CircKEAP1 Suppresses the Progression of Lung Adenocarcinoma via the miR-141-3p/KEAP1/NRF2 Axis

Abstract

Background: Lung cancer is the leading cause of death from cancer, and lung adenocarcinoma (LUAD) is the most common form. Despite the great advances that has been made in the diagnosis and treatment for LUAD, the pathogenesis of LUAD remains unclear. In this study, we aimed to identify the function of circKEAP1 derived from the exon of KEAP1 in LUAD.

Methods: The expression profiles of circRNAs in LUAD tissues and adjacent non-tumor tissues were analyzed by Agilent Arraystar Human CircRNA microarray. The levels and prognostic values of circKEAP1 in tissues and cancer cell lines were determined by quantitative real-time PCR (qRT-PCR). Subsequently, the effects of circKEAP1 on tumor growth were investigated by functional experiments in vitro and in vivo. Mechanistically, the dual luciferase reporter assay, RNA pull-down, and RNA immunoprecipitation experiments were performed to confirm the interaction between circKEAP1 and miR-141-3p in LUAD.

Results: We found circKEAP1 was significantly downregulated in LUAD tissues and repressed tumor growth both in vitro and in vivo. Mechanistically, circKEAP1 competitively binds to miR-141-3p and relive miR-141-3p repression for its host gene, which activated the KEAP1/NRF2 signal pathway, and finally suppresses the tumor progress. Our findings suggest that circKEAP1 inhibits LUAD progression through circKEAP1/miR-141-3p/KEAP1 axis and it may serve as a novel method for the treatment of LUAD.

Keywords: KEAP1; cell proliferation; circKEAP1; circRNA; lung adenocarcinoma.

Figure 1

circKEAP1 was significantly downregulated in LUAD by circRNA microarray. (A, B) The heatmap (A) and volcano plot (B) of circRNA profiles in lung adenocarcinoma tumor tissues (C) and adjacent normal tissues (N). (C) Venn diagrams presenting circRNAs downregulated in our circRNA expression profile and circRNA expression profile of GSE112214 and GSE101586. (D) qRT-PCR for the abundance of hsa_circRNA_104126 and hsa_circRNA_102442 in the seven paired lung adenocarcinoma tumor tissues (C) and adjacent normal tissues (N). Data are shown as the means ± standard error of the mean (n = 3), statistical analysis was performed by two-tailed Student’s t test; ***P < 0.001.

Figure 2

Characterization of circKEAP1 in LUAD. (A) Genomic loci of circKEAP1 gene. CircKEAP1 is produced at the KEAP1 gene locus containing exon 2. The back-splice junction of circKEAP1 was identified by Sanger sequencing. (B) Northern blot analysis showed the abundance of circKEAP1 in one paired sample of LUAD cancer tissue (C) and adjacent normal tissues (N). (C) PCR analysis for circKEAP1 and its linear isoform KEAP1 in cDNA and genomic DNA (gDNA) in one paired sample of LUAD cancer tissue (N) and adjacent normal tissues (C). (D) qRT-PCR for the abundance of circKEAP1 in 105 paired samples of LUAD cancer tissues (cancer) and adjacent normal tissues (normal). (E) The association of circKEAP1 expression with the overall survival of LUAD patients was analyzed by Kaplan-Meier method. Log-rank test. (F) qRT-PCR revealed the expression of circKEAP1 in LUAD cells (A549, PC9, H1975) relative to normal cells (BEAS-2B). (G) PCR analysis for circKEAP1 and its linear isoform KEAP1 in cDNA and genomic DNA (gDNA) in human LUAD cell lines A549 and PC9, and normal human bronchial epithelial cell line BEAS-2B. (H) qRT-PCR for the abundance of circKEAP1 and KEAP1 in BEAS-2B and PC9 cells treated with actinomycin D at the indicated time point. Data are shown as the means ± standard error of the mean (n = 3), statistical analysis was performed by two-tailed Student’s t test; ***P < 0.001.

Figure 3

circKEAP1 inhibits tumor growth. (A) The expression level of circKEAP1 in A549 and PC9 cells transfected with control plasmid or circKEAP1 plasmid. (B) The volume of subcutaneous xenograft tumors of A549 and PC9 cells isolated from nude mice. (C, D) The weight of subcutaneous xenograft tumors of A549 and PC9 cells isolated from nude mice. The graduated scale is 1 cm. (E) HE staining and IHC staining for Ki-67 in xenografted tumors. (F) Expression levels of circKEAP1 in xenografted tumors. (G) Cell proliferation analysis for A549 and PC9 cells transfected with control plasmid or circKEAP1 overexpression plasmid by EDU assay. (H) Cell proliferation analysis for A549 and PC9 cells transfected with control plasmid or circKEAP1 overexpression plasmid by CCK-8 assay. (I) Cell migration analysis for A549 and PC9 cells transfected with control plasmid or circKEAP1 overexpression plasmid by Transwell assay. (J) Cell viability analysis for A549 and PC9 cells transfected with control plasmid or circKEAP1 overexpression plasmid by MTT assay. Data are shown as the means ± standard error of the mean (n = 3), statistical analysis was performed by two-tailed Student’s t-test; **P < 0.01, ***P < 0.001.

