Exosome-derived circKIF20B suppresses gefitinib resistance and cell proliferation in non-small cell lung cancer

Abstract

Background: The gefitinib resistance mechanism in non-small cell lung cancer (NSCLC) remains unclear, albeit exosomal circular RNA (circRNA) is known to possibly play a vital role in it.

Methods: We employed high-throughput sequencing techniques to detect the expressions of exosomal circRNA both in gefitinib-resistant and gefitinib-sensitive cells in this study. The circKIF20B expression was determined in serum exosomes and tissues of patients by qRT-PCR. The structure, stability, and intracellular localization of circKIF20B were verified by Sanger sequencing, Ribonuclease R (RNase R)/actinomycin D (ACTD) treatments, and Fluorescence in situ hybridization (FISH). The functions of circKIF20B were investigated by 5-Ethynyl-20-deoxyuridine (EdU), flow cytometry, Cell Counting Kit-8 (CCK-8), oxygen consumption rate (OCR), and xenograft model. Co-culture experiments were performed to explore the potential ability of exosomal circKIF20B in treating gefitinib resistance. The downstream targets of circKIF20B were determined by luciferase assay, RNA pulldown, and RNA immunoprecipitation (RIP).

Results: We found that circKIF20B was poorly expressed in the serum exosomes of gefitinib-resistant patients (n = 24) and the tumor tissues of patients with NSCLC (n = 85). CircKIF20B was negatively correlated with tumor size and tumor stage. Decreasing circKIF20B was found to promote gefitinib resistance by accelerating the cell cycle, inhibiting apoptosis, and enhancing mitochondrial oxidative phosphorylation (OXPHOS), whereas increasing circKIF20B was found to restore gefitinib sensitivity. Mechanistically, circKIF20B is bound to miR-615-3p for regulating the MEF2A and then altering the cell cycle, apoptosis, and mitochondrial OXPHOS. Overexpressing circKIF20B parental cells can restore sensitivity to gefitinib in the recipient cells by upregulating the exosomal circKIF20B expression.

Conclusions: This study revealed a novel mechanism of circKIF20B/miR-615-3p/MEF2A signaling axis involving progression of gefitinib resistance in NSCLC. Exosomal circKIF20B is expected to be an easily accessible and alternative liquid biopsy candidate and potential therapeutic target in gefitinib-resistant NSCLC. The schematic diagram of mechanism in this study. Exosomal circKIF20B inhibits gefitinib resistance and cell proliferation by arresting the cell cycle, promoting apoptosis, and reducing OXPHOS via circKIF20B/miR-615-3p/MEF2A axis in NSCLC.

Keywords: Apoptosis; Cell cycle; CircKIF20B; Exosome; Gefitinib resistance.

The schematic diagram of mechanism in this study. Exosomal circKIF20B inhibits gefitinib resistance and cell proliferation by arresting the cell cycle, promoting apoptosis, and reducing OXPHOS via circKIF20B/miR-615-3p/MEF2A axis in NSCLC.

Fig. 1

Hsa_circ_0019079 was confirmed to be a downregulated circRNA molecule in NSCLC. A Detection of IC50 values in PC9, PC9GR, HCC827, and HCC827GR cell lines using CCK-8 assay. B The exosomes of PC9 and PC9GR were extracted, and the exosomes were verified by TEM, NTA, and Western blotting. C The heatmap showing the significantly differentially expressed circRNAs in the exosomes from PC9 and PC9GR. The volcano plot indicates the degree of difference in the expression of circRNAs. The hsa_circ_0019079 was significantly downregulated, logFC = − 4.728, p < 0.05. D Electron microscopy graphs of the serum exosomes in the gefitinib-sensitive group and the gefitinib-resistant group from patients with NSCLC. E and G The relative expression of circ_0019079 in the serum exosomes of patients with gefitinib sensitivity and gefitinib resistance (n = 24), as measured by qRT-PCR with β-actin serving as the internal reference. F and H The relative expression of circ_0019079 in the NSCLC tissues and the matched normal tissues (n = 85). I The expression of circ_0019079 in NSCLC cell lines (PC9, HCC827, H1975, A549). A normal airway epithelial cell line (Beas-2B) as control. J The relative expression of circ_0019079 and mRNA KIF20B in PC9, PC9GR, HCC827, and HCC827GR. K Schematic representation of the formation of circKIF20B by circularization of exons 24 to 29 in the KIF20B mRNA. L The circular closed construction of circKIF20B was verified by divergent and convergent primers in cDNA and gDNA. M Sanger sequencing of circKIF20B. N The expression of circKIF20B and KIF20B mRNA in PC9GR and HCC827GR treated with RNase R was detected by qRT-PCR. O The stability of circKIF20B and KIF20B mRNA in PC9GR treated with ACTD at different times was analyzed by qRT-PCR. P qRT-PCR analysis of nucleoplasm fractionation products. β-actin served as an internal reference in the cytoplasm, and U6 served as an internal reference in the nucleus. Q Intracellular localization of circKIF20B was detected by FISH, and the nuclei were labeled with DAPI. CircKIF20B was enriched in PC9GR and the HCC827GR cytoplasm

