Identification of targets of viral and cellular miRNAs
Among the many factors that cause a normal cell to proliferate and become malignant, viral infections represent one of the major contributors, as up to 15% of all human cancers involve a virus infection, such as the Epstein-Barr virus (EBV) associated cancers. Viruses have developed multiple strategies to prevent infected cells to enter apoptosis and to hide themselves from the immune system through the use of many immunomodulatory molecules. The discovery of viral miRNAs shows that in mammals, viruses can use the host RNA silencing machinery to their own advantage, and provides new insights as to how viruses can cause cancer. In that respect, the identification of cellular targets of viral and/or cellular miRNAs is an important challenge. To address this point, we use global and unbiased approaches such as transcriptomic and proteomic analyses of cell lines expressing individual or clustered viral miRNAs. We have generated such data for Kaposi’s sarcoma associated herpesvirus (KSHV) miRNAs. We use stable cell lines expressing all KSHV miRNAs either constitutionally or in an inducible manner. By analyzing the Affymetrix microarray profiles of these cells, it is possible to generate lists of potential targets. This is feasible because miRNAs do sometimes destabilize targeted transcripts. Candidate targets that are retrieved by this approach are then validated by reporter assays. We are also using a biochemical approach relying on the immunoprecipitation of argonaute complexes to isolate cellular targets.This project also contains a clinical aspect in which we are analyzing the miRNA profiles of tumor samples isolated from patients that are latently infected by EBV or KSHV.