Abstract 6697: Role of the miR-101-3p/CALCRL/NEAT1 axis in immune modulation and tumorigenesis in non-small cell lung cancer

Abstract Lung cancer (LC), particularly Non-Small Cell Lung Cancer (NSCLC), remains a global health challenge with a five-year survival rate of 17.8%, largely due to delayed diagnosis and limited understanding of tumor progression. MicroRNAs (miRNAs), key regulators of gene expression, play critical roles in immune modulation, metabolism, and tumorigenesis, but their role in immunometabolic reprogramming in LC is underexplored. This study investigated novel molecular targets in LC using integrated bioinformatics and in vitro approaches, focusing on the regulatory interplay between miRNAs, mRNAs, and long non-coding RNAs (lncRNAs). RNA-Seq datasets of lung adenocarcinoma (LUAD) were analyzed to identify differentially expressed genes (DEGs) and miRNAs (DEMs). Immune-related genes (IRGs) were curated and intersected with DEGs to identify differentially expressed immune-related genes (DEIRGs). A protein-protein interaction (PPI) network was constructed, and enrichment analyses were performed to identify hub DEIRGs. A ceRNA regulatory network involving miRNA (miR-101-3p), mRNA (CALCRL), and lncRNA (NEAT1) was constructed and analyzed using survival (KM Plotter) and tumor immune infiltration (TIMER) analyses. Experimental validation included expression analysis of miR-101-3p, CALCRL, and NEAT1 using Real-Time PCR and functional studies involving overexpression of NEAT1 and miR-101-3p via transfection. Real-time PCR, Western Blotting & Dual-luciferase assays confirmed CALCRL as a direct target of miR-101-3p. Functional assays—including cell proliferation (MTT assay), ROS production, mitochondrial membrane potential, ATP content, and apoptosis analysis (FACS)—were performed to assess the biological impact of this regulatory network. This study revealed a novel ceRNA axis involving miR-101-3p, CALCRL, and NEAT1. miR-101-3p was significantly downregulated, correlating with increased expression of CALCRL and NEAT1. Functional studies revealed that NEAT1 overexpression suppressed miR-101-3p while enhancing CALCRL expression, promoting tumor progression. Restoration of miR-101-3p suppressed tumor cell proliferation, induced ROS accumulation, impaired mitochondrial function, reduced ATP levels, and triggered apoptosis, underscoring its tumor-suppressive potential. In conclusion, this study provides a detailed framework for the miR-101-3p/CALCRL/NEAT1 axis, offering novel insights into immunometabolic reprogramming in LC. These findings highlight miR-101-3p as a promising therapeutic target, paving the way for miRNA-based therapies in NSCLC Citation Format: Gulnaz Tabassum, Mansoor A. Syed, Kapil Dev. Role of the miR-101-3p/CALCRL/NEAT1 axis in immune modulation and tumorigenesis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6697.