Fig. 2

Knockdown of circKIF20B promotes gefitinib resistance and NSCLC proliferation. A and C The effect of circKIF20B knockdown on the viability of PC9, PC9GR, HCC827, and HCC827GR were analyzed by CCK-8 assay. B and D The effect of circKIF20B knockdown on the gefitinib IC50 values of PC9, PC9GR, HCC827, and HCC827GR were analyzed by CCK-8 assay. E The cell cycle in HCC827 and HCC827GR were detected by flow cytometry. F In HCC827 and HCC827GR, the apoptosis levels of the circKIF20B-KD groups were analyzed by flow cytometry in the presence or absence of gefitinib treatment. G and H In HCC827 and HCC827GR, EdU analyzed the proliferation rate of the circKIF20B-KD groups with or without gefitinib. I and J Western blotting assays verified the BAX and CDK4 protein expression in the HCC827 circKIF20B-KD group

Fig. 3

Overexpression of circKIF20B inhibits gefitinib resistance and proliferation of NSCLC. A and C The effect of circKIF20B overexpressed on the viability of PC9, PC9GR, HCC827, and HCC827GR were analyzed by CCK-8 assay. B and D The effect of circKIF20B overexpressed on the gefitinib IC50 values of PC9, PC9GR, HCC827, and HCC827GR were analyzed by CCK-8 assay. E With or without gefitinib, cell cycle assay of circKIF20B-OE groups was detected using flow cytometry in PC9 and PC9GR. F With or without gefitinib, the apoptosis level of the circKIF20B-OE groups was analyzed by flow cytometry in PC9 and PC9GR. G and H In PC9 and PC9GR, EdU analyzed the proliferation rate of circKIF20B-OE groups with or without gefitinib. I and J Western blot assays verified the BAX and CDK4 protein expression in the PC9 circKIF20B-OE group

Fig. 4

The miR-615-3p was regulated by circKIF20B to affect gefitinib IC50 value and cell proliferation. A MiR-615-3p was predicted as a target of circKIF20B in four databases. B The expression of miR-615-3p in serum exosomes of gefitinib-resistant and sensitive patients (n = 23/19), measured by qRT-PCR with U6 serving as an internal reference. C The expression of miR-615-3p in NSCLC tissues and matched normal tissues (n = 85). D The expression of miR-615-3p in the circKIF20B-KD group of PC9GR, the PC9GR transfected with LV3-NC served as control. E The expression of miR-615-3p in PC9GR circKIF20B-OE group, control was the cell transfected with GV689-NC. F The luciferase activity of HEK293T cell co-transfected with WT or MUT vectors and miR-615-3p mimics. G The expression of miR-615-3p was detected by qRT-PCR in PC9GR circKIF20B-OE cell lysates after pulldown with circKIF20B probe or oligo probe. The oligo probe group served as the control. H The gefitinib IC50 values of PC9GR co-transfected LV3-NC and inhibitors-NC, PC9GR circKIF20B-KD transfected inhibitors-NC, and PC9GR circKIF20B-KD transfected the inhibitors of miR-615-3p were detected using CCK-8 assay, respectively. I The gefitinib IC50 values of PC9GR co-transfected GV689-NC and mimics-NC, PC9GR circKIF20B-OE transfected mimics-NC, PC9GR circKIF20B-OE transfected the mimics of miR-615-3p were detected using CCK-8 assay, respectively. L The intracellular co-localization of circKIF20B and miR-615-3p in PC9, PC9GR, and Beas-2B cell lines by FISH. Nuclei were labeled with DAPI. J and M The cell cycle and apoptosis assays of PC9GR co-transfected with LV3-NC and inhibitor-NC, the circKIF20B-KD groups transfected with inhibitor-NC, and the circKIF20B-KD groups transfected with miR-615-3p inhibitors. K and N The cell cycle and apoptosis assays of PC9GR co-transfected with GV689-NC and mimic-NC, circKIF20B-OE groups transfected with mimics-NC, and the circKIF20B-OE groups transfected with miR-615-3p mimics. O and P The cell proliferation of the abovementioned groups was analyzed by EdU. Q and R The MEF2A, CDK4, and BAX protein expression of the abovementioned groups were analyzed using Western blotting

