Circ_0008035 contributes to cell proliferation and inhibits apoptosis and ferroptosis in gastric cancer via miR-599/EIF4A1 axis

Abstract

Background: Currently, multiple circular RNAs (circRNAs) have been verified to act as essential regulators in the progression of gastric cancer (GC). We aimed to investigate the role of circ_0008035 in GC progression.

Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to measure the expression of circ_0008035 and miR-599. 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was employed to evaluate cell proliferation and ferroptosis. Western blot assay was performed to measure the levels of cyclin D1, proliferating cell nuclear antigen (PCNA) and eukaryotic initiation factor 4A1 (EIF4A1). Flow cytometry analysis was conducted to assess cell apoptosis. The iron accumulation, lipid peroxidation and mitochondrial membrane potential were examined by relevant kits. Dual-luciferase reporter assay was conducted to determine the targeting relationship between miR-599 and circ_0008035 or EIF4A1. A murine xenograft model was established to investigate the function of circ_0008035 in vivo.

Results: Circ_0008035 was up-regulated in GC tissues and cells. Silencing of circ_0008035 repressed cell proliferation and induced cell apoptosis and ferroptosis in GC cells. Circ_0008035 acted as a sponge of miR-599. The effects of circ_0008035 knockdown on GC cell proliferation, apoptosis and ferroptosis were abolished by miR-599 inhibition. EIF4A1 was confirmed to be a target gene of miR-599. Circ_0008035 knockdown inhibited EIF4A1 expression by targeting miR-599. Moreover, the suppressive role of circ_0008035 deficiency in GC progression could be restored by EIF4A1. Additionally, circ-0008035 knockdown hampered tumorigenesis in vivo.

Conclusion: Circ_0008035 promoted GC cell growth and repressed apoptosis and ferroptosis by up-regulating EIF4A1 through sponging miR-599.

Keywords: Apoptosis; Circ_0008035; EIF4A1; Ferroptosis; Gastric cancer; Proliferation; miR-599.

Fig. 1

Circ_0008035 was elevated in GC tissues and cells. a The expression of circ_0008035 in tumor tissues and normal tissues was determined using qRT-PCR. b Circ_0008035 expression in GES-1, HGC-27 and AGS cells was measured by qRT-PCR. c, d The nuclear and cytoplasm of HGC-27 and AGS cells were isolated and then the expression of circ_0008035 was measured by qRT-PCR. *P < 0.05

Fig. 2

Knockdown of circ_0008035 repressed cell proliferation and induced cell apoptosis and ferroptosis in GC cells. a, b Si-NC or si-circ_0008035 was transfected into HGC-27 and AGS cells and then circ_0008035 expression was examined by qRT-PCR. c, d Cell proliferation in HGC-27 and AGS cells transfected with si-NC or si-circ_0008035 was evaluated by MTT assay. e, f The protein levels of cyclin D1 and PCNA in HGC-27 and AGS cells transfected with si-NC or si-circ_0008035 were determined by western blot assay. g, h Cell apoptosis in HGC-27 and AGS cells transfected with si-NC or si-circ_0008035 was analyzed by flow cytometry analysis. i–l HGC-27 and AGS cells were treated with erastin (10.0 μM)/RSL3 (2.0 μM), erastin (10.0 μM)/RSL3 (2.0 μM) plus ferrostain-1 (2.0 μM), erastin (10.0 μM)/RSL3 (2.0 μM) plus ZVAD-FMK (10.0 μM) or erastin (10.0 μM)/RSL3 (2.0 μM) plus necrosulfonamide (0.5 μM) for 48 h and then cell death was evaluated by MTT assay. m, n HGC-27 and AGS cells were transfected with si-NC or si-circ_0008035 and treated with erastin (10.0 μM) or RSL3 (2.0 μM), and then cell death was evaluated by MTT assay. *P < 0.05

Fig. 3

Circ_0008035 knockdown promoted iron accumulation, lipid peroxidation and suppressed mitochondrial membrane potential in GC cells. HGC-27 and AGS cells were transfected with si-NC and si-circ_0008035 and then treated with or without erastin or RSL3 for 48 h. a, b Total iron level, c, d Fe²⁺ accumulation, e, f MDA level, g, h lipid ROS level, i, j mitochondrial superoxide level and k, l mitochondrial membrane potential in HGC-27 and AGS cells were investigated by specific kits. *P < 0.05

