| Background and Objective: Musashi-2(MSI2)is an evolutionarily conserved RNA-binding protein of the Musashi gene family,which has important physiological significance for the maintenance of stem cell function and state.Studies have confirmed that MSI2 is closely related to the occurrence and development of various tumors.MSI2 also plays an important role in the pathogenesis of leukemia.The role of MSI2 in promoting cell growth and maintaining cell stemness has been demonstrated in chronic myeloid leukemia(CML),acute myeloid leukemia(AML),and normal hematopoietic stem cells.Our previous studies have shown that MSI2 is highly expressed in B lymphocytes.The poor prognosis of cell-based leukemia(B-ALL)may be closely related to the primary drug resistance of T-ALL.However,the function and specific molecular mechanism of MSI2 in ALL are still unclear.Studies have confirmed that MSI2 not only binds to the protein coding gene mRNA involved in translational regulation,but also binds to linear long non-coding RNA(LncRNA)and participates in regulation.However,whether MSI2 can regulate circRNA still needs further research to confirm.Methods: 1.MSI2 shRNA lentivirus infected ALL cells NALM6,and verified by PCR and gene down verified by PCR and western blotting.2.To detect changes in growth,cycle and apoptosis of NALM6 cells after down-regulation of MSI2.3.In the NALM6 cells,the MSI2 gene was interfered,and the change of NUMB,p53 and LEF1 was detected by western blotting.To further explore the relationship between MSI2 and p53,we down regulate MSI2 gene in p53-deleted cell HL-60 and observe its effect on the expression of NUMB and LEF1.5.The HL-60 with the most significant small interference of MSI2 was selected and detected by circRNA microarray after interference.The cirRNA with significant difference in expression was screened.6.According to the chip detection results,16 circRNAs with the most significant difference and high expression were selected,and the expression differences were verified by RQ-PCR in HL-60 and NALM6 cells.Results: 1.After MSI2 shRNA lentivirus infection of ALL cells NALM6,the mRNA and protein levels of MSI2 in shRNA group were significantly down regulated by PCR and western blotting.2.After down-regulation of MSI2,the growth rate of NALM6 cells was observed at 1,3,and 5 days.The relative cell growth rate in the control group was 1.27±0.012,2.48±0.035,4.21±0.023,and in the small interference group was 1.24±0.038,2.35±0.049 and 3.8±0.036,respectively.On the 5th day,the growth and irrelevant sequences of the small-interference group were significantly different,P<0.05.3.In the NALM6 cells,the apoptotic rate of the unrelated sequence group was 0.96±0.21%.The apoptotic rate of shRNA group was3.1±0.46%,P<0.05,and the PARP cleavage of apoptotic protein was higher than that of NC group.The percentage of apoptotic cells in the shRNA group was increased by flow cytometry.The down regulation of MSI2 blocked NALM6 cells in G0/G1 phase,MSI2 can down regulate the expression of cyclin CDK4 and up regulate the expression of p21 protein.In the NALM6 cells,after silencing the MSI2 gene,NUMB and p53 were found to be up regulated,and the LEF1 protein level in the Wnt signal was down regulated.5.In the p53-deleted HL-60 cells,after silencing the MSI2 gene,observe the growth rate on days 1,2,3,and 4,the relative cell growth rates of the control group were 1.931±0.064,3.07±0.09,4.017±0.058 and4.215±0.032,respectively,the small-interference group was 1.927±0.012,2.564±0.035,2.825±0.023,and3.223±0.042,respectively.There was a significant difference in the growth and irrelevant sequences of the small-interference group from the third day,P<0.05.After the down regulation of MSI2,the cell apoptosis increased,the cell cycle arrested in G0/G1 phase,NUMB was up regulated,and LEF1 levels also decreased.6.Silencing the MSI2 gene of HL-60 cells revealed that 183 circRNAs were up-regulated(FC≥ 2)and 86 circRNAs were down-regulated(FC ≥ 2).We selected 16 circRNAs with the most significant difference and high abundance on the chip,and verified by RQ-PCR.The results showed that in HL-60 cells,the circRNAs that were consistent with the microarray results and whose expression difference was greater than 2-fold were only ci-001214,the difference between the shRNA group and the control group was 3.48 times.In the NALM6 cells,the shRNA group showed a difference of 1.57 times compared with the control group.Conclusions: Our results show that MSI2 shRNA can inhibit the growth of ALL cell line NALM6,up regulate the expression of p53 and NUMB,and down regulate the expression of LEF1,but p53-wnt does not play a key role in the molecular mechanism of MSI2.Silencing MSI2 can dramatically up regulate the expression of the intron circRNA,ci-001214.These results further reveal the molecular mechanism of MSI2 in ALL and provide basis for MSI2 regulation of circRNA. |
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