Circular RNA KIF4A promotes cell migration, invasion and inhibits apoptosis through miR-152/ZEB1 axis in breast cancer

A total of 41 BC tissues and normal tissues were harvested from patients who were diagnosed with BC at College of Nursing and Health of Henan University. All tissues were immediately placed in liquid nitrogen after removing from patients and saved at − 80 °C prior to use. The research was permitted by the Ethics Committee of College of Nursing and Health of Henan University and written informed consents were signed by all patients before surgery. None of the experimental subjects had received treatment before operation. The clinical and pathological characteristics of 41 patients with BC were shown in Table 1.

Human BC cells (MCF-7 and MDA-MB-231) and human mammary gland epithelial cells (MCF-10A) were bought from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-7 and MDA-MB-231 cells were kept in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) including 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). MCF-10A cells were kept in DMEM: Nutrient Mixture F-12 (DMEM/F12; Gibco) including 5% Horse Serum (Gibco), 10 μg/mL insulin (Gibco), 20 ng/mL epidermal growth factor (Gibco) and 0.5 μg/mL hydrocortisone (Gibco) and 1% penicillin/streptomycin (Gibco). These cells were maintained in an incubator containing 5% CO₂ at 37 °C.

Small interfering RNA (siRNA) against circKIF4A (si-circKIF4A; GCCUGGAUCUAUAACGUAUTT) and its control (si-NC; AAGTCGGGTCAAGAGAAGC), miR-152 mimics (miR-152; 5′-UCAGUGCAUGACAGAACUUGG-3′) and its control (miR-NC; 5′-GGAACUUAGCCACUGUGAAUU-3′), miR-152 inhibitors (anti-miR-152; 5′-CCAAGUUCUGUCAUGCACUGA-3′) and its control (anti-miR-NC; 5′-UCGCUUGGUGCAGGUCGGGAA-3′), pcDNA3.1-circKIF4A overexpression vector (pcDNA-circKIF4A), pcDNA3.1-ZEB1 overexpression vector (pcDNA-ZEB1) and pcDNA were bought from GenePharma (Shanghai, China). Then MCF-7 and MDA-MB-231 cells were plated into 6-well plates at a density of 1.0 × 10⁵ cells/well and transfected with indicated synthetic oligonucleotides or vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers’ instructions. After 48 h of transfection, cells were harvested for subsequent experiments.

Total RNA was isolated using TRIzol reagent (Invitrogen). RNA concentration was quantified by NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Then cDNA was synthesized by M-MLV Reverse Transcriptase Kit (Promega, Madison, WI, USA) or miRNA 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). QRT-PCR was carried out using an iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Philadelphia, PA, USA). The expression of circKIF4A, ZEB1, Caspase-3 and miR-152 was analyzed using the 2^-ΔΔCt method with GAPDH or U6 as an internal control [23]. The primers used in our study were purchased from GeneCopoeia (Guangzhou, China) and primers sequences were: circKIF4A: (F: 5′-GAGGTACCCTGCCTGGATCT-3′ and R: 5′-TGGAATCTCTGTAGGGCACA-3′); ZEB1: (F: 5′-TCCTCGAGGCACCTGAAGAGG-3′ and R: 5′-CAGAGAGGTAAAGCGTTTATAGCC-3′); Caspase-3: (F: 5′-TGGAACGAACGGACCTGTG-3′ and R: 5′-CGGGTGCGGTAGAGTAAGC-3′); miR-152: (F: 5′-GTGCAGGGTCCGAGGT-3′ and R: 5′- TGACAGAACTTGGGTCGT-3′); GAPDH: (F: 5′-TGTTCGTCATGGGTGTGAAC-3′ and R: 5′-ATGGCATGGACTGTGGTCAT-3′); U6: (F: 5′-CGCTTCGGCAGCACATATAC-3′ and R: 5′-TTCACGAATTTGCGTGTCAT-3′).

BC cells were re-suspended in DMEM (Gibco) at a density of 5.0 × 10⁴ cells. For the detection of cell migration, 300 μL cell suspension was placed into the upper chamber of a transwell (8 μm pore; Corning Incorporated, Corning, NY, USA) and the bottom chamber was filled with 300 μL DMEM (Gibco) including 10% FBS (Gibco). After incubation for 24 h at 37 °C, cells that passed through the membranes were treated with 4% para-formaldehyde (PFA) and stained with 0.1% crystal violet (Solarbio, Beijing, China). Then the migrated cells were counted under a microscope (Olympus, Tokyo, Japan). For the detection of cell invasion, the steps were the same as above, except that the upper chamber was pre-coated Matrigel (Corning Life Sciences, Corning, NY, USA).

Cell apoptosis was evaluated by an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Vazyme). Briefly, collected cells were washed, re-suspended and then stained with 5 μL Annexin V-FITC and PI for 15 min at room temperature in the dark. Apoptotic cells were analyzed within 1 h by the flow cytometry (BD Biosciences, San Jose, CA, USA).

The sequences of circKIF4A (or 3′ UTR of ZEB1) including the putative complementary sequences of wild-type or mutant miR-152 were cloned into pmirGLO vectors (Promega) to generate luciferase reporter plasmids circKIF4A-WT, circKIF4A-MUT, ZEB1-WT and ZEB1-MUT, respectively. MiR-152 or miR-NC and corresponding vector were co-transfected into BC cells. After 48 h, the luciferase activity was examined using the Dual-Luciferase Reporter Assay Kit (Promega).

RNA immunoprecipitation (RIP) assay was conducted using an EZMagna RIP kit (Millipore, Billerica, MA, USA). MiR-152 or miR-NC transfected BC cells were harvested and lysed in RIP buffer. Then cell lysates were interacted with magnetic beads conjugated with Ago2 or IgG antibody. After RNAs were isolated, the enrichment of circKIF4A and ZEB1 was examined via qRT-PCR.

Total protein was extracted from BC tissues and cells with RIPA buffer (Beyotime, Shanghai, China) and determined using a BCA Protein Assay Kit (Beyotime). Equal amount of proteins was separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Then the membranes were blocked with 5% nonfat milk at room temperature for 1 h and incubated corresponding primary antibodies against ZEB1 (1:2000; Santa Cruz, Dallas, TX, USA) or β-actin (1:5000; Santa Cruz) overnight at 4 °C with and horseradish peroxidase-conjugated secondary antibody (1:5000; Santa Cruz) for 1 h at room temperature. At last, protein bands were visualized by an enhanced chemiluminescence chromogenic substrate (Beyotime).

All data were exhibited as the means ± standard deviation (SD) from at least three independent experiments. The differences were analyzed by Student’s t-test or one-way analysis of variance (ANOVA). The association between circKIF4A level and clinicopathologic features of patients was analyzed by χ² test. It was defined as statistically difference if P value less than 0.05.

To explore the potential role of circKIF4A in BC progression, we first determined the level of circKIF4A in 41 BC tissues and corresponding normal tissues by qRT-PCR. As we observed, circKIF4A was markedly elevated in BC tissues in reference to normal tissues (Fig. 1a). Subsequently, the level of circKIF4A in two BC cell lines and one normal breast cell line was detected. The results exhibited that circKIF4A was obviously raised in MCF-7 and MDA-MB-231 cells compared to that in MCF-10A cells (Fig. 1b). Thus, we thought that the dysregulation of circKIF4A might be involved in the development of BC.

In order to investigate the functions of circKIF4A in the development of BC in vitro, si-circKIF4A was transfected into BC cