Kaposi's sarcoma-associated herpesvirus (KSHV) is the responsible agent for kaposi's sarcoma (KS), primary effusion lymphoma and multicentric Castlemans disease. A recent discovery revealed KSHV expresses multiple microRNAs with the potential to modulate host gene expression. Most microRNAs repress target gene expression by destabilizing the mRNA transcript and decreasing translational efficiency. The general path of research in our group starts with target prediction using expression profiling data, followed by target validation using luciferase reporters and protein expression measurements, and finally we conduct target significance assays. The significance assays attempt to address the question of why the virus has selected specific target genes for inhibition by viral microRNAs. We hope to discover new functions of human genes as they relate to viral infection and cancer. Using previously generated expression profiling data, we constructed a new dataset utilizing a different method of expression analysis to integrate the expression data from multiple gain and loss of microRNA function experiments. After making new unbiased systematic microRNA target predictions based on the new method of expression analysis and disregarding whether these targets are predicted by commonly used microRNA target prediction programs, we cloned around fifty 3'untranslated regions (these are common microRNA target regions) from the predicted target genes into luciferase reporter plasmids. Our group acquired a luminometer that handles 96-well plates in order to test more reporters genes in a more efficient and reproducible fashion. We have validated an additional fourteen microRNA target genes using luciferase assays this year. The luciferase validation experiments are ongoing, but we have a 50% success rate in accurate microRNA target prediction. A subset of these target genes has been further validated by looking at protein expression of endogenous target genes in response to viral microRNA expression, microRNA inhibition and KSHV infection. To assess changes in protein expression, we utilize an acquired near-infrared scanner to perform simultaneous two-color quantitative western blotting assays. In addition, we have recently optimized inhibition of viral microRNAs using 96-well electroporation system and locked nucleic acids. These optimized conditions will be used for future microRNA target validation assays, but also in addressing the functional significance on specific viral microRNA expression during infection. In addition, we have mapped at least one functional microRNA target site by site directed mutagenesis. Furthermore, we have begun processing KS biopsies from patients that we will use in western blotting assays to determine if the microRNA target genes that are inhibited in our cell culture systems are reproduced at sites of infection in patients. In order to complement our mRNA expression profiling dataset, we have also begun a proteomic approach to assess changes in protein expression as a result of KSHV microRNA expression. In the near future, we will investigate changes in protein expression as an additional filter in our microRNA target prediction method. While most of our work this year has been focused on microRNA target prediction and validation, we plan to devote more resources and establish collaborations to determine the functional significance of specific microRNA targets genes in the future.
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