Molecular Mechanism Of MBNL1 Regulation Of MLL-r Leukemia

The MLL gene is located in the long arm 2 region 3 band(11q23)of chromosome 11.The abnormality of 11q23 chromosome can cause various types of MLL gene rearrangement and thus lead to MLL rearrangement(MLL-rearranged,MLL-r)leukemia.MLL-r leukemia generally has a poor prognosis and there is still a lack of effective and less toxic treatments.MBNL1,as a member of the MBNL family of tissue-specific RNA metabolism regulators,is evolutionarily conservative and multifunctional,and can participate in the regulation of alternative splicing,alternative polyadenylation,translation,m RNA localization,mi RNA processing,and other biological processes.In our previous research,we found that MBNL1 is relatively highly expressed in MLL-r leukemia cells,while it is low expressed in non-MLL-r leukemia cells through the analysis in the Oncomine database and the ATCG database.Compared with leukemia patients with low MBNL1 expression,patients with high MBNL1expression have lower survival rate and poorer prognosis.We speculate that MBNL1gene in MLL-r leukemia may be associated with the occurrence,development and prognosis of leukemia,and MBNL1 may be one of the potential therapeutic targets for MLL-r leukemia.First,we constructed and obtained MBNL1 overexpressing cell lines:THP1-MBNL1 and RS4:11-MBNL1 in two MLL-r leukemia cell lines through MSCV and MSCV-MBNL1 plasmid-mediated gene overexpression technology.Next,we examined cell proliferation,cell cycle,apoptosis,and cell differentiation of MBNL1overexpressing cells.The results showed that,compared with the control group,the cell proliferation and cell cycle of THP1-MBNL1 and RS4:11-MBNL1 were not significantly different,and the number of consecutive clones was not significantly changed.After the overexpression of MBNL1,the proportion of Annexin V~+cells and the proportion of apoptosis were decreased significantly,and the proportion of CD11b-APC~+and CD14-PE~+cells was significantly reduced,and the differentiation of leukemia cells into mononuclear cells was reduced.We also studied the resistance of THP1-MBNL1 and RS4:11-MBNL1 to the chemotherapeutic drugs cytarabine(Ara-c)and daunorubicin(DNR).MTT assay results showed that,compared with the control group,after treatment with chemotherapy drugs THP1-MBNL1 and RS4:11-MBNL1,the optical density(OD)value of cells was increased,the cell inhibition rate was significantly reduced,and the IC50 value was increased.The apoptosis detection showed that the proportions of early and late apoptotic cells in the two MLL-r leukemia cells overexpressing MBNL1 were significantly reduced,and the proportion of living cell population was significantly increased.Overexpression of MBNL1makes MLL-r leukemia cells more resistant to chemotherapy drugs.We also constructed MOLM13-sh MBNL1 and MOLM13-sh Con,MLL-r leukemia cells with MBNL1 knockdown.The results of series colony formation experiments showed that after the MBNL1 was knocked down,the number of colonies was significantly reduced as compared with the control group,and the colony formation ability of the cells was significantly weakened.To explore whether the effect of MBNL1 on MLL-r leukemia cells also works in vivo,we transplanted MLL-r leukemia cells MOLM13-sh Con and MOLM13-sh MBNL1,into the subcutaneous layer of the back of nude mice and treated with chemotherapeutic drugs(100μg/g cytarabine for 5 days and 3μg/g epirubicin hydrochloride for 3 days)to observe the trend of solid tumor size.We found that there was no significant difference in tumor volume between the sh MBNL1 group and the sh Con group.To explore the molecular mechanism of MBNL1 in regulating MLL-r leukemia,we performed RNA-seq sequencing on MOLM13-sh Con and MOLM13-sh MBNL1cells.Sequencing results showed that there were multiple significantly differentially expressed genes in MOLM13-sh MBNL1 cells,and the vast majority of these genes were closely related to cell metabolism,cell differentiation,cancer signaling pathway,etc.Some of these genes differ significantly,such as BCL2L11,APC,and SPTAN1,which are associated with leukemia.We further verified the expression levels of BCL2L11,APC and SPTAN1 by q RT-PCR.The results showed that the expression level of BCL2L11 did not change significantly,while the expression level of APC was increased significantly,while the expression level of SPTAN1 was decreased significantly.SPAG9 is one of the downstream genes of MBNL1.Studies have shown that inhibition of the SAPK/JNK pathway can delay the onset of leukemia.Through the verification of RNA-seq data and q RT-PCR,we found that the expression of SPAG9 was significantly down-regulated in the MBNL1 knockdown cell line.We also detected the expression levels of JNK and c-jun related to SAPK/JNK signaling pathway proteins by western blot.The results showed that the expression levels of p-JNK and p-c-jun proteins were significantly increased in the MBNL1 overexpression cell line,while the expression levels of p-JNK and p-c-jun proteins were significantly decreased in the MBNL1 knockdown cell line,indicating that the activation of SAPK/JNK pathway was closely related to MBNL1.In summary,our results indicate that overexpression of MBNL1 in MLL-r leukemia cells can reduce the proportion of cell apoptosis and differentiation and enhance the resistance of MLL-r leukemia cells to chemotherapeutic drugs.The mechanism of MBNL1 in the development of MLL-r leukemia may be that it regulates the expression of its downstream gene SPAG9 and activates the SAPK/JNK pathway to affect the survival of leukemia cells.In the future studies,we will further verify through in vivo experiments in mice how MBNL1 affects the occurrence and development of leukemia through the SAPK/JNK pathway.Our study has provided certain experimental data for in-depth understanding of the pathogenesis of MLL-r leukemia and the development of gene targeted drugs for the treatment of MLL-r leukemia.