Differentially Regulated Splice Variants and Systems Biology Analysis of Kaposi’s Sarcoma-Associated Herpesvirus-Infected Lymphatic Endothelial Cells

• Main Authors: Ting-Yu Chang, 張庭毓
• Other Authors: Hsei-Wei Wang
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/38757941591034555286

博士 === 國立陽明大學 === 微生物及免疫學研究所 === 99 === Alternative RNA splicing greatly increases proteome diversity and thereby contributes to species-specific and tissue-specific functionality. The abnormal regulation of alternative splicing (AS) is known to lead to cellular pathogenesis and cancer. The possibility to study AS events on a genomic scale using splicing-sensitive microarrays, including the Affymetrix™ GeneChip® Exon 1.0 ST microarray (exon array), has appeared very recently. Kaposi’s sarcoma (KS) associated herpesvirus (KSHV) is the etiological agent of KS, a tumor of lymphatic endothelial cell (LEC) lineage. Earlier transcriptomic studies based solely on mRNA expression levels have suggested that gene expression is important to KSHV pathogenesis. However, little is known about the AS variations induced by KSHV. In this dissertation, we performed a genome-wide analysis of KSHV-controlled AS isoforms in virus-infected LEC to study the interaction between the host and this virus, and the mechanisms of both angiogenesis and lymphangiogenesis. Nevertheless, the application of this new exon array is hindered by the lack of free and user-friendly software devoted to these platforms. To solve this obstruction, a Java-based freeware, easyExon (http://microarray.ym.edu.tw/easyexon) has been developed for processing, filtrating and visualizing exon array data with an analysis pipeline. This tool implements the most commonly used probe set summarization methods as well as AS-orientated filtration algorithms, e.g. MIDAS and PAC, for the detection of AS events. EasyExon includes a biological filtration function according to Gene Ontology terms, and provides a module to visualize and interpret the selected exons and transcripts. Furthermore, easyExon can integrate with other related programs, such as Integrate Genome Browser (IGB) and Affymetrix™ Power Tools (APT), to make the whole analysis more comprehensive. EasyExon was applied to analyze KSHV infected LECs. This analysis covered ~20,000 known transcript clusters (~genes) and concluded AS is a key feature of KSHV infection. 542 genes with significant transcript isoform changes were identified. Splicing variants and altered exon-intron usage were found and these correlated with protein domain modification; this may result in function alternations that affect the encoded proteins. In addition, the different 3’ UTR used in these new transcripts will help the KSHV-induced transcript isoforms to escape microRNA-mediated surveillance and this will result in enhanced translation and protein synthesis. Exome-level analysis further revealed information that cannot be disclosed using classical gene-level profiling. For example, a significant exon usage difference existed between LECs and CD34+ endothelial precursor cells, and KSHV infection resulted in LEC-to-precursor, dedifferentiation-like exon level reprogramming. Systems biology analysis showed that KSHV-affected isoforms form a major genetic network consisting of several submodules, with genes such as IL1B, INSR and FOXC2 as hubs. The pathways identified showed that genes involved in cell motility, adhesion, proliferation, GNRH and RAR signaling were deregulated, which provide novel insights into KSHV-induced lymphangiogenesis. The results demonstrate that the regulatory effects of genetic variation in endothelial cells are far more complex than previously observed. This extra layer of molecular diversity helps to account for various aspects of endothelial biology, the KSHV life cycle and the disease pathogenesis that until now have been unexplored.