Anastasija Bubanja*, Tamara Babic and Aleksandra Nikolic
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia
anastasija.bubanja [at] imgge.bg.ac.rs
Abstract
Cancer is characterized by a highly dynamic transcriptional landscape, in which alternative promoter usage plays a key role in regulating gene expression and contributes to transcript diversity. Recent large-scale transcriptomic studies have demonstrated differential promoter activity between malignant and non-malignant rectal tissues, including pronounced activation of the promoter driving the LDLRAD4-217 transcript (ENST00000592657.1).
In silico mutagenesis was performed by generating all possible single nucleotide variants across the transcript, followed by RNA secondary structure prediction and minimum free energy (MFE) calculation using RNAfold. Structural sensitivity was assessed as ΔMFE relative to the wild-type sequence, enabling identification of regions with high structural impact. Potential functional targets were identified by correlating LDLRAD4-217 expression with other transcripts in the Rectal Adenocarcinoma dataset from The Cancer Genome Atlas. Significantly correlated transcripts were selected, and RNA–RNA interactions were predicted using IntaRNA, with binding strength assessed based on interaction energy (ΔG). Cell-type deconvolution of TCGA READ data was performed using CIBERSORT (LM22 and custom single-cell-derived signatures) followed by correlation with LDLRAD4-217 expression and stromal stratification.
In silico mutagenesis revealed a non-uniform distribution of structural sensitivity across the transcript sequence, suggesting the presence of structurally important domains. Notably, prominent hotspot was identified within the region spanning nucleotides 238-302 and two small hotspots around position 165 and 460. Interaction hotspots were predominantly localized around 200 and 300 nt, corresponding to the unique exon of LDLRAD4-217. Among candidates a transcript DUOXA2-203, showed the strongest predicted interaction. Deconvolution results revealed a significant positive correlation with epithelial cells and immune cell populations, with divergent patterns observed within immune subsets, including positive and negative associations for mast cells, NK cells and CD4 T cells.
Our results suggest that LDLRAD4-217 represents a structurally constrained lncRNA with potential regulatory interactions and cell-type-specific expression, implicating its role in shaping the tumor microenvironment in rectal cancer.
Keywords: Rectal Cancer, LncRNA, Prediction, Transcriptome