The Functional And Mechanistic Study Of NFATC4 And TRPV2 In Acute Myeloid Leukemia

Objective:Acute myeloid leukemia(AML)is the most common acute myeloid leukemia in adults,caused by the development of hematopoietic stem cells arrested in the early stage coupled with malignant proliferation.A variety of gene mutations and expression changes,abnormal immune responses are involved in the development of AML.Transient receptor potential channel(TRPV)is a newly discovered calcium channel consisted of six subtypes,in which TRPV2 is involved in the normal physiological process of cells,oncogenesis and tumor development,while its role in AML is still unclear.Our previous data showed high-expression of TRPV2 in AML cells,suggesting it might participate in the progression of AML.Considering the importance of immune responses in cancer progression,the first part of this study explored the function and mechanism of immune related genes in AML based on bioinformatics methods.In the second part,we intend to deeply explore the role and mechanism of TRPV2 in AML through in vitro and in vivo experiments,and preliminarily understand whether it participates in regulating the immune response in AML.Methods:1.Bioinformatics methods were used to investigate and analyze key immune genes,transcription factors(TFs),downstream KEGG pathways and immune gene sets,immune cells associated with AML prognosis.2.RT-qPCR and Western blot were used to detect the expression of TRPV2 in AML cell lines.And the effects of TRPV2 on AML cells proliferation were observed by adding specific inhibitors or knocking down TRPV2 expression.3.RNA sequencing was performed on THP-1 infected with shControl(Ctr)or sh TRPV2lentivirus to identify the downstream pathways of TRPV2.And the expression level of key genes in downstream pathway was validated in proteins of AML cell lines infected with sh Ctr or sh TRPV2 lentivirus.4.In vitro rescue experiments were performed to confirm that TRPV2 affected phenotype through above genes and pathways.5.In vivo experiments in mice were conducted to further confirm the role of TRPV2 in oncogenesis and progression of AML.Results:1.NFATC4 was key immune gene related to AML poor prognosis and is positively regulated by RAG1(R=0.248,P<0.01).2.NFATC4 was associated with AML poor prognosis through affecting the downstream ABC transporters signaling pathway(R=0.309,P<0.001,positive),check-point immune gene set(R=0.300,P<0.001,positive)and Tregs(R=0.526,P<0.001,positive).3.TRPV2 is highly expressed in AML cells,and knockdown of TRPV2 by lentivirus or inhibitor inhibited the proliferation of AML cells and promote their apoptosis at 24h,48h,72h(P<0.05).4.In AML cells,knockdown of TRPV2 inhibited the downstream PI3K/Akt signaling pathway.The addition of PI3K-IN inhibited Akt phosphorylation and promoted cell apoptosis.The over-expression of TRPV2 could reverse this phenotype.5.In vivo experiments confirmed that knocking down TRPV2 could slow down the progression of AML and prolong survival of AML mice(64±25d vs 26±5d),along with increase of CD4~+lymphocytes(27.998%±3.703%vs 21.998%±4.728%)and decrease of Tregs(4.863%±0.873%vs 7.583%±0.918%)in peripheral blood.Conclusions:1.Immune gene NFATC4 is associated with AML poor prognosis through positively affecting ABC transporters signaling pathway,check-point immune gene set and Tregs.2.Highly expressed TRPV2 in leukemia cells inhibits self-apoptosis through activating PI3K/Akt pathway and affects immune microenvironment,thus contributing to AML progression.