Integrated Transcriptomic, In Silico, and In Vitro Characterization of lncRNA ENST00000615487.1 Reveals Epithelial-Specific Expression, Differential Subcellular Distribution Between Normal and Colorectal Cancer Cells, and Potential Regulatory Functions

Background/Objectives: Long non-coding RNAs (lncRNAs) are important regulators of tumor biology through their interactions with DNA, proteins, and non-coding RNAs. Although ENST00000615487.1 (also known as CTD-2396E7.11/AC010503.4) has been associated with multiple malignancies, its biological role in colorectal cancer (CRC) remains poorly characterized. This study aimed to investigate the expression pattern, cellular and subcellular localization, and potential functional role of ENST00000615487.1 in CRC using integrated in vitro and in silico approaches. Methods: Molecular characteristics of the transcript were obtained with the CPC2 and RNA Analyzer 3 tools. Differential expression of ENST00000615487.1 across 10 tumor types was analyzed using the UCSC Xena Browser. Transcript expression was experimentally evaluated in normal, tumor, and fibroblastic colon cell lines by PCR, while subcellular localization was assessed through the lncATLAS, lncLocator, and iLoc-LncRNA tools, and experimentally confirmed by qRT-PCR. Single-cell RNA sequencing data from the GSE161277 dataset were analyzed to determine cell type-specific expression patterns. Potential interactions with DNA, miRNAs, and proteins were investigated using Fasim-LongTarget, miRDB, and AnnoLnc2, followed by functional enrichment analyses using STRING and Enrichr. Results: ENST00000615487.1 was identified as a structurally stable non-coding transcript with a highly organized secondary structure. Differential expression analysis demonstrated significant downregulation in CRC compared with that in normal colon tissue. Single-cell transcriptomic analysis revealed predominantly epithelial-specific expression. In silico and experimental analyses demonstrated predominant nuclear localization in normal colon cells, whereas cytoplasmic enrichment was observed in CRC cells. Functional analyses identified potential interactions with HIP1R, RPH3AL, specific miRNAs, and proteins involved in transcriptional regulation and RNA processing pathways, as well as functional connections with proteins involved in vesicular transport. Conclusions: ENST00000615487.1 is a structurally stable lncRNA exhibiting context-dependent expression and localization patterns in CRC, suggesting a potential shift from nuclear transcriptional regulation toward cytoplasmic post-transcriptional functions during colorectal carcinogenesis.