Identification of key lncRNAs associated with oxaliplatin resistance in colorectal cancer cells and isolated exosomes: From In-Silico prediction to In-Vitro validation

Abstract

One of the critical challenges in managing colorectal cancer (CRC) is the development of oxaliplatin (OXP) resistance. Long non-coding RNAs (lncRNAs) have a crucial role in CRC progression and chemotherapy resistance, with exosomal lncRNAs emerging as potential biomarkers. This study aimed to predict key lncRNAs involved in OXP-resistance using in-silico methods and validate them using RT-qPCR methods in CRC cells and their isolated exosomes. Two public datasets, GSE42387 and GSE119481, were downloaded from the GEO database to identify differentially expressed genes (DEGs) and miRNAs (DEmiRNAs) associated with OXP-resistance in the HCT116 cell line. The analysis of GSE42387 revealed 210 DEGs, and GSE119481 identified 73 DEmiRNAs. A protein-protein interaction (PPI) network analysis of the DEGs identified 133 interconnected genes, from which the top ten genes with the highest degree scores were selected. By intersecting predicted miRNAs targeting these genes with the DEmiRNAs, 38 common miRNAs were found. Subsequently, 224 lncRNAs targeting these common miRNAs were predicted. LncRNA-miRNA-mRNA network were constructed and the top five lncRNAs with the highest degree scores were identified. Analysis using the Kaplan-Meier plotter database revealed that the key lncRNAs NEAT1, OIP5-AS1, and MALAT1 are significantly associated with the overall survival of CRC patients. To validate these lncRNAs, OXP-resistant HCT116 sub-cell line (HCT116/OXR) was developed by exposing parental HCT116 cells to gradually increasing concentrations of OXP. Exosomes derived from both HCT116 and HCT116/OXR cells were isolated and characterized utilizing dynamic light scattering (DLS), transmission electron microscopy (TEM), and Western blotting. RT-qPCR confirmed elevated levels of NEAT1, OIP5-AS1, and MALAT1 in HCT116/OXR cells and their exosomes compared to parental HCT116 cells and their exosomes. This study concludes that NEAT1, OIP5-AS1, and MALAT1 are associated with the OXP-resistance in CRC. The high levels of these lncRNAs in exosomes of resistant cells suggest their involvement in intercellular communication and resistance propagation. This positioning makes them promising biomarkers for OXP-resistance in CRC.

Fig 1. Bioinformatics analysis flowchart.

This flowchart illustrates the stepwise bioinformatics procedure utilized for identifying key lncRNAs associated with OXP-resistance in CRC. OXP, Oxaliplatin; CRC, Colorectal Cancer.

Fig 2. Heatmaps and volcano plots illustrating gene expression patterns associated with OXP-resistance in HCT116 cells, identified from the GSE42387 dataset (|log2 fold change (FC)| > 1, p-value < 0.05).

(A) Heatmap displaying gene expression patterns, with sample names on the horizontal axis and fold-change on the vertical axis. Up-regulated genes are indicated in green, while down-regulated genes are shown in red. (B) Volcano Plot demonstrating the association between alterations in gene expression fold change and their statistical significance. Green dots indicate up-regulated genes, while red dots represent down-regulated genes. Among the genes, HCLS1, EHF, KIRREL2, FGF9, ATG4A, CALB2, COL13A1, PMEPA1, BST2, and AKR1C3 were identified as the top 10 up-regulated genes, whereas IAH1, SUSD2, S100A4, FMR1, KRT23, HDGF, ZNF266, STXBP6, CYB5B, and GNE were the top 10 down-regulated genes. Additionally, the up-regulated lncRNAs LINP1, LINC00326, LINC00707, LINC00992, and CCDC144NL-AS1 were identified, while TMEM132D-AS1, SFTA1P, RARA-AS1, and LINC01405 were found to be down regulated. OXP, Oxaliplatin.

Fig 3. The PPI network of DEGS associated with OXP-resistance.

The PPI network consists of 133 nodes (genes) and 297 edges (interactions). Orange rectangles display up-regulated genes, while green rectangles display down-regulated genes. DEG, Differentially expressed gene; PPI, Protein-protein interaction; OXP, Oxaliplatin.

Fig 4. Functional enrichment analysis of genes in the PPI network.

