The Role Of Human Cytomegalovirus MiRNA In Viral Latent Infection And SIRPα In Arsenic Trioxide-Induced Promyelocytic Leukemia Cell Apoptosis

The thesis consists of two parts,focusing on the role of hcmv-miR-UL148D in viral latent infection and SIRPα in arsenic trioxide-induced promyelocytic leukemia cell apoptosis,respectively.1.Human cytomegalovirus hcmv-miR-UL148D facilitates HCMV latent infection via targeting progenitor cell immediate early response gene 5.Human cytomegalovirus(HCMV),a member of the β-herpesvirus subfamily,is a ubiquitous human virus with up to 90%adult population infected around the world.Although HCMV infection rarely causes clinically symptomatic disease in immunocompetent healthy hosts,HCMV can establish a latent infection in the hosts and the reactivation of HCMV from latency in immunocompromised people,for example in AIDS patients,solid organ transplant recipients and neonates,can lead to severe morbidity and mortality.Although previous evidences have suggested that various viral factors are involved in establishing of HCMV latent infection,the mechanism underlying the HCMV latent infection remains incompletely understood.Here we show that HCMV-encoded miRNA,hcmv-miR-UL148D,is highly expressed at the late stage of HCMV infection in human bone marrow progenitor cells and facilitates HCMV latency through modulating progenitor cell immediate early response gene 5(IER5)-cell division cycle 25B(CDC25B)axis.HCMV-miR-UL148D inhibits IER5 by directly targeting the three prime untranslated region(3’UTR)of IER5 and thus prevents IER5-induced CDC25B reduction.Infection with NR-1ΔmiR-UL148D,an HCMV clinical strain NR-1 with specific hcmv-miR-UL148D knockout,results in sustained IER5 induction but decreased CDC25B expression in progenitor cells.The mechanistic study further shows that CDC25B can suppress HCMV IE1 expression through activating cyclin-dependent kinase 1(CDK-1).Both gain-of-function and lose-of-function assays demonstrate that hcmv-miR-UL148D facilitates HCMV latency in progenitor cells via maintaining high CDC25B activity.Interestingly,our data also indicate that hcmv-miR-UL148D secreted from HCMV-infected progenitor cells can be delivered into non-infected progenitor cells where it upregulates cellular CDC25B expression and thus predisposes the recipient cells for HCMV latency.2.Role of SIRPα in arsenic trioxide-induced promyelocytic leukemia cell apoptosisSIRPa,an immunoglobulin superfamily member,is a receptor-like membrane protein mainly present on mature myeloid leukocytes including neutrophils,monocytes,and macrophage.SIRPa consists of three extracellular IgV-like loops and a cytoplasmic region with two immunoreceptor tyrosine-based inhibitory motifs(ITIMs).Previous studies have demonstrated that ligation of SIRPα by its ligand CD47,a ubiquitous cell membrane protein,leads to phosphorylation of its ITIMs,which in turn,recruits SH2 domain-containing protein tyrosine phosphatases SHP-1 or SHP-2 to initiate downstream inhibitory signal.As a critical signal transduction protein,SIRPa has been shown to be involved in regulating many aspects of cellular responses,particularly the inflammatory responses of leukocytes,including activation,chemotaxis and phagocytosis.A correlation between SIRPa and cell growth and survival was reported recently.Arsenic trioxide(ATO),a compound therapeutically used in Chinese traditional medicine,is effective in the treatment of patients with APL.ATO is a potent inducer of APL cell apoptosis,however,the mechanism underlying the ATO-induced APL cell apoptosis remains incompletely understood.In the present study,we investigated the general pro-apoptotic effect of SIRPa on tumor cells,and as an extension,we studied the role of SIRPα in the ATO-induced apoptosis of APL cells.We found that expression of SIRPa resulted in apoptosis both of APL HL-60 cells and hepatocellular carcinoma Huh7 cells possibly by suppressing β-catenin signal pathway and upregulating Foxo3a.SIRPa-induced apoptosis could be reversed by pharmacological activation of β-catenin signal pathway.We also observed that ATO treatment induced SIRPa expression in APL cells in a time-dependent manner and abrogation of SIRPa induction prevented APL cell apoptosis and left the β-catenin signaling pathway unperturbed by the ATO treatment.The present study also further explored the miRNA-based mechanism that governs the induction of SIRPa by ATO in APL cells.As our previous study showed that SIRPα was post-transcriptionally regulated by a cluster of miRNAs,viz.,miR-17,miR-20a,and miR-106a.In this study,we also found that induction of SIRPa by ATO is through suppression of miR-17,miR-20a and miR-106a.A miRNA-based regulation of SIRPa expression may play an important role in ATO-induced apoptosis of APL cells...