Figure 4

circKEAP1 acts as a sponge for miR-141-3p. (A) Levels of circKEAP1 in the nuclear and cytoplasmic fractions of BEAS-2B cells. (B) The volcano plot of miRNA in seven paired lung adenocarcinoma tumor tissues and adjacent normal tissues. (C) A schematic model shows the putative binding sites of nine predicted miRNAs on circKEAP1. (D) Luciferase activity of circKEAP1 in A549 cells transfected with miRNA mimics which are putative binding to the circKEAP1 sequence. Luciferase activity was normalized by Renila luciferase activity. (E) The binding sites of miR-141-3p with circKEAP1 were predicated via targetScan. (F) Luciferase reporter activity of circKEAP1 in A549 cells co-transfected with miR-141-3p mimics and circKEAP1 luciferase reporter plasmid. (G) RIP was performed using AGO2 antibody in BEAS-2B cells transfected with miR-141-3p mimics or mimics NC, then the enrichment of circKEAP1 was detected. (H) circKEAP1 was pulled down and enriched with 3'-end biotinylated miR-141-3p in BEAS-2B cells. (I) Expression levels of miR-141-3p in A549 and PC9 cells co-transfected with miR-141-3p mimics and circKEAP1 overexpression plasmid. (J) qRT-PCR for the abundance of miR-141-3p in 105 paired LUAD cancer tissues (C) and adjacent normal tissues (N). (K) Pearson analysis the relationship between miR-141-3p and circKEAP1 in expression in LUAD tissues (K). Data are shown as the means ± standard error of the mean (n = 3), statistical analysis was performed by two-tailed Student’s t test; ***P < 0.001.

Figure 5

miR-141-3p inhibits KEAP1 expression. (A) Putative target genes of miR-141-3p were predicated by trargetScan, miRDB, and miRDana. (B) Luciferase activity of miR-141-3p in A549 cells transfected with luciferase reporter plasmid containing the putative binding of target genes to miR-141-3p. Luciferase activity was normalized by Renila luciferase activity. (C) The binding sites of miR-141-3p with KEAP1 were predicated via targetScan. (D) Luciferase reporter activity of KEAP1 in A549 cells co-transfected with miR-141-3p mimics and KEAP1 luciferase reporter plasmid. (E) KEAP1 was pulled down and enriched with 3'-end biotinylated miR-141-3p in BEAS-2B cells. (F) Expression levels of KEAP1 protein in 105 paired LUAD cancer tissues (C) and adjacent normal tissues (N) by IHC staining. (G) Expression levels of KEAP1 protein in 12 paired LUAD cancer tissues (C) and adjacent normal tissues (N) by western blotting. (H) The protein levels of KEAP1 in BEAS-2B cells transfected with mimics of miR-141-3p and A549 cells transfected with inhibitors of miR-141-3p. Data are shown as the means ± standard error of the mean (n = 3), statistical analysis was performed by two-tailed Student’s t test; ***P < 0.001.

Figure 6

circKEAP1 regulate KEAP1 signaling pathway by sponging miR-141-3p. (A) Luciferase activity in A549 cells co-transfected with miRNA mimics or circKEAP1 overepression plasmid and luciferase reporter plasmid which have putative binding site of KEAP1 to miR-141-3p. Luciferase activity was normalized by Renila luciferase activity. (B) Western blot analysis of KEAP1, NRF2 and HDAC4 levels in A549 cells co-transfected with mimic nc or miR-141-3p mimic or circKEAP1 overexpression plasmid. (C) The expression level of miR-1 and miR-206 in A549 cells co-transfected with mimic nc or miR-141-3p mimic or circKEAP1 overexpression plasmid. (D) Cell proliferation analysis for A549 cells co-transfected with mimic nc or miR-141-3p mimic or circKEAP1 overexpression plasmid by CCK-8 assay. (E) Cell proliferation analysis for A549 cells co-transfected with mimic nc or miR-141-3p mimic or circKEAP1 overexpression plasmid. (F) Cell migration analysis for A549 cells co-transfected with mimic nc or miR-141-3p mimic or circKEAP1 overexpression plasmid by Transwell assay. (G) Cell viability analysis for A549 cells co-transfected with mimic nc or miR-141-3p mimic or circKEAP1 overexpression plasmid MTT assay. Data are shown as the means ± standard error of the mean (n = 3), statistical analysis was performed by two-tailed Student’s t test; **P < 0.01, ***P < 0.001.

Figure 7

Hypothesis diagram illustrates function and mechanism of circKEAP1 in LUAD progress.