AAV-HOTAIR Delivery in Colorectal Cancer Cells: Preliminary Insights into Gene Silencing and Epigenetic Influences

INTRODUCTION: Colorectal cancer (CRC) remains a common and aggressive malignancy worldwide, highlighting the need for novel strategies to study and modulate disease-relevant pathways. This study aims to evaluate the feasibility of Adeno-Associated Virus-mediated HOTAIR silencing (AAV-HOTAIR) in CRC cells, focusing on delivery efficiency, gene expression modulation, and the influence of epigenetic context. METHODS: Caco-2 (CRC) and WI-38 (normal fibroblast) cell lines were used to assess AAV-HOTAIR penetration, cell viability, and expression of key oncogenes and tumor suppressors (KRAS, NF-κB, and p53). DNA methylation effects were explored using bisulfite-mediated CpG island analyses. RESULTS: AAV-HOTAIR transduction was higher in Caco-2 cells (84%) than in WI-38 cells (16%), and was associated with a 21% reduction in CRC cell viability, while WI-38 cells remained largely unaffected. Gene expression analysis showed a 77% decrease in KRAS, an 83% decrease in NF-κB, and an 85% increase in p53 in Caco-2 cells. CpG demethylation conditions influenced these trends, suggesting that epigenetic background can modulate cellular responses to HOTAIR silencing. Consistently, cleaved caspase-3 activity increased in transfected Caco-2 cells, confirming induction of apoptosis, while remaining unchanged in WI-38 cells. DISCUSSION: These findings provide preliminary evidence that AAV-mediated HOTAIR silencing is feasible and can selectively modulate oncogenic and tumor suppressor pathways in CRC cells. The observed effects were influenced by epigenetic context, underscoring the importance of accounting for DNA methylation in future studies. This work establishes a foundational framework for evaluating lncRNA-targeted delivery strategies in CRC. CONCLUSION: AAV-mediated HOTAIR silencing represents a promising approach for modulating CRC-associated molecular pathways. Further mechanistic and functional investigations, including apoptosis assays and expanded cell line panels, are warranted to validate and extend these findings.