LINC02418 upregulates EPHA2 by competitively sponging miR-372-3p to promote 5-Fu/DDP chemoresistance in colorectal cancer

Abstract

Chemoresistance is a huge clinical challenge in the treatment of advanced colorectal cancer (CRC). Non-coding RNAs (ncRNAs) and messenger RNA (mRNA) are involved in CRC chemoresistance. However, the profiles of long ncRNAs (lncRNAs), microRNAs (miRNAs), mRNAs and competing endogenous RNA (ceRNA) networks in CRC chemoresistance are still largely unknown. Here, we compared the gene expression profiles in chemosensitive (HCT8) and chemoresistant [HCT8/5-fluorouracil (5-Fu) and HCT8/cisplatin (DDP)] cell lines by whole-transcriptome sequencing. The common differentially expressed RNAs in two drug-resistant cells were selected to construct lncRNA-miRNA-mRNA networks. The ceRNA network closely related to chemoresistance was further established based on the widely accepted drug resistance-associated genes enriched in three signaling pathways involved in chemoresistance. In total 52 lncRNA-miRNA-mRNA pathways were screened out, among which EPHA2 and LINC02418 were identified as hub genes; thus, LINC02418/miR-372-3p/EPHA2 were further selected and proved to affect the 5-Fu and DDP resistance of CRC. Mechanistically, LINC02418 upregulated EPHA2 by functioning as a 'sponge' of miR-372-3p to modulate the chemoresistance of CRC. Collectively, our study uncovered the underlying mechanism of LINC02418/miR-372-3p/EPHA2 in 5-Fu and DDP resistance of CRC, which may provide potential therapeutic targets for improving the chemosensitivity of CRC.

Graphical abstract

Figure 1.

Expression profiles of lncRNAs, miRNAs and mRNAs. The volcano plot of DE lncRNAs, miRNAs and mRNAs in (A) HCT8/5-Fu cell lines and (B) HCT8/DDP cell lines. (C) Cluster analysis of DE lncRNAs, miRNAs and mRNAs. (D) Venn diagram displayed DE and overlapping lncRNAs, miRNAs and mRNAs between HCT8/5-Fu and HCT8/DDP cell lines.

Figure 2.

The clinical significance of key genes in chemoresistant related lncRNA–miRNA–mRNA regulatory networks. (A) CeRNA networks are based on the genes related to chemoresistance. The expression of LINC02418 (B) and EPHA2 (C) in CRC tissues and normal tissues from TCGA database. The expression of LINC02418 (D) and EPHA2 (E) in CRC cell lines was detected by qRT-PCR or Western blotting. Data were shown as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Kaplan-Meier survival analysis revealed that CRC patients with high LINC02418 (F) and EPHA2 (G) expression showed poorer overall survival compared to those with low expression.

Figure 3.

LINC02418 is upregulated in CRC chemoresistant cell lines and responsible for the chemoresistance of CRC. (A) LINC02418 was upregulated in HCT8/5-Fu and HCT8/DDP cells detected and validated by RNA-sequencing and qRT-PCR, respectively. (B) The expression of LINC02418 was knocked down by three different siRNAs in two chemoresistant cell lines. Cell viability and IC₅₀ values of HCT8/5-Fu (C) and HCT8/DDP cells (D) with LINC02418 knockdown were assessed by MTT assay. (E) Knockdown LINC02418 in chemoresistant cells increased cell apoptosis rates. (E) LINC02418 knockdown led to a decrease in the S phase in HCT8/5-Fu (F) and HCT8/DDP cells (G). Data are shown as mean ± SD, and the results represent three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

MiR-372-3p is downregulated in CRC chemoresistant cell lines and responsible for the chemoresistance of CRC. (A) MiR-372-3p was lowly expressed in HCT8/5-Fu and HCT8/DDP cells detected and validated by RNA-sequencing and qRT-PCR, respectively. (B) The expression of miR-372-3p was detected when transfected with miR-372-3p mimics or NC mimics in two chemoresistant cell lines. Cell viability and IC₅₀ values of HCT8/5-Fu (C) and HCT8/DDP cells (D) transfected with miR-372-3p and NC mimics were detected. (E) The proliferation capacities were inhibited in miR-372-3p overexpressing chemoresistant cells. (F) Flow cytometry assays suggested that the percentage of apoptotic chemoresistant cells was increased by miR-372-3p overexpression. (G) Overexpressed miR-372-3p led to the changes in cell cycle distribution in chemoresistant cells. Data are shown as mean ± SD, and the results represent three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

EPHA2 is upregulated in CRC chemoresistant cell lines and related to the chemoresistance of CRC. (A) EPHA2 was highly expressed in HCT8/5-Fu and HCT8/DDP cells detected and validated by RNA-sequencing and qRT-PCR and western blotting, respectively. (B) The activity of EPHA2 was inhibited when treated with ALW-II-41-27 for 72 h. Cell viability and IC₅₀ values of HCT8/5-Fu (C) and HCT8/DDP cells (D) treated with 1 µM of ALW-II-41-27 and dimethyl sulfoxide (DMSO) for 72 h were assessed by MTT assay. (E) The proliferation capacities, (F) Apoptotic rates and (G) distributions of the cell cycle were assessed in HCT8/5-Fu and HCT8/DDP cells treated with either DMSO or 1 µM of ALW-II-41-27 for 72 h. Data are shown as mean ± SD, and the results represent three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

LINC02418 functions as a molecular sponge for miR-372-3p to regulate EPHA2. (A) The expression levels of miR-372-3p in HCT8/5-Fu and HCT8/DDP cells following knockdown of LINC02418 were evaluated by qRT-PCR. (B) The potential interaction sites between miR-372-3p and LINC02418 were predicted with bioinformatics analysis. Dual-luciferase reporter assay was applied to demonstrate the combination between miR-372-3p and LINC02418 in 293T cells. The expression levels of EPHA2 in HCT8/5-Fu (C) and HCT8/DDP cells (D) transfected with miR-372-3p mimics or NC mimics were evaluated by qRT-PCR and western blotting. (E) Schematic diagram of putative base pairing between miR-372-3p and EPHA2. Luciferase activity was measured in 293T cells cotransfected with pmirGLO-EPHA2-WT or pmirGLO-EPHA2-MUT and miR-372-3p mimics. The mRNA and protein levels of EPHA2 in HCT8/5-Fu (F) and HCT8/DDP cells (G) transfected with si-LINC02418 were detected by qRT-PCR and western blotting assay. (H) EPHA2 mRNA levels in HCT8/5-Fu and HCT8/DDP cells transfected with si-NC + miR-NC inhibitor, si-LINC02418 + miR-NC inhibitor, si-NC + miR-372-3p inhibitor and si-LINC02418 + miR-372-3p inhibitor were detected by qRT-PCR. Data are shown as mean ± SD, and the results represent three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.