Classic Hodgkin lymphoma (cHL) is a B-cell malignancy marked by Hodgkin and Reed/Sternberg (HRS) cells that highly express CD30. We explored the roles of microRNAs (miRNAs) in cHL pathogenesis and CD30-targeting therapy using Brentuximab Vedotin (BV) through high-throughput screening techniques. Review of miRNA-related studies on known and potential miRNA-target gene interactions identified multiple miRNA-gene pairs with potential relevance for cHL pathogenesis. This highlighted the relevance of miRNAs in cHL, warranting further studies to deepen our understanding.
A microRNA overexpression library screen revealed 11 miRNA-target gene pairs implicated in cHL pathogenesis and 66 miRNA constructs that regulate BV treatment response. Among these, miR-590 was found to inhibit CD30 expression and induce BV resistance. Additionally, miR-191-5p not only conferred BV resistance but also promoted cell growth. By using AGO2-immunoprecipitation, we identified a network of involved target genes, including PTEN.
To investigate endogenous miRNAs involved in BV resistance mechanisms, we established two BV-resistant cHL cell lines and observed dynamic changes in CD30 expression. Moreover, we utilized a miRNA-focused CRISPR-Cas9 screen to identify endogenous miRNAs that regulate CD30 expression and/or BV sensitivity. To this end, we generated two inducible Cas9 monoclonal cell lines and optimized the entire screening process. These screens await further data analysis.
In summary, this thesis of Penny Pan highlights the functional importance of specific miRNAs in cHL and provides detailed procedures of the miRNA-focused screening approaches, from initial setup to optimization and validation of results.
