the cell proliferation rate of Msi2 siRNA2 group was 1.343±0.064,lower than the unrelated sequence group1.533±0.097;after seven days,the cell viability proliferation rate of Msi2 siRNA2 group was 2.380±0.503,lower than the unrelated sequence group(3.553±0.628,P=0.038).(4)The Msi2 siRNA induced the cell apoptosis: the apoptosis rate of siRNA2 group was3.6±0.46%,which was higher than that of the unrelated sequence group 0.96±0.21%,P= 0.015.Caspase 3 and PARP protein bands was deepened but not obviously.(5)The Msi2 siRNA blocked the cell cycle: the proportion of G0/G1 phase cells in siRNA group(72.53±1.28%)was higher than the control group(65.44±2.33%,P=0.0073).Meanwhile,in the Msi2 siRNA group Cyclin D1 decreased and P21 increased.(6)The Msi2 siRNA induced differentiation of NALM6 cells: The percent of CD38+CD10-cells in siRNA group was 62.87±2.10%,CD38-CD10+ cells was 0.67±0.21%;and group CD38+CD10-in unrelated sequence group was0.5±0.1%,CD38-CD10+ cells was 47.43±1.68%.(7)The expression of LEF1 gene in the siRNA2 group was significantly lower than that of the unrelated sequence group(up to 63.4±8.1%)and the corresponding protein was also decreased significantly.Conclusion:(1)Down regulation of Msi2 expression could decrease the activities of leukemia cells,induce cell cycle arrest and promote cell differentiation.But there was no obvious effect on apoptosis.(2)The expression of LEF1 level decreased after Msi2 expression down-regulated,which indicated that the high level of Msi2 in the B-ALL could activate the Wnt signaling pathway to enhance proliferation of cells. |
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