MicroRNA profile of Marek's disease virus-transformed T-cell line MSB-1: predominance of virus-encoded microRNAs - PubMed (original) (raw)

MicroRNA profile of Marek's disease virus-transformed T-cell line MSB-1: predominance of virus-encoded microRNAs

Yongxiu Yao et al. J Virol. 2008 Apr.

Abstract

Research over the last few years has demonstrated the increasing role of microRNAs (miRNAs) as major regulators of gene expression in diverse cellular processes and diseases. Several viruses, particularly herpesviruses, also use the miRNA pathway of gene regulation by encoding their own miRNAs. Marek's disease (MD) is a widespread lymphomatous neoplastic disease of poultry caused by the highly contagious Marek's disease virus type 1 (MDV-1). Recent studies using virus-infected chicken embryo fibroblasts have identified at least eight miRNAs that map to the R(L)/R(S) region of the MDV genome. Since MDV is a lymphotropic virus that induces T-cell lymphomas, analysis of the miRNA profile in T-cell lymphoma would be more relevant for examining their role in oncogenesis. We determined the viral and host miRNAs expressed in MSB-1, a lymphoblastoid cell line established from an MDV-induced lymphoma of the spleen. In this paper, we report the identification of 13 MDV-1-encoded miRNAs (12 by direct cloning and 1 by Northern blotting) from MSB-1 cells. These miRNAs, five of which are novel MDV-1 miRNAs, map to the Meq and latency-associated transcript regions of the MDV genome. Furthermore, we show that miRNAs encoded by MDV-1 and the coinfected MDV-2 accounted for >60% of the 5,099 sequences of the MSB-1 "miRNAome." Several chicken miRNAs, some of which are known to be associated with cancer, were also cloned from MSB-1 cells. High levels of expression of MDV-1-encoded miRNAs and potentially oncogenic host miRNAs suggest that miRNAs may have major roles in MDV pathogenesis and neoplastic transformation.

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Figures

FIG. 1.

FIG. 1.

Secondary structures of MDV-1 pre-miRNAs predicted using the MFOLD algorithm (68). The mature miRNA strands are indicated in red.

FIG. 2.

FIG. 2.

Genomic locations of MDV-1 miRNAs. The schematic diagram shows where the MDV-1 miRNAs (small arrowheads) identified in this report map. The five novel miRNAs identified in this report are shown in bold. The TRL and IRL regions flanking the unique long region and the TRS and IRS regions flanking the unique short regions are shown. Genomic positions and orientations of MDV ORFs contained in the miRNA loci are indicated.

FIG. 3.

FIG. 3.

(a) Northern blotting analysis for determining the expression of MDV-1 miRNAs. Twenty micrograms of total RNA from MSB-1 cells, the MDV-negative lymphoid cell line AVOL-1, MDV-1-infected CEF, uninfected CEF, or MD lymphoma tissues was separated in a 15% denaturing polyacrylamide gel, blotted, and probed with end-labeled antisense oligonucleotides to the indicated miRNAs. Size markers to indicate the positions of the pre-miRNA and the mature miRNA are shown. The cellular U6 snRNA served as the loading control, and a representative blot of this set is shown. (b) Analysis of tissue-specific expression of Gallus gallus (gga-) miRNAs identified in the MSB-1 library. Total RNAs (20 μg) extracted from different tissues of chickens were separated in polyacrylamide gels and probed with end-labeled antisense oligonucleotides specific for the individual miRNAs indicated. The cellular U6 snRNA served as the loading control.

FIG. 4.

FIG. 4.

Pie chart showing proportions of MDV-1, MDV-2, and chicken miRNAs and other molecules cloned from the MSB-1 library.

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