The Role And Mechanism Of CPSF6 Regulating Alternative Polyadenylation In Acute Myeloid Leukemia

Background:During the process of m RNA maturation,the 3? end of the m RNA precursor selects sites with different cleavage and poly A sequences,thereby producing m RNA transcripts of different lengths.This regulatory mechanism is called alternative polyadenylation(Alternative Polyadenylation,APA).APA causes a gene to produce transcripts of different lengths,greatly increasing the diversity and complexity of the transcriptome and proteome.Abnormal APA is closely related to tumor development and prognosis,but the mechanism is not clear.CF?m complex subunit CPSF6(cleavage and polyadenylation specific factor 6),as an important subunit regulating 3?UTR cleavage and polyadenylation,is upregulated in acute myeloid leukemia(AML)and negatively correlated with differentiation.However,the role of CPSF6 in hematopoietic differentiation and its mechanism remain unclear.In this study,we proved that CPSF6 is a key gene for maintaining the survival and differentiation arrest of leukemia cells from multiple perspectives in vivo and in vitro,and explored the mechanism of CPSF6 in the development of leukemia through APA analysis and dual luciferase reporter experiments.Methods:We first analyzed the expression of CPSF6 in different tumor cell lines,AML patients,normal peripheral blood mononuclear cells,and myeloid cells by analyzing datasets from the Cancer Cell Line Encyclopedia(CCLE)and Gene Expression Omnibus(GEO)databases.At the same time,the normal bone marrow single cell sequencing data was obtained by using the single cell database of European EMBLEBI,and the expression of CPSF6 in normal hematopoietic development was observed.Then,in order to clarify the function of CPSF6 in AML,after inhibiting the expression of CPSF6 by sh RNA in NB4 and NB4-R2 cell lines,we verified the differentiation level by detecting CD11 b,STAT1,PU.1 and other indicators.The proliferation ability of leukemia cells was verified by cell counting,cycle and apoptosis experiments,and verified by luciferase imaging,immunohistochemistry and survival cycle in vitro.In order to explore the mechanism by which CPSF6 regulates AML,we searched for downstream pathways through RNA-seq,GSEA analysis,and APA analysis,and verified them by dual luciferase reporter experiments and western blot.Result:In this study,we found that CPSF6 was overexpressed in AML,and the expression level was independent of MICM type and negatively correlated with myeloid differentiation markers.Meanwhile,in normal bone marrow single-cell sequencing data,CPSF6 expression decreased with differentiation and maturation.In the process of inducing leukemia cell differentiation in vitro,the expression of CPSF6 also decreased with the increase of differentiation level.Further studies found that inhibition of CPSF6 in vivo and in vitro could lead to leukemia cell differentiation,proliferation arrest,apoptosis,and significantly prolong the survival of mice.Through sequencing,it was found that the deletion of CPSF6 caused the shortening of a wide range of gene transcripts,including CD11 b,CD15,PTEN,etc.Among them,PTEN(phosphatase and tensin homolog)up-regulates expression by shortening transcripts and inhibits PIK3/AKT signaling pathway.Furthermore,we found that targeting CPSF6 enhanced the differentiation-induced effect of ATRA and reversed drug resistance.Conclusion:1.CPSF6 regulates selective polyadenylation,which is a key factor for maintaining the malignant proliferation of AML cells and inhibiting differentiation and apoptosis.Its absence will greatly inhibit the progress of AML.2.Inhibition of CPSF6 will enhance the sensitivity of ATRA to AML cells and reverse ATRA drug resistance.