(A–C) Top 20 enriched GO Terms for CC, BP, and MF, respectively (D) Top 20 Significant KEGG pathway terms (p-value < 0.05). GO, Gene Ontology; CC, Cellular Component; BP, Biological Process; MF, Molecular Function; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Fig 5. Heatmaps and volcano plots illustrating miRNA expression patterns associated with OXP-resistance in HCT116 cells, identified from the GSE119481 dataset (|log2 fold change (FC)| > 0.6, p-value < 0.05).

(A) Heatmap displaying miRNA expression patterns, with sample names on the horizontal axis and fold change on the vertical axis. Up-regulated miRNA are presented in green, while down-regulated miRNA are in red. (B) Volcano Plot demonstrating the association between alterations in expression fold change and their statistical significance. Green dots indicate up-regulated miRNA, while red dots represent down-regulated miRNA. OXP, Oxaliplatin.

Fig 6. The Venn diagram of overlapped data.

(A) The intersection between the predicted miRNAs and the DEmiRNAs to identify common miRNAs. (B) The intersection between the predicted lncRNAs from the StarBase and DIANA-lncBase databases to identify common lncRNAs. DEmiRNA, Differentially Expressed miRNAs.

Fig 7. The lncRNA-miRNA-mRNA networks.

This network contains 278 nodes and 2012 edges. Orange and green rectangles display up- and down-regulated genes, respectively; yellow ellipses represent lncRNAs, and blue hexagons represent miRNAs.

Fig 8. Kaplan–Meier overall survival curves for key lncRNAs in CRC patients.

The horizontal axis of the graph shows the overall survival time in years, while the vertical axis indicates the corresponding survival probability. (A) NEAT1, (B) MALAT1, (C) XIST and (D) OIP5-AS1.

Fig 9. The pathway enrichment analysis for DEGs associated with NEAT1, MALAT1, and OIP5-AS1.

This pathway performed using ClusterProfiler.

Fig 10. Establishment and characterization of HCT116/OXR sub-cell line.

(A) The process of establishing OXP-resistant cells involved gradually exposing parental HCT116 cells to increasing concentrations of OXP over a period. (B) Cell viability dose-response curves for parental HCT116 and HCT116/OXR cells exposed to different concentrations of OXP (0.1–1000 μM). (C) Growth curve of parental HCT116 and HCT116/OXR cells. (D) Morphology of parental HCT116 and HCT116/OXR cells. OXP, Oxaliplatin; HCT116/OXR, Oxaliplatin-resistance HCT116.

Fig 11. Apoptosis and cell cycle assay induced by OXP in parental HCT116 and HCT116/OXR cells using flow cytometry.

(A) Left: Flow cytometric histograms of apoptosis induced by OXP in cells. Apoptosis was analyzed using Annexin V/PI staining. The diagram represents live cells (lower left), early apoptotic cells (lower right), late apoptotic cells (upper right), and necrotic cells (upper left). Right: Bar diagrams, representing the average counts of apoptotic cells obtained from at least two independent experiments. The percentage of early and late apoptotic cells determined the apoptosis rate. The data are presented as means ± SD (*p < 0.05). (B) Left: Flow cytometric histograms of cell cycle phase distribution under treatment with OXP in parental HCT116 and HCT116/OXR cells. Right: Stacked bar graphs showing the average counts of cells at cell cycle phase obtained from at least two independent experiments. OXP, Oxaliplatin; HCT116/OXR, Oxaliplatin-resistance HCT116.

Fig 12. Characterization of exosomes derived from HCT116 and HCT116/OXR cells.

(A) The process of exosome isolation, including stepwise centrifugation and the use of an isolation kit. (B) Representative TEM images showing the presence and morphology of exosomes. (C) Size distribution graphs of exosomes detected by DLS analysis. (D) Western blot analysis demonstrating the detection of exosomal markers CD9 and CD63 in isolated exosomes.

Fig 13. Relative expression levels of NEAT1, OIP5-AS1 and MALAT1 in parental HCT116 and HCT116/OXR cells and their isolated exosomes.

(A) Bar diagrams present the expression levels of key lncRNAs in parental and OXP-resistance cells (B) Bar diagrams present the expression levels of key lncRNAs in isolated exosomes from parental and OXP-resistance cells. The data are presented as means ± SD. (* p < 0.05, **p < 0.01, and ***p < 0.001). OXP, Oxaliplatin; HCT116/OXR, Oxaliplatin-resistance HCT116; Exo, Exosome.