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AAV-HOTAIR Delivery in Colorectal Cancer Cells: Preliminary Insights into Gene Silencing and Epigenetic Influences
Mohammed Y I Al-Hamadani1, Firas H Khathayer2, Mohammed N Al-Khafaf3
1Department of Pathological Analysis, College of Applied Science, University of Fallujah, Fallujah, Iraq.
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.
Insights
Adeno-Associated Virus-mediated HOTAIR silencing (AAV-HOTAIR) effectively targets colorectal cancer (CRC) cells, reducing viability and modulating key oncogenes. Epigenetic context influences these promising results for CRC therapy.
Area of Science:
- Molecular Biology
- Cancer Research
- Epigenetics
Background:
- Colorectal cancer (CRC) is a prevalent and aggressive malignancy globally.
- Novel therapeutic strategies are crucial for modulating CRC-associated pathways.
- Long non-coding RNAs (lncRNAs) like HOTAIR are implicated in CRC pathogenesis.
Purpose of the Study:
- To evaluate the feasibility of Adeno-Associated Virus-mediated HOTAIR silencing (AAV-HOTAIR) in CRC.
- To assess AAV-HOTAIR's delivery efficiency and gene expression modulation in CRC cells.
- To investigate the influence of epigenetic context, specifically DNA methylation, on AAV-HOTAIR efficacy.
Main Methods:
- Utilized Caco-2 (CRC) and WI-38 (normal fibroblast) cell lines.
- Assessed AAV-HOTAIR transduction efficiency, cell viability, and expression of KRAS, NF-κB, and p53.
- Analyzed DNA methylation using bisulfite-mediated CpG island analysis and measured apoptosis via cleaved caspase-3 activity.
Main Results:
- AAV-HOTAIR showed higher transduction in Caco-2 cells (84%) versus WI-38 cells (16%).
- CRC cell viability decreased by 21% with AAV-HOTAIR, while normal cells were unaffected.
- Significant modulation of oncogenes/tumor suppressors observed: KRAS (-77%), NF-κB (-83%), p53 (+85%) in Caco-2 cells.
- Apoptosis was induced in CRC cells, confirmed by increased cleaved caspase-3 activity.
- Epigenetic context, particularly CpG demethylation, influenced HOTAIR silencing effects.
Conclusions:
- AAV-mediated HOTAIR silencing is a feasible strategy for selectively targeting CRC cells.
- This approach modulates key oncogenic and tumor suppressor pathways in CRC.
- Epigenetic factors, like DNA methylation, play a significant role and must be considered in future CRC therapies.
- This study provides a foundation for lncRNA-targeted delivery strategies in CRC treatment.
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