In-Silico and In-Vitro Investigation of Key Long Non-coding RNAs Involved in 5-Fluorouracil Resistance in Colorectal Cancer Cells: Analyses Highlighting NEAT1 and MALAT1 as Contributors.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:39246994", "kind": "science-record", "revision": 4, "content_hash": "sha256:80aef0a6a37e9cfab5e8321f417a837ec8d896152a8ace6b270cd909aa10ff73", "created_at": "2026-11-01T20:29:23Z", "updated_at": "2026-11-01T22:55:07Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "In-Silico and In-Vitro Investigation of Key Long Non-coding RNAs Involved in 5-Fluorouracil Resistance in Colorectal Cancer Cells: Analyses Highlighting NEAT1 and MALAT1 as Contributors.", "doi": "10.7759/cureus.66393", "pmid": "39246994", "authors": ["Roxana Sahebnasagh", "Zahra Azizi", "Tahereh Komeili-Movahhed", "Kazem Zendehdel", "Mohammad Hossein Ghahremani"], "journal": "Cureus", "year": 2024, "abstract": "Background Acquired resistance to 5-fluorouracil (5-FU) frequently results in chemotherapy failure and disease recurrence in advanced colorectal cancer (CRC) patients. Research has demonstrated that dysregulation of long non-coding RNAs (lncRNAs) mediates the development of chemotherapy resistance in cancerous cells. The present study aims to identify key lncRNAs associated with 5-FU resistance in CRC using bioinformatic and experimental validation approaches. Methods The Gene Expression Omnibus (GEO) dataset GSE119481, which contains miRNA expression profiles of the parental CRC HCT116 cell line (HCT116/P) and its in-vitro established 5-FU-resistant sub-cell line (HCT116/FUR), was downloaded. Firstly, differentially expressed microRNAs (DEmiRNAs) between the parental and 5-FU resistance cells were identified. LncRNAs and mRNAs were then predicted using online databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to uncover relevant biological mechanisms and pathways. Networks integrating lncRNAs, miRNAs, and mRNAs interactions were constructed, and topological analyses were used to identify key lncRNAs associated with 5-FU resistance. An in-vitro model of the HCT116/FUR sub-cell line was developed by exposing the HCT116/P cell line to increasing concentrations of 5-FU. Finally, real-time quantitative PCR (RT-qPCR) was performed on total RNA extracted from the HCT116/P cell line and the HCT116/FUR sub-cell line to validate the in-silico predictions of key lncRNAs. Results A total of 32 DEmiRNAs were identified. Enrichment analysis demonstrated that these DEmiRNAs were mainly enriched in several cancer hallmark pathways that regulate cell growth, cell cycle, cell survival, inflammation, immune response, and apoptosis. The predictive analysis identified 237 unique lncRNAs and 123 mRNAs interacting with these DEmiRNAs. The pathway analysis indicated that most of these predicted genes were enriched in the cellular response to starvation, protein polyubiquitination, chromatin remodeling, and negative regulation of gene expression. Topological analyses of the lncRNA-miRNA-mRNA network highlighted the nuclear enriched abundant transcript 1 (NEAT1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and Opa interacting protein 5 antisense RNA 1 (OIP5-AS1) as central lncRNAs. Experimental analysis by RT-qPCR confirmed that the expression levels of NEAT1 and MALAT1 were significantly increased in HCT116/FUR cells compared to HCT116/P cells. However, no significant difference was observed in the OIP5-AS1 expression level between the two cells. Conclusion Our findings specifically highlight MALAT1 and NEAT1 as significant contributors to 5-FU resistance in CRC. These lncRNAs are promising biomarkers for diagnosing and predicting outcomes in CRC.", "claims": [{"id": "claim-method", "statement": "Differentially expressed miRNAs (DEmiRNAs) were identified by comparing the HCT116/FUR sub-cell line with HCT116/P cells, using a threshold of |log2 fold change (FC)| > 0.6 and a p-value < 0.05. A student's t-test was employed to compare the two groups.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:39246994", "locator": "sections[2] \"Materials and methods\" paragraph 4", "quote": "Differentially expressed miRNAs (DEmiRNAs) were identified by comparing the HCT116/FUR sub-cell line with HCT116/P cells, using a threshold of |log2 fold change (FC)| > 0.6 and a p-value < 0.05."