ZIA BC 011176 (ZIA) | |||
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Title | Functions of KSHV microRNAs | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Ziegelbauer, Joseph | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $574,853 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2017 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Herpes - Other (100.0%) |
Kaposi Sarcoma (50.0%) Sarcoma (50.0%) |
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Research Type | |||
Cancer Initiation: Oncogenes & Tumor Suppressor Genes Interactions of Genes and/or Genetic Polymorphisms with Exogenous and/or Endogenous Factors |
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Abstract | |||
Kaposi's sarcoma-associated herpesvirus (KSHV) is the responsible agent for Kaposi's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV expresses multiple microRNAs that modulate human gene expression. Most microRNAs (miRNAs) repress target gene expression by destabilizing the mRNA transcript and decreasing translational efficiency. A goal of the project is to determine targets of viral miRNAs and understand why the virus has selected specific human target genes for inhibition. We hope to discover new functions of human genes as they relate to viral infection and cancer. Using a variety of expression profiling data, we constructed a dataset to integrate the expression data from multiple gain and loss of microRNA function experiments. We have tested over fifty predicted target genes and over thirty microRNA target genes were significantly inhibited by viral miRNAs using a variety of validation methods. 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 in infected cells and KSHV infection. In addition, we have mapped functional microRNA target sites in multiple human genes using site-directed mutagenesis. Furthermore, using KS biopsies from patient enrolled in clinical trials, we have determined multiple microRNA target genes that are inhibited in our cell culture systems are also inhibited at sites of KSHV infection in patients. We are currently investigating the functional roles of a couple of selected miRNA targets identified in these approaches. Below are some of the selected miRNA targets and their functional significance. From various expression screens to identify miRNA targets, we found that Kaposi's sarcoma-associated herpesvirus (KSHV) viral microRNAs (miRNAs) target several enzymes in the mevalonate/cholesterol pathway. 3-Hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR a rate-limiting step in the mevalonate pathway), and farnesyl-diphosphate farnesyltransferase 1 (FDFT1 a committed step in the cholesterol branch) are repressed by multiple KSHV miRNAs. Transfection of viral miRNA mimics in primary endothelial cells (human umbilical vein endothelial cells, HUVECs) reduced intracellular cholesterol levels. We also found that cholesterol levels were decreased in de novo-infected HUVECs after seven days. This reduction is at least partially due to viral miRNAs, since the mutant form of KSHV lacking 10 of the 12 miRNA genes had increased cholesterol compared to wild-type KSHV infections. We hypothesized that KSHV is downregulating cholesterol to suppress the antiviral response by a modified form of cholesterol, 25-hydroxycholesterol (25HC). We found that the cholesterol 25-hydroxylase (CH25H) gene, which is responsible for generating 25HC, had increased expression in de novo-infected HUVECs, but was strongly suppressed in long-term latently infected cell lines. We found that 25HC inhibits KSHV infection when added exogenously prior to de novo infection. In conclusion, we found that multiple KSHV viral miRNAs target enzymes in the mevalonate pathway to modulate cholesterol in infected cells during latency. This repression of cholesterol levels could potentially be beneficial to viral infection by decreasing the levels of antiviral 25HC. These results suggest a new virus-host relationship and indicate a previously unidentified viral strategy to lower cholesterol levels. We were interested in identifying cellular networks that were targeted by KSHV-miRNAs and employed network building strategies using validated KSHV miRNA targets and gene-gene associations based on published literature. Here, we report the identification of a network of genes repressed by KSHV miRNAs that center on the transcription factor- signal transducer and activator of transcription 3 (STAT3) that is also targeted by KSHV miRNAs. KSH |