Trancriptome- based identification of circular RNA (circRNA) and its functional role in osimertinib-resistant non-small cell lung cancer (NSCLC) cells

Nalini, Devi Verusingam (2024) Trancriptome- based identification of circular RNA (circRNA) and its functional role in osimertinib-resistant non-small cell lung cancer (NSCLC) cells. PhD thesis, UTAR.

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Abstract

Recent GLOBOCAN 2022 report identifies lung cancer as the leading cause of cancer mortality worldwide, with 85% of cases being non-small cell lung cancer (NSCLC). Patients with classical EGFR mutations (E19 Delins/L858R) respond to first- and second-generation EGFR-TKIs, while those with the secondary EGFR-T790M mutation initially benefit from Osimertinib (AZD9291). However, unceasing resistance in these patients, poses ongoing challenges in NSCLC treatment. Recent advancements in RNA biomarkers have shown that circular RNAs (circRNAs) significantly contribute to NSCLC progression. Remarkable stability in blood plasma makes circRNAs promising cancer biomarkers, though their roles in Osimertinib-resistant (OR) remain unclear. In this study, we aimed to identify a putative circRNA biomolecule that regulates OR and elucidate its functional and mechanism involvements to reveal potential therapeutic targets for OR lung adenocarcinoma. Transcriptome-wide profiling via circRNA sequencing identified circSPINT2 (hsa_circ_0050818) as significantly downregulated in OR cell lines against parental H1975 cell line (fold change [FC] ≥ 2.0; P < 0.05). Characterization and validation of circSPINT2 were performed using qRT-PCR, Sanger sequencing (Back Splicing Junction: TATGAAGAGAATGC), ddPCR, actinomycin D, and RNase R assays (n=3, P<0.0001). Overexpression of circSPINT2 increased sensitivity to Osimertinib, reducing tumoursphere formation, colony formation, and migration, while knockdown induced resistance and enhanced tumorigenicity in the targeted cells. Biotinylated-RNA probe pull-down, miRNA sequencing, and in�silico analysis identified hsa-miR-1296-3p as a target of circSPINT2, regulating RBP1 expression. In-vivo validation using an Osimertinib induced resistant H1975 xenograft tumour model confirmed these findings. This study concludes that circSPINT2 has a tumour-suppressive effect and enhances Osimertinib sensitivity in OR cells by sponging oncogenic miR-1296-3p, leading to increased expression of tumour-suppressive RBP1. These findings may have a substantial influence on detection and treatment of Osimertinib resistance, potentially leading to tailored treatment options for patients with NSCLC. Keywords: biomarkers; drug resistant; epithelial growth factor receptor; lung cancer; RNA therapeutics; transcriptomic profiling Subject Area: Subclass RM Therapeutics. Pharmacology; RC254-282 Neoplasms. Tumours. Oncology including cancer and carcinogens

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