Publication: Elucidating IFI6 and RSAD2 protein expression in oral squamous cell carcinoma using immunohistochemistry and aptahistochemistry with novel in silico DNA-aptamers
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Date
2025-07
Authors
Butt, Danial Qasim
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Abstract
Emerging research highlights interferon-stimulated genes (ISGs) such as IFI6 and
RSAD2 as potential protumorigenic biomarkers, contributing to cancer proliferation and
survival. While their role in apoptotic dysregulation has been documented in various
malignancies, their involvement in evading immune evasion, particularly in oral
squamous cell carcinoma (OSCC), remains largely unexplored. Understanding their
influence within the tumour microenvironment is, therefore, imperative. Advances in
biomarker detection technologies offer substantial advantages in improving specificity,
sensitivity, and reproducibility while mitigating cost and batch variation challenges in
antibody-based OSCC diagnostics. This study aims to examine IFI6 and RSAD2 protein
expression in cancer and immune cells (neutrophils, plasma cells, and lymphocytes)
within the OSCC tumour microenvironment using immunohistochemistry (IHC) and
aptahistochemistry (AHC), employing in silico-derived DNA aptamers. Additionally, it
seeks to design, characterise, and validate these DNA aptamers for potential application
in AHC. IFI6 and RSAD2 protein expression was analysed in OSCC (n=23) and healthy
(n=7) tissue samples via IHC and AHC. The correlation between protein expression,
cancer, immune cell presence, and histological tumor grades was statistically assessed.
DNA aptamers for IFI6 and RSAD2 were designed in silico, and their binding interactions
were characterised using molecular docking (binding energy), PyMOL, and Protein Ligand Interaction Profiler (PLIP) for hydrogen bonding analysis. Molecular dynamics
simulations in GROMACS assessed complex stability (RMSD) and aptamer flexibility
(RMSF). Stem-hairpin loop DNA aptamers (35–50 meters) were successfully developed
for IFI6 and RSAD2. Among them, 35- and 50-mer IFI6 and 35- and 45-mer RSAD2
aptamers exhibited high binding affinity (-15.7 to -18.7 kcal/mol), strong hydrogen
bonding (<4 Å), stable RMSD (<0.4 nm), and flexible loop regions (RMSF >0.4 nm). IHC
and AHC analyses revealed significant IFI6 and RSAD2 expression in OSCC tissues
while absent in healthy samples (p<0.05). Expression levels were markedly elevated in
poorly differentiated tumour grades and tumour-associated immune cells (p<0.05). This
study confirms IFI6 and RSAD2 expression in OSCC cancer and immune cells,
correlating with higher tumour grades. These findings support their potential as prognostic
biomarkers in the tumour microenvironment. The in silico-derived DNA aptamers
demonstrate strong binding characteristics and hold promise for AHC-based OSCC
detection, offering a cost-effective and reliable diagnostic alternative.