Fig. 4

Circ_0008035 directly interacted with miR-599 and negatively regulated miR-599 expression in GC cells. a The predicted binding sites between circ_0008035 and miR-599 were shown. b, c Dual-luciferase reporter assay was performed to determine the luciferase activity in HGC-27 and AGS cells co-transfected with circ_0008035 WT or circ_0008035 MUT and miR-599 or miR-NC. d, e The expression of miR-599 in GC tissues, cells (HGC-27 and AGS) and corresponding normal tissues and cells (GES-1) was measured by qRT-PCR. f, g The expression of miR-599 in HGC-27 and AGS cells transfected with si-NC or si-circ_0008035 was examined with qRT-PCR. *P < 0.05

Fig. 5

The influences of circ_0008035 silencing on GC cell proliferation, apoptosis and ferroptosis were weakened by miR-599 knockdown. HGC-27 and AGS cells were assigned to si-NC, si-circ_0008035, si-circ_0008035 + anti-miR-NC and si-circ_0008035 + anti-miR-599 groups. a, b The expression of miR-599 in HGC-27 and AGS cells was determined by qRT-PCR assay. c, d The proliferation of HGC-27 and AGS cells was analyzed by MTT assay. e, f The protein levels of cyclin D1 and PCNA were examined by western blot assay. g, h The apoptosis of HGC-27 and AGS cells was measured through flow cytometry analysis. i, j Cell survival rate was assessed by MTT assay after HGC-27 and AGS cells were treated with erastin or RSL3. *P < 0.05

Fig. 6

Circ_0008035 interacted with miR-599 to promote iron accumulation, lipid peroxidation and reduce mitochondrial membrane potential in the process of ferroptosis in GC cells. Si-NC, si-circ_0008035, si-circ_0008035 + anti-miR-NC or si-circ_0008035 + anti-miR-599 was transfected into HGC-27 and AGS cells and then these cells were treated with or without erastin or RSL3. a, b Total iron level, c, d Fe²⁺ accumulation, e, f MDA level, g, h lipid ROS level, i, j mitochondrial superoxide concentration and k, l mitochondrial membrane potential in HGC-27 and AGS cells were examined by specific kits. *P < 0.05

Fig. 7

Knockdown of circ_0008035 decreased EIF4A1 expression by targeting miR-599 in GC cells. a The potential binding sites between miR-599 and EIF4A1 3′ UTR were predicted by starBase 2.0. b, c The association between miR-599 and EIF4A1 was verified by dual-luciferase reporter assay. d, e The protein level of EIF4A1 in GC tissues, cells and corresponding normal tissues and cells was determined using western blot assay. f, g The protein level of EIF4A1 in HGC-27 and AGS cells transfected with miR-NC or miR-599 was measured by western blot assay. h, i HGC-27 and AGS cells were transfected with si-NC, si-circ_0008035, si-circ_0008035 + anti-miR-NC or si-circ_0008035 + anti-miR-599 and then the protein expression of EIF4A1 was examined by western blot assay. *P < 0.05

Fig. 8

The influences of circ_0008035 knockdown on cell proliferation, apoptosis and ferroptosis were rescued by EIF4A1 in GC cells. HGC-27 and AGS cells were transfected with si-NC, si-circ_0008035, si-circ_0008035 + vector or si-circ_0008035 + EIF4A1. a, b The level of EIF4A1 in HGC-27 and AGS cells was measured by western blot assay. c, d The proliferation of HGC-27 and AGS cells was assessed by MTT assay. e, f The protein levels of cyclin D1 and PCNA in HGC-27 and AGS cells were detected using western blot assay. g, h The apoptosis of HGC-27 and AGS cells was evaluated by flow cytometry analysis. i, j Transfected HGC-27 and AGS cells were treated with erastin or RSL3 for 48 h and then cell death was assessed by MTT assay. *P < 0.05

Fig. 9

EIF4A1 abated the effects of circ_0008035 silencing on iron accumulation, lipid peroxidation and mitochondrial membrane potential in GC cells. HGC-27 and AGS cells were transfected with si-NC, si-circ_0008035, si-circ_0008035 + vector or si-circ_0008035 + EIF4A1 and then treated with or without erastin or RSL3. a, b Total iron level, c, d Fe²⁺ accumulation, e, f MDA level, g, h lipid ROS level, i, j mitochondrial superoxide concentration and k, l mitochondrial membrane potential in HGC-27 and AGS cells were examined by specific kits. *P < 0.05

Fig. 10

Circ_0008035 knockdown blocked tumorigenesis in vivo. AGS cells were transfected with sh-NC or sh-circ_0008035 and then injected into the mice. a Tumor volume was monitored every 5 days. b Tumor weight was detected 30 days later. c, d The levels of circ_0008035 and miR-599 in the collected tumor samples were examined by qRT-PCR. e The protein level of EIF4A1 in the collected tumor samples was measured by western blot assay. *P < 0.05