Abstract
Human cytomegalovirus (HCMV) infection can cause inflammatory tissue-invasive end-organ diseases upon lytic replication. In humans, mature miR-200b-3p and -200c-3p suppress the synthesis of HCMV immediate early 2 (IE2) protein by binding to the 3′-UTR of the mRNA encoded by the unique long (UL) 122-123 region in human foreskin fibroblasts and pre-transplant peripheral blood mononuclear cells stimulated with HCMV. The present study aimed to quantitate the expression of Homo sapiens (hsa)-miR-200b-3p and 200c-3p in HCMV-infected tissues. We collected 240 HCMV-infected and 154 HCMV-non-infected, formalin-fixed, paraffin-embedded tissue samples of the gastrointestinal (GI) tract and bronchi/lungs. MiRNAs, HCMV, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantitated by quantitative reverse transcription-PCR (qRT-PCR) and quantitative PCR (qPCR) on the basis of standard curves generated using miRNA mimics, the HCMV strain from National Institute for Biological Standards and Control (NIBSC) 09/162, and GAPDH control. To avoid the effect of cell counts on the qRT-PCR and qPCR results, the data were normalized to GAPDH levels. HCMV-infected tissues had significantly lower levels of 200b-3p/GAPDH (3.03 ± 1.50 compared with 3.98 ± 1.08 log10 copies/μl, P<0.001) and 200c-3p/GAPDH (4.67 ± 1.84 compared with 6.35 ± 1.47 log10 copies/μl, P<0.001) than normal tissues. The values for 200b-3p/GAPDH (r = −0.51, P<0.001) and 200c-3p/GAPDH (r = −0.54, P<0.001) were significantly inversely correlated with HCMV load. Low tissue levels of 200b-3p and 200c-3p in humans are associated with cytopathic inflammation due to HCMV infection.
Highlights
Human cytomegalovirus (HCMV) replication can cause serious harm in solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT) recipients [1,2,3]
This finding is in-line with those in two previous studies that evaluated the levels of two miRNAs in pre-transplant peripheral blood mononuclear cells (PBMCs) from SOT recipients with in vitro HCMV stimulation, and performed co-transfection assays using a laboratory HCMV strain and two miRNA mimics in fibroblasts [12,17]
The suppressive role of hsa-miR-200b-3p and -200c-3p on HCMV lytic replication was supported by two stepwise objective bases: (i) accurate prediction through in silico analysis of miRNAs binding to the 3 -UTR in the HCMV UL122-123 rather than high-throughput screening methods, such as oligonucleotide microarrays, in which expression profiles could be biased due to sample characteristics, platforms, or statistical analyses [24,25], (ii) verification of the binding of identical seed sequences in hsa-miR-200b-3p and -200c-3p to the HCMV UL122-123 by luciferase reporter assay using wild-type and mutant recombinant vectors, the results of which precisely corresponded with in silico prediction [12,17]
Summary
Human cytomegalovirus (HCMV) replication can cause serious harm in solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT) recipients [1,2,3]. HCMV reactivation has been associated with increased mortality and prolonged treatment of mechanical ventilation in critically ill intensive care unit patients without prior objective immunocompromised conditions [4,5,6,7]. This worsening of outcomes by HCMV infection has led to the implementation of regular HCMV monitoring as well as well-organized and patient-tailored preventive strategies with risk stratification in routine clinical practice of SOT and HSCT recipients [1,2,3]. In vitro experiments on cultured cells have shown that epigenetic regulation through chromatin structural changes in the major immediate-early promoter (MIEP) and miRNA originating from the human host and/or HCMV
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