}, {"asset": "source:pmid:39246994", "locator": "sections[2] \"Materials and methods\" paragraph 31", "quote": "A student's t-test was employed to compare the two groups."}], "state": "complete"}, {"id": "claim-data-access", "statement": "Data associated with this study are available upon request.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:39246994", "locator": "sections[6] \"Acknowledgments\" paragraph 2", "quote": "Data associated with this study are available upon request."}], "state": "complete"}, {"id": "claim-target-result", "statement": "Experimental analysis by RT-qPCR confirmed that the expression levels of NEAT1 and MALAT1 were significantly increased in HCT116/FUR cells compared to HCT116/P cells.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:39246994", "locator": "sections[0] \"Abstract\" paragraph 6", "quote": "Experimental analysis by RT-qPCR confirmed that the expression levels of NEAT1 and MALAT1 were significantly increased in HCT116/FUR cells compared to HCT116/P cells."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "Experimental analysis by RT-qPCR confirmed that the expression levels of NEAT1 and MALAT1 were significantly increased in HCT116/FUR cells compared to HCT116/P cells.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:39246994"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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Finally, real-time quantitative PCR (RT-qPCR) was performed on total RNA extracted from t", "as central lncRNAs. Experimental analysis by RT-qPCR confirmed that the expression levels of NEAT", "-FU. Finally, real-time quantitative PCR (RT-qPCR) was performed on total RNA extracted from t", "central lncRNAs. Experimental analysis by RT-qPCR confirmed that the expression levels of NEAT"], "order_basis": "paper_text_order:1505", "id": "assay:1"}, {"role": "data_generation", "definition_id": "apoptosis_assay", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["survival, inflammation, immune response, and apoptosis. The predictive analysis identified 237 uniq", "city assay and resistance index measurement, apoptosis evaluation, cell cycle analysis, and morphol", "nal cell number) − ln(Initial cell number)). Apoptosis assay 5-FU-induced apoptosis in the HCT116/P", "cell number)). Apoptosis assay 5-FU-induced apoptosis in the HCT116/P cell and HCT116/FUR sub-cell"], "order_basis": "paper_text_order:1940", "id": "assay:2"}, {"role": "data_generation", "definition_id": "cell_culture", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["ration over 10 months. Briefly, the HCT116/P cell line was cultured in a drug-free medium for 24 hours. After th", "itute of Iran, Tehran, Iran). The cells were cultured in RPMI medium, supplemented with 100 U/mL peni", "months. Briefly, the HCT116/P cell line was cultured in a drug-free medium for 24 hours. After that,", "re trypsinized, and the remaining cells were cultured in a fresh, drug-free medium. The treatment was"], "order_basis": "paper_text_order:7461", "id": "assay:3"}, {"role": "data_generation", "definition_id": "elisa", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.93, "evidence": ["well was measured at 570 nm/690 nm using an ELISA Plate Reader (Anthos, UK). The resistance in"], "order_basis": "paper_text_order:10195", "id": "assay:4"}, {"role": "data_generation", "definition_id": "flow_cytometry", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["HCT116/FUR sub-cell line was evaluated using flow cytometry and annexin V-FITC/propidium iodide (PI) sta", "d to 5-FU was assessed using PI staining and flow cytometry. Cells (5×104 cells/well) were seeded in 12-", "After staining, cells were analyzed using a fluorescence-activated cell sorting Calibur flow cytometer (BD Biosciences, Madr", "es (G0/G1, S, and G2/M) was analyzed using a fluorescence-activated cell sorting Calibur flow cytometer. RNA extraction and r"], "order_basis": "paper_text_order:10946", "id": "assay:5"}, {"role": "analysis", "definition_id": "group_comparison", "definition_version": "f2132e96ae6c7a4e", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"dsl_catalogue": "f2132e96ae6c7a4e"}, "confidence": 1.0, "evidence": ["Differentially expressed miRNAs (DEmiRNAs) were identified by comparing the HCT116/FUR sub-cell line with HCT116/P cells, using a threshold of |log2 fold change (FC)| > 0.6 and a p-value < 0.05. A student's t-test was employed to compare the two groups."], "order_basis": "consumes_earlier_output", "id": "assay:6"}]}