Assessment of Epstein-Barr virus miR-BARTs effects on in vitro properties of human-derived lymphomas and the expression of cell signaling pathways involved in lymphomagenesis

Abstract

Burkitt Lymphoma (BL) is a highly aggressive B-cell non-Hodgkin's lymphoma, and virtually all cases of endemic BL are associated with infection by the Epstein-Barr virus (EBV). Formally designated Human Gammaherpervirus 4 (HHV4), EBV is a ubiquitous virus that causes lifelong latent infection in over 90% of the adult human population. EBV infection is recognized as carcinogenic to human and the virus infects neoplastic cells also in cancers other than BL, such as nasopharyngeal carcinomas and some cases of gastric carcinomas, classical Hodgkin's lymphoma and other lymphoproliferative disorders, notably in immunosuppressed patients. EBV was the first virus in which small non-coding RNAs (microRNAs, or miRs) were found encoded by the viral genome. Worthy to note, latently infected B-cells abundantly express EBV miRs from the BamHI A rightward transcripts locus (aka, EBV miR-BARTs). In the last years, several studies showed that human (endogenous) miRs play a critical regulatory role in B-cell differentiation and oncogenesis. Nonetheless, little is known about the role of EBV miR-BARTs in lymphomagenesis. This study aims to investigate whether the expression of EBV-miR-BARTs affect selected parameters of cell health and behavior in vitro, as well as key molecules for signaling pathways relevant to lymphomagenesis. For this purpose, the CRISPR/CAS9 system will be used for EBV genome edition in order to abrogate the expression of EBV-miR-BARTs -7 and -9 in EBV-positive human lymphoma-derived cell lines. Briefly, cell lines expressing a humanized form of Cas9 will be produced and transduced with lentiviral vectors for expression of gRNAs targeting EBV-miR-BARTs -7 or -9. Upon successful generation of EBV miR-BARTs-7 and -9 knock-out cell lines (CRISPR/Cas9-edited), they will be assayed vitro for assessment of cell proliferation, cell viability, apoptosis, cell migration and cell invasion rates. Finally, the cells will be investigated regarding expression of selected molecules in key intracellular signaling pathways involved in lymphomagenesis, such as NFkB, PI3K/AKT, and JAK/STAT.