The efficacy of the prophylactic vaccine against types 16 and 18 for preventing cervical cancer would be close to 80% in Liaoning Province, China. Human papillomavirus types 16, 18, 58, 33, and 31 may be predominant high-risk factors for CC and its precursors in this region.
Edited by Hans-Dieter KlenkKeywords: DNA synthesis Down-regulation eIF4A1 HCMV-miR-US25-2-3p Human cytomegalovirus a b s t r a c t It has been reported that human cytomegalovirus (HCMV) miR-US25-2 reduces DNA viral replication including HCMV. However, the mechanism remains unknown. In our study, eukaryotic translation initiation factor 4A1 (eIF4A1) was identified to be a direct target of miR-US25-2-3p. Small interfering RNA (siRNA) and miR-US25-2-3p mediated eIF4A1 knockdown experiments revealed that high level of miR-US25-2-3p in MRC-5 cells decreased HCMV and host genomic DNA synthesis, and inhibited cap-dependent translation and host cell proliferation. However, eIF4A1 up-regulation induced by miR-US25-2-3p inhibitor increased HCMV copy number. Therefore, the over-expression of miR-US25-2-3p and consequent lower expression of eIF4A1 may contribute to the inhibition of HCMV replication.
Abstract. Human cytomegalovirus (HCMV) is a herpesvirus that causes congenital diseases and opportunistic infections in immunocompromised individuals. Its functional proteins and microRNAs (miRNAs) facilitate efficient viral propagation by altering host cell behavior. The identification of functional target genes of miRNAs is an important step in the study of HCMV pathogenesis. HCMV encodes at least 14 miRNAs, including hcmv-mir-UL148D, which resides in the HCMV UL/b' region. hcmv-mir-UL148D is the only miRNA encoded by the HCMV UL/b' region; however, its targets and functional effects have not yet been eludidated. In this study, hybrid-PCR screening was used to identify target genes and dual luciferase reporter assay was used to evaluate the binding effect of hcmv-miR-UL148D to the 3' untranslated region (3'UTR) of IEX-1. In addition, western blot analysis was used to detect the expression kinetics of IEX-1 protein and apoptosis assay was used to identify the effects of hcmv-miR-UL148D on cell apoptosis. The hybrid-PCR results showed that only one binding site in the 3'UTR of the cellular gene, human immediate early gene X-1 (IEX-1), was completely complementary to an 11 nucleotide (nt) sequence in the 5' terminus of hcmvmir-UL148D, including the entire seed region. The binding site was demonstrated to be functional by dual luciferase reporter assay with a 47% repression of the relative luciferase activity. In an in vitro system of human embryonic kidney 293 (HEK293) cells, the ectopically expressed hcmv-mir-UL148D exhibited a downregulatory effect on IEX-1 expression, and decreased the cell apoptosis induced by transfected IEX-1. Our data demonstrate that hcmv-mir-UL148D targets the cellular gene, IEX-1, downregulating its expression and thus results in anti-apoptotic effects.
BackgroundmicroRNAs (miRNAs) are a group of regulatory RNAs that regulate gene expression by binding to specific sequences on target mRNAs. However, functional identification of mRNA targets is usually difficult and time consuming. Here we report hybrid-PCR as a new and rapid approach to screen putative mRNA targets in vitro.ResultsFifteen putative target mRNAs for human cytomegalovirus (HCMV) miR-UL112-1, including previously confirmed HCMV IE72, were identified from mRNA-derived cDNAs using hybrid-PCR. Moreover, we randomly validated six different target candidates by luciferase reporter assays, and confirmed that their luciferase activities were down-regulated with co-transfection of HCMV miR-UL112-1.ConclusionsOur study demonstrated that hybrid-PCR is an effective and rapid approach for screening putative miRNA targets, with much more advantage of simplicity, low cost, and ease of implementation.
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