Fig. 5

CircKIF20B regulated gefitinib resistance and cell proliferation by MEF2A. A The PSMD11 and MEF2A were predicted as the downstream target of miR-615-3p from 5 databases. B The expression of MEF2A in serum exosomes of gefitinib-resistant and sensitive patients (n = 24/23) as measured by qRT-PCR, with β-actin serving as an internal reference. C The expression of MEF2A in the NSCLC tissues and matched normal tissues (n = 85). D MEF2A and miR-615-3p expression of the anti-Ago2 group was detected by qRT-PCR in the complex from PC9 lysates co-immunoprecipitation, with IgG group serving as the control. E The luciferase activity of HEK293T cell co-transfected with WT or MUT vectors and the mimics of miR-615-3p. F and H The RNA and protein expressions of MEF2A using qRT-PCR and Western blotting in the circKIF20B-KD and circKIF20B-OE groups, with the cells transfected with LV3-NC and GV689-NC serving as control. G and I The RNA and protein expressions of MEF2A in PC9GR transfected with miR-615-3p mimics or inhibitors were detected using qRT-PCR and Western blotting, the cells transfected with mimics-NC or inhibitor-NC serving as control. J The gefitinib IC50 values of PC9GR co-transfected LV3-NC and pEX3-NC, PC9GR circKIF20B-KD transfected pEX3-NC, PC9GR circKIF20B-KD transfected pEX3-MEF2A were detected by CCK-8 assay. K The IC50 values of gefitinib for PC9GR co-transfected GV689-NC and siRNA-NC, PC9GR circKIF20B-OE transfected siRNA-NC, circKIF20B-OE groups transfected siRNA1-MEF2A were detected using CCK-8 assay, respectively. L and N The cell cycle and apoptosis assays of PC9GR co-transfected with LV3-NC and pEX3-NC, the circKIF20B-KD groups transfected with pEX3-NC, and the circKIF20B-KD groups transfected with pEX3-MEF2A. M and O The cell cycle and apoptosis assays of PC9GR co-transfected with GV689-NC and siRNA-NC, the circKIF20B-OE groups transfected with siRNA-NC, and the circKIF20B-OE groups transfected with siRNA1-MEF2A. P and Q EdU analyzed the cell proliferation of the abovementioned groups. R and S The MEF2A, CDK4, and BAX protein expression of the abovementioned groups were analyzed by Western blotting

Fig. 6

CircKIF20B regulated mitochondrial function and OXPHOS by MEF2A in NSCLC. A GSEA analysis found that the expression of MEF2A was significantly negatively correlated with mitochondrial function and OXPHOS. B ATP assay of circKIF20B-KD cells and circKIF20B-OE cells. C Mitochondrial membrane potential assay of circKIF20B-KD cells and circKIF20B-OE cells by TMRE. CCCP is a positive control. D and E The OCR analysis of PC9GR cells co-transfected with LV3-NC and pEX3-NC, circKIF20B-KD cells transfected with pEX3-NC, and circKIF20B-KD cells transfected with pEX3-MEF2A via Seahorse. F and G The OCR analysis of PC9GR cells co-transfected with GV689-NC and siRNA-NC, circKIF20B-OE cells transfected with siRNA-NC, and circKIF20B-OE cells transfected with siRNA1-MEF2A via Seahorse

Fig. 7

Exosome-derived circKIF20B transfers the malignant state and gefitinib resistance of NSCLC in vitro. A The identification of exosomes extracted from Beas-2B, PC9, and PC9GR by electron microscopy. B The expression of circKIF20B, miR-615-3p, and MEF2A in exosomes of Beas-2B, PC9, and PC9GR, as detected by qRT-PCR. C The DiO-labeled exosomes extracted from the circKIF20B-OE PC9 cells were co-cultured with PC9 for 24 h. D Dio-labeled overexpressed circKIF20B PC9 cell-derived exosomes were endocytosed by the recipient PC9 cells. E The expression of circKIF20B, miR-615-3p, and MEF2A in the PC9 cells co-cultured with exosomes from the circKIF20B-OE cells, as measured by qRT-PCR. F and G EdU assay of PC9 cells co-cultured with the exosomes (without gefitinib or with 0.05µM gefitinib). H Cell cycle assay of PC9 cells co-cultured with exosomes from circKIF20B-OE PC9 cells. I Gefitinib IC50 values of PC9 cells co-cultured with exosomes from circKIF20B-OE PC9 cells. J and K The OCR analysis of PC9 cells co-cultured with the exosomes from circKIF20B-OE PC9 cells

Fig. 8

CircKIF20B acts as a tumor suppressor in NSCLC in vivo. A and B Images of the xenograft tumors from the circKIF20B-OE group, the GV689-NC, served as the control. C and D Growth curves and weight analysis of tumors from the circKIF20B-OE and GV689-NC groups. E The expression of circKIF20B, miR-615-3p, and MEF2A in xenograft tumors from the circKIF20B-OE and GV689-NC groups. F The protein expression of MEF2A in the circKIF20B-OE groups in vivo. G IHC and HE is staining of the tumor sections in the circKIF20B-OE group with MEF2A and Ki67 antibodies