Kaposi's Sarcoma-associated Herpesvirus Encodes an Ortholog of miR-155

MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression by binding to 3’UTRs of target mRNAs. Kaposi’s sarcoma-associated herpesvirus (KSHV), a virus linked to malignancies including primary effusion lymphoma (PEL), encodes 12 miRNA genes, but only a few regulatory targets are known. We found that KSHV-miR-K12-11 shares 100% seed-sequence homology with hsa-miR-155, a miRNA frequently found up-regulated in lymphomas and critically important for B cell development. Based on this seed-sequence homology, we hypothesized that both miRNAs regulate a common set of target genes and as a result, could have similar biological activities. Examination of five PEL lines showed that PELs do not express miR-155, but do express high levels of miR-K12-11. Bioinformatics tools predicted the transcriptional repressor BACH-1 to be targeted by both miRNAs and ectopic expression of either miR-155 or miR-K12-11 inhibited a BACH-1 3'UTR containing reporter. . Furthermore, BACH-1 protein levels are low in cells expressing either miRNA. Gene expression profiling of miRNA-expressing stable cell lines revealed 66 genes that were commonly down-regulated. For select genes, miRNA targeting was confirmed by reporter assays. Thus, based on our in silico predictions, reporter assays, and expression profiling data, miR-K12-11 and miR-155 regulate a common set of cellular targets. Given the role of miR-155 during B cell maturation, we speculate that miR-K12-11 may contribute to the distinct developmental phenotype of PEL cells, which are blocked in a late stage of B cell development. Together, these findings indicate that KSHV miR-K12-11 is an ortholog of miR-155. Keywords: comparison, experiemental versus control 12 samples, 4 experiemental (miR-155 ), 4 experimental (miR-K12-11) and 4 reference controls (pCDNA3.1)

微小RNA（MicroRNAs）是一类小型非编码RNA，可通过结合靶mRNA的3’非翻译区（3’UTR）在转录后水平调控基因表达。卡波西肉瘤相关疱疹病毒（KSHV）是一种与原发性渗出性淋巴瘤（PEL）等多种恶性肿瘤相关的病毒，其基因组编码12个微小RNA基因，但目前已知的调控靶点极少。本研究发现，KSHV编码的miR-K12-11与hsa-miR-155的种子序列完全同源；后者是一种在淋巴瘤中常见高表达的微小RNA，对B细胞发育至关重要。基于该种子序列同源性，我们推测二者可调控共同的靶基因集，进而发挥相似的生物学活性。对5株PEL细胞系的检测显示，PEL细胞不表达miR-155，但高表达miR-K12-11。生物信息学工具预测转录抑制因子BACH-1是二者共同的靶基因；异位表达miR-155或miR-K12-11均可抑制携带BACH-1 3’UTR的报告基因活性。此外，在表达任一微小RNA的细胞中，BACH-1的蛋白水平均显著降低。对稳定表达微小RNA的细胞系进行基因表达谱分析，共筛选出66个共同下调的基因。针对部分候选基因，我们通过报告基因实验验证了其受miRNA靶向调控的特性。综上，结合计算机预测结果、报告基因实验及表达谱数据分析，miR-K12-11与miR-155可调控一组共同的细胞靶基因。鉴于miR-155在B细胞成熟过程中的关键作用，我们推测miR-K12-11可能参与PEL细胞独特的发育表型调控——PEL细胞的发育被阻断于B细胞发育的晚期阶段。上述研究结果表明，KSHV编码的miR-K12-11是miR-155的直系同源物。关键词：比较分析；实验组与对照组；共12个样本，包括4个miR-155实验组、4个miR-K12-11实验组及4个pCDNA3.1空白对照。