FOXA2 Activates RND1 to Regulate Arachidonic Acid Metabolism Pathway and Suppress Cisplatin Resistance in Lung Squamous Cell Carcinoma

Abstract

Background: The primary cause of cancer-related fatalities globally is lung cancer. Although the chemotherapy drug cisplatin (DDP) has brought certain benefits to patients, the rapid development of drug resistance has greatly hindered treatment success.

Methods: We used the lung squamous cell carcinoma (LUSC) mRNA data set to explore the differentially expressed gene (RND1) in LUSC and detected RND1 expression in LUSC cells and DDP-resistant cells by qRT-PCR. Meanwhile, we performed abnormal expression treatment on RND1 and conducted CCK8, colony formation, and flow cytometry to evaluate the impact of RND1 expression on cell proliferation, apoptosis, and DDP resistance. In addition, we analyzed metabolism pathways involving RND1 using GSEA. We also used online tools such as hTFtarget and JASPAR to screen for the upstream transcription factor FOXA2 of RND1 and verified their relationship through CHIP and dual luciferase experiments. Finally, we validated the role of FOXA2-RND1 in DDP resistance in LUSC through the above experiments.

Results: RND1 was downregulated in LUSC, and overexpression of RND1 repressed proliferation and DDP resistance of LUSC cells and facilitated cell apoptosis. RND1 modulated the arachidonic acid (AA) metabolism pathway, and FOXA2 positively manipulated RND1 expression. By activating FOXA2, stabilizing RND1, and regulating AA levels, the sensitivity of LUSC cells to DDP could be enhanced.

Conclusion: Our study suggested that FOXA2 positively modulated the RND1-AA pathway, which repressed the resistance of LUSC cells to DDP.

Keywords: DDP resistance; RND1; arachidonic acid; lung squamous cell carcinoma.

FIGURE 1

RND1 expression in LUSC and effects of abnormal expression on cancer cell DDP resistance, proliferation, and apoptosis. (A) Violin plot showing the expression levels of RND1 in LUSC tumor tissues and adjacent tissues. (B) qPCR detection of RND1 expression levels in human normal lung epithelial cells (BESA‐2B), LUSC cell line (SK‐MES‐1), and human non–small cell lung cancer cell lines (H1299 and A549). (C) qPCR and western blot detection of RND1 mRNA and protein expression levels in DDP‐resistant and sensitive SK‐MES‐1 cells. (D) CCK8 analysis of DDP IC₅₀ values in SK‐MES‐1/DDP and SK‐MES‐1 cells. (E) qPCR detection of transfection efficiency of overexpressed RND1 in SK‐MES‐1/DDP cells. (F) CCK8 analysis of DDP IC₅₀ values for oe‐RND1 and oe‐NC group. (G, H) Images and quantitative analysis of colony formation assay in LUSC cells transfected with oe‐NC and oe‐RND1. (I, J) Cell apoptosis rate and quantification graphs of LUSC cells transfected with oe‐RND1 as assayed by flow cytometry. * represents p < 0.05.

FIGURE 2

RND1 affects the AA pathway and regulates DDP resistance in LUSC. (A) GSEA results show enrichment of RND1 in the AA metabolism pathway. (B, C) qPCR and western blot analysis of mRNA and protein expression levels of AA metabolism‐related genes PTGS1, PTGS2, and PLA2G4A in drug‐resistant cells overexpressing RND1. (D) CCK8 analysis of IC₅₀ values in different treatment groups (oe‐NC+PBS, oe‐RND1+PBS, oe‐NC+AA, and oe‐RND1+AA) after treatment of drug‐resistant cells. * represents p < 0.05.

FIGURE 3

FOXA2 positively activates transcription of RND1. (A) hTFtarget predicted potential transcription factors upstream of RND1. (B) Pearson's analysis of RND1 and 13 potential transcription factors. (C) Motif prediction of the 2000‐bp region upstream of RND1 promoter using the JASPAR website. (D) Violin plot showing the expression levels of FOXA2 in normal and LUSC tissues by bioinformatics analysis. (E) qPCR detection of FOXA2 expression levels in human normal lung epithelial cells (BESA‐2B), LUSC cell line (SK‐MES‐1), and non–small cell lung cancer cell lines (H1299 and A549). (F) qPCR and western blot detection of FOXA2 mRNA and protein expression levels in DDP‐resistant and sensitive LUSC cell lines. (G) Co‐IP experiment of the relationship between protein of FOXA2 and protein of RND1. (H) CHIP experiment of the relationship between FOXA2 and RND1. (I) Dual‐luciferase assay of effect of FOXA2 knockdown on luciferase activity in PGL3‐RND1‐WT and PGL3‐RND1‐MUT treated groups. (J, K) qRT‐PCR and western blot detection of RND1 mRNA and protein expression levels after overexpression of FOXA2 in SK‐MES‐1/DDP and SK‐MES‐1 cells. * represents p < 0.05.

FIGURE 4

FOXA2 positively regulates RND1 to affect AA metabolism and inhibit DDP resistance in LUSC. (A) mRNA expression levels of RND1 in resistant cells were detected in different groups (oe‐NC+si‐NC, oe‐NC+si‐FOXA2, and oe‐RND1+si‐FOXA2). (B) Protein expression levels of RND1 in the above groups were detected by western blot. (C) IC₅₀ values of the above groups were measured by CCK8 assay. (D, E) Colony formation experiments were performed to assess cell proliferation in the above groups, and images were taken and quantified. (F, G) Flow cytometry was used to measure cell apoptosis levels in different treatment groups, and a quantification graph was generated. (H, I) mRNA and protein levels of AA pathway‐related genes in different treatment groups were detected by qPCR and western blot, respectively. * represents p < 0.05.