The Role Of MBNL1 In The Development And Resistance Of MLL-r Leukemia

The abnormality of 11q23 causes multiple types of rearrangement of MLL gene,resulting in MLL rearrangement(MLL-r)leukemia.It can occur in people of any age,most of which are characterized by high degree of malignancy,insensitivity to chemotherapy,low remission rate,poor prognosis and high recurrence rate.The World Health Organization(WHO)lists it as a unique subtype of acute leukemia,the 11q23/MLL leukemia group.As a member of the family of tissue-specific RNA metabolism regulators,MBNL1 is evolutionarily conserved and versatile,and can be involved in the regulation of alternative splicing,translation,alternative polyadenylation,miRNA processing,and mRNA localization.MBNL1 is one of the potential candidate genes in the study of the molecular genomics of hematological malignancies.However,at present,no research reports have clearly pointed out the specific role of MBNL1 in MLL-r leukemia and its mechanism of action.Our previous analysis through the Oncomine database showed that MBNL1 was relatively highly expressed in patients with MLL-r leukemia,while low in other non-MLL-r leukemia patients.Compared with leukemia patients with low MBNL1 expression,patients with high MBNL1 expression had lower survival rate and poorer prognosis,which was found by analysis of ATCG database.We speculated that the poor prognosis and low survival rate of MLL-r leukemia might be related to the high expression of MBNL1 gene.We first detected the expression of MBNL1 gene in non-MLL-r and MLL-r leukemia cells by qRT-PCR experiment,and confirmed that MBNL1 was relatively highly expressed in MLL-r leukemia cells,especially in MOLM13 and KOPN8 cells.Through shRNA mediated gene knockdown,we successfully constructed cell lines: MOLM13-shCon,MOLM13-shMBNL1,KOPN8-shCon,KOPN8-shMBNL1.We examined the cell proliferation,cell cycle,cell differentiation and clone formation of MOLM13 and KOPN8 cells after MBNL1 knockdown.After MBNL1 was knocked down by MOLM13 and KOPN8 cells,the proliferation was accelerated,and the proportion of s-phase cells significantly increased,indicating that DNA synthesis was promoted and cell cycle from G1 to S-phase was promoted.After MBNL1 was knocked down by MOLM13 cells,the expression of CD14 and CD11 b decreased.However,after knocking down MBNL1 by KOPN8 cells,the expressions of CD14 and CD11 b were not significantly changed,and the differentiation degree was not significantly changed.After two kinds of leukemia cells knocked down MBNL1,the clone morphology becomed larger and the number of clones increased.Then we studied the role of MBNL1 in drug resistance of MLL-r leukemia cells.We treated MLL-r leukemia cells and MLL-r leukemia cells knocked down MBNL1 gene with chemotherapy drugs cytarabine and daunorubicin.The cell inhibition rate was detected by MTT assay,which confirmed that MLL-r leukemia cells were more sensitive to chemotherapeutic drugs,the cell inhibition rate increased significantly,the IC50 value decreased,and the number of clones decreased significantly after knocking down MBNL1.There was no significant difference between these two cells in the apoptosis experiment.This may be because the killing of the chemotherapy drugs was not related to apoptosis but through other death pathways,or because the concentration of chemotherapy drugs is low.Finally,we investigated the role of MBNL1 in the development of MLL-r leukemia.We transplanted MOLM13-shCon and MOLM13-shMBNL1 cells into immunodeficient NOD/SCID mice,and each cell was set up with a control group and a drug group.The mice in the control group were injected with PBS intraperitoneally,and the mice in the drug group were injected with 100 ?g/g cytarabine for 5 days and 3 ?g/g epirubicin hydrochloride for 3 days to observe the incidence of the mice.Because NOD/SCID mice could not withstand the effects of chemotherapy drugs,the weight of the mice in the drug group suddenly dropped on the fourth day of the drug treatment,and the mice began to die the next day.The control mice did not die within 45 days of the observation period.These results indicate that the in vivo transplantation protocol needs further improvement.Through RNA-seq assay to sequence MOLM13-shCon and MOLM13-shMBNL1 cells,we initially explored the mechanism of MBNL1 function.Compared with MOLM13-shCon cells,MOLM13-shMBNL1 cells had 90 significantly up-regulated genes and 84 down-regulated genes,and most of these genes were involved in cell metabolism,cell differentiation and cancer signaling pathways.MBNL1 may play its role by regulating these signal pathways.To sum up,we found that MBNL1 was relatively high expression in MLL-r leukemic cells for the first time.Knocking down MBNL1 in MLL-r leukemic cells can promote cell proliferation and enhance the sensitivity of MLL-r leukemic cells to chemotherapy drugs.MBNL1 may play an important role in the occurrence and development of MLL-r leukemia,and the specific mechanism needs to be further studied.Our research provides an important theoretical basis for further understanding the pathogenesis of leukemia and the development of new anti leukemia drugs.