The Regulation Of MiR-218 On Slam Receptor Expression In Goat PBMCs

Peste des petits ruminants(PPR)is an acute,highly contagious fetal disease,thecause agent is Peste des Petits Ruminants virus(PPRV),which belongsto the genus Morbillivirus of the family Paramyxoviridae.PPRV and other members of the measles virus have a well-established receptor-dependent lymphotropism and epitheliotropism.PPRV,due to its lymphocyte tropism,leads to persistent immunosuppression after infection.It has been confirmed that the Signaling lymphocyte activation molecule(SLAM),also known as CD150,is the receptor of the measles virus(MV,CDV,RPV,PPRV)in lymphocyte,and can be expressed in activated and memory T cells,B cells,monocytes,NKT cells,and mature DC cells.In recent years,more and more evidence indicates that the host cell microRNA(mi RNA)is an indispensable regulatory factor in the complex network of host pathogen interactions.The purpose of this study is to explore the mechanism of cell source miRNA regulation on SLAM receptor and its effect on virus replication during PPRV infection in goat PBMCs in vitro.The main results obtained are as follows:1)We employed deep sequencing technology to determine cellular miRNAs expression profile in goat peripheral blood mononuclear cells(PBMCs)infected with Nigeria 75/1vaccine virus strain.Expression analysis demonstrated that PPRV infection can elicit 316 significantly differentially expressed(DE)miRNAs including 103 known and 213 novel miRNAs candidates in infected PBMCs at 24 h post infection as compared with mock control.Target prediction and functional analysis of these DEmiRNAs revealed significant enrichment for several signaling pathways including TLR signaling pathways,PI3K-Akt,endocytosis,viral carcinogenesis,and JAK-STAT signaling pathways.2)Compared to mock-infected cells,CPE of the PPRV-infected cells exhibited ballooning and clumping,with the prolongation of infection time,the degree of cytopathy was gradually enhanced.PBMCs was infected with different MOI(0.1,1,10)PPRV,the expression levels of miR-218 and SLAM were detected by qRT-PCR and Western blot.The results showed that there was a dose-dependent decrease in miR-218 and increase in SLAM expression in PBMCs by the increase dose of PPRV infection.In addition,the expression ofmiR-218 decreased first and then increased with the prolongation of PPRV infection,the expression of SLAM increased first and then decreased with the prolongation of PPRV infection,these findings showed that the expression levels of miR-218 and SLAM in PPRV-infected goat PBMCs were always negatively correlated.To further investigate the regulatory mechanism of miR-218 on SLAM receptor expression in PPRV-infected goat PBMCs,different concentrations of miR-218 mimic,miR-218 inhibitor and their corresponding control sequences were transfected.The results showed that miR-218 negatively regulates the expression of SLAM receptor in PBMCs infected with PPRV in a dose-dependent manner.In addition,dual luciferase reporter assays demonstrated that miR-218 can directly target the 3'UTR of the SLAM gene.Western blot and ELISA were used to detect the protein expression and secretion of different cytokines in goat PBMCs after transfection and PPRV infection.It was found that miR-218 affected the replication of PPRV by regulating the Th1 immune response mediated by SLAM pathway.3)Infection of goat PBMCs with UV-inactivated PPRV(UV-PPRV),qRT-PCR,Western blot,and flow cytometry revealed that PPRV replication is an important factor affecting miR-218 mediated SLAM expression.In summary,in this study,goat PBMCs were infected with the PPRV N75-1 vaccine strain,confirming the posttranscriptional regulation mechanism of miR-218 on the expression of SLAM receptor in goat PBMCs infected with PPRV and its effect on virus replication.