Identification of Cellular Genes Targeted by KSHV-Encoded MicroRNAs

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Abstract
MicroRNAs (miRNAs) are 19 to 23 nucleotide–long RNAs that post-transcriptionally regulate gene expression. Human cells express several hundred miRNAs which regulate important biological pathways such as development, proliferation, and apoptosis. Recently, 12 miRNA genes have been identified within the genome of Kaposi sarcoma–associated herpesvirus; however, their functions are still unknown. To identify host cellular genes that may be targeted by these novel viral regulators, we performed gene expression profiling in cells stably expressing KSHV-encoded miRNAs. Data analysis revealed a set of 81 genes whose expression was significantly changed in the presence of miRNAs. While the majority of changes were below 2-fold, eight genes were down-regulated between 4- and 20-fold. We confirmed miRNA-dependent regulation for three of these genes and found that protein levels of thrombospondin 1 (THBS1) were decreased >10-fold. THBS1 has previously been reported to be down-regulated in Kaposi sarcoma lesions and has known activity as a strong tumor suppressor and anti-angiogenic factor, exerting its anti-angiogenic effect in part by activating the latent form of TGF-β. We show that reduced THBS1 expression in the presence of viral miRNAs translates into decreased TGF-β activity. These data suggest that KSHV-encoded miRNAs may contribute directly to pathogenesis by down-regulation of THBS1, a major regulator of cell adhesion, migration, and angiogenesis. Kaposi sarcoma–associated herpesvirus (KSHV) is a gamma-herpesvirus associated with Kaposi sarcoma, primary effusion lymphoma, and a subset of muticentric Castleman disease. Recently, it was found that KSHV encodes 12 microRNAs (miRNAs) within its latency-associated region. miRNAs are small ∼22 nucleotide-long single-stranded RNA molecules that act to inhibit gene expression by binding to target messenger RNAs (mRNAs). Because miRNAs bind to these targets with limited base pairing, it has been difficult to find targets. The goal of our study was to identify cellular mRNAs targeted by KSHV-encoded miRNAs. Microarray analysis of cells expressing the KSHV miRNAs revealed a set of 81 genes that were changed. Several genes are regulators of important functions such as blood vessel growth, cell proliferation, and cell death. One target, thrombospondin 1, is a potent inhibitor of blood vessel growth and is known to be down-regulated in Kaposi sarcoma tumors. Thrombospondin 1, which is targeted by multiple miRNAs, also showed reduced protein levels in our study. To our knowledge, our data describe the first targets for tumorvirus-encoded miRNAs and suggest that these novel regulators may have roles in pathogenesis.