Studying the role of KSHV-encoded miRNAs

描述(申请人提供)：卡波西肉瘤相关疱疹病毒(KSHV)编码12个前miRNAs，在KS皮损和KSHV相关淋巴增生性恶性肿瘤中高表达。异位表达和miRNA抑制方法，结合基因表达谱，已经确定了参与调控血管生成、细胞凋亡、内皮细胞分化和免疫监视等关键生物学过程的靶向mRNAs。此外，一些miRNAs可能通过靶向病毒mRNAs来调节潜伏期。使用HITS-CLIP技术对KSHV miRNAs在PEL细胞中靶向的mRNAs进行了初步分析，以丰富RISC相关的RNA，已经确定了数百个新的mRNAs靶点，并确认了先前已确定的靶点。此外，我们的分析表明，PEL细胞内的RISC复合体中有很高比例包含KSHV编码的miRNAs。基于这些数据，我们假设KSHV miRNAs导致了显著的和组织特异性的基因表达变化，这些变化有助于发病机制和肿瘤的发生。为了直接解决这一假设，我们建议1)通过HITS-CLIP在感染KSHV的PEL和内皮细胞中全面鉴定KSHV miRNA靶标；2)通过使用缺乏单个或多个miRNAs的KSHV突变体，在病毒复制、潜伏期的建立和维持、细胞周期控制和抗凋亡方面表征体外表型及其相关的调控途径。总之，将核基因组学方法用于miRNA靶标识别与病毒遗传学以及在培养细胞中确定病毒表型的能力相结合，将提供对KSHV编码的miRNA功能的详细分析，并可能导致抗病毒/抗肿瘤策略的发展。

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs that are highly expressed in KS lesions and KSHV- associated lymphoproliferative malignancies. Ectopic expression and miRNA inhibition approaches, combined with gene expression profiling, have identified target mRNAs that are involved in the regulation of critical biological processes such as angiogenesis, apoptosis, endothelial cell differentiation, and immune surveillance. In addition, several miRNAs may regulate latency by targeting viral mRNAs. Preliminary analyses of the mRNAs targeted by KSHV miRNAs in PEL cells using HITS-CLIP technology to enrich RISC-associated RNAs have identified hundreds of new mRNA targets and confirmed previously identified targets. Moreover, our analysis showed that a high percentage of RISC complexes within PEL cells contain KSHV- encoded miRNAs. Based on these data, we hypothesize that the KSHV miRNAs cause significant and tissue-specific changes in gene expression that contribute to pathogenesis and tumorigenesis. To directly address this hypothesis we propose to 1) Comprehensively identify KSHV miRNA targets by HITS-CLIP in KSHV-infected PEL and endothelial cells; 2) Characterize in vitro phenotypes and their associated regulatory pathways with respect to viral replication, the establishment and maintenance of latency, cell cycle control and resistance to apoptosis by using KSHV mutants lacking single or multiple miRNAs. In summary, combining ribonomics approaches for miRNA target identification with viral genetics, and the ability to determine viral phenotypes in cultured cells, will provide a detailed analysis of KSHV-encoded miRNA functions and may lead to the development of antiviral/antitumor strategies.