Dual role of gga-miR-155 in modulating immunogenic and oncogenic responses in avian leukosis virus (ALV) induced B cell lymphomas

Abstract

MicroRNAs (miRNAs) are small, single stranded RNA molecules ranging between 19-22nt in length, that are mainly responsible for maintaining cellular homoeostasis by epigenetically regulating the mRNA translation. Each individual miRNA possesses a signature seed sequence spanning between 2-8nt in length. The seed sequence specifically binds to the untranslated regions (UTR) occurring at the either end of functional mRNA molecules based on Watson and Crick complementarity which decides the translational fate of target mRNAs into functionally active proteins. Amongst the many miRNAs discovered, miRNA-155 (miR-155) is one of the most studied miRNAs due to its diverse functional roles in several biological processes. For example, miR-155 expression is reported to be essential in maintaining germinal centers as well as in biogenesis of immunologically functional B cell lineages. Suppression of miR-155 causes immunological impairment in B cells. Interestingly, miR-155 is found to be over-expressing in several human lymphomas including those caused by oncogenic viruses. Some of the examples include diffuse large B cell lymphoma, chronic lymphocytic leukemia, as well as in Epstein–Barr virus (EBV)-induced B cell transformation. All mis-express the miR-155 pathway in human cancers. Furthermore, the occurrence of viral orthologues of miR-155 in oncogenic viruses for e.g., kshv-miR-k12 in Kaposi sarcoma human virus (KSHV), along with their induced expression in human cancers highlights the functional involvement of this molecule in oncogenesis. A similar condition of neoplastic transformation is observed in lymphoid organs of Galliformes which is mainly caused by different avian oncogenic viruses such as Marek’s disease virus (MDV), avian leukosis virus (ALV) and reticuloendotheliosis virus (REV), that utilize the gga-miR-155 pathway. MDV, possesses a functional orthologue of gga-miR-155 known as the mdv1-miR-M4 that is upregulated and is proven to be essential for neoplastic transformation. The main goal of my PhD project is to perform an in-depth investigation of the underlying pathways regulated by gga-miR-155 in chicken tumour cell lines. I have employed CRISPR/Cas9 based genome editing for genetic ablation of gga-miR-155 in several chicken cell lines, including DF-1 fibroblast cells, HP45 (ALV subtype-A-induced B cell lymphoma cell line), AVOL-1 (REV-T strain transformed T cell lymphoma cell line) and chicken primordial germ cells (chPGCs). First, I have generated purified single cell clones of DF-1 cell line with ablated gga-miR-155 locus using CRISPR/Cas9 and demonstrated the feasibility and efficiency of CRISPR/Cas9 gene editing in a chicken cell line. I utilized the same gRNAs for genetic editing of gga-miR-155 locus in the ALV-transformed. B-cell lymphoma-derived HP45 cell line, as well as in the primary chicken primordial germ cells (PGC). Successful generation of gga-miR-155 deleted clones of HP45 and chicken PGCs demonstrated that expression of gga-miR-155 is not essential for the maintenance and proliferation of these cell types. Furthermore, gga-miR-155 deletion in chicken PGCs will allow the generation of gga-miR-155 knockout transgenic chickens in the future to study and understand effects of viral replication as well as oncogenesis in in vivo environment lacking gga-miR-155 expression. Derivation of purified single cell clones from genetically edited HP45 cell line allowed me to explore the functionality of gga-miR-155 in the following aspects – 1) In understanding involvement of gga-miR-155 on the biology, morphology, viability, and proliferation of the HP45 cell line. 2) In exploring differentially regulated genes (DRGs) using genome-wide high-throughput analysis: RNA sequencing as well as mass spectrometry and in analyzing effect of genetic modification on downstream genetic pathways by a bio-informatic approach. 3) Using the DRGs for characterizing new targets of gga-miR-155 by luciferase reporter assays. 4) And, in understanding the impact of gga-miR-155 ablation on the ALV replication which was analyzed by ELISA based ALV quantification. ALV, an alpha retrovirus of the family Retroviridae, induces activation of host genes by insertional mutagenesis. However, the specificity and selectivity of the insertion sites in insertional upregulation of host genes is not known. A duplicated long terminal repeat region (LTR) on either end of the virus, with strong promoter-enhancer function, is responsible for inducing the host gene expression. In the HP45 cell line, the ALV subgroup-A upregulates the host genes by integration of its LTR. According to earlier reports, ALV LTR showed integration and upregulation of MYC and BIC (gga-miR-155 host gene) in addition to other genes such as MYB and TERT that are oncogenic in nature. I confirmed that the HP45 cell line also showed induced expression of both MYC and BIC. Further, I have mapped for new integration sites by the Targeted Locus Amplification (TLA) (a high-throughput technique) in the same cell line. As a result, 8 new genes are identified, of which I successfully characterized 5 which showed successful upregulation.