Oral Presentations - Pathology and Laboratory Medicine - University ...

Graduate StudentXin Ye 1 , Mary Zhang, Zhen Liu, Maged Hemida, Decheng YangDepartment of Pathology and Laboratory Medicine, University of British Columbia, The Heartand Lung Institute, St. Paul’s Hospital, Vancouver, CanadaAbstract # 17microRNA(mir)-126 promotes coxsackievirus b3replication by enhancing erk signaling pathwayXin YeBackround/ObjectivesCoxsackievirus B3 (CVB3) is the primary pathogen of viral myocarditis, a life threatening heart disease in infantsand children. Unfortunately, there is no effective or specific treatment to this disease yet. More studies on themechanism of CVB3 infection and pathogenesis of viral myocartidis are necessary to searching for new therapeutictargets. MciroRNAs(miRNAs) are small endogenous non-coding RNA molecules regulating the gene expression atthe post-transcriptional level. To date, hundreds of miRNAs have been found to participate in various physiologicaland pathological processes including heart diseases such as myocarditis and cardiomyopathy. Mir-126 is one ofthe miRNAs enriched in the heart and plays an important role in vascular development and function. It regulatesseveral fundamental signal pathways including the ERK (extracellular signal-related kinase) signal pathway. Here weinvestigated the effect of mir-126 on the CVB3 infection and pathogenesis of viral myocarditis.MethodsPre-mir-126 or negative control was transfected to HeLa cells for 48 hours. During the transfection, 24 h serumstarvation or non-serum starvation was conducted to determine the effect of serum on signal activation. The livecell numbers and mir-126 level of the transfected cells were measured. The transfected cells were then infected with10MOI CVB3 for 0-8h and the replication of CVB3 was determined by microscopy analysis of cell morphology,Western blotting to detect viral protein and viral plaque assay to measure viral titer. The ERK pathway signal proteinswere also detected by Western blotting to elucidate the mechanism by which the mir-126 regulates CVB3 replication.ResultsIn this study, we demonstrated the following: i). Compared with the control group, there was a 15% decrease inthe total number of live cells in the pre-mir-126 transfected group. ii). The pre-mir-126 transfected cells exhibitedsignificant cell elongation and membrane protrusions. iii). Increased levels of mir-126 led to more cell rounding anddetaching as well as enhanced pro-Caspase-3 cleavage than the control cells during the CVB3 infection, indicating anincreased cell death. The viral protein 1 (VP1) synthesis was also enhanced by the mir-126 transfection. The release ofthe viral particles was increased more than 100 times in the mir-126 group. iv). Infection of CVB3 downregulated theexpression levels of SPRED1, a negative regulator of ERK signal pathway, correlating to the upregulation of phospho-ERK signal. Finally, v). The transfection of pre-mir-126 significantly enhanced the activation of ERK pathway byfurther reducing the expression levels of SPRED1 during the CVB3 infection in both serum-starvation and nonstarvationconditions.ConclusionOur findings indicated that i) mir-126 may affect the cell proliferation and cell morphology; ii) Mir-126 plays animportant role in facilitating the replication of CVB3 through the ERK signal.Oral Presentations * 2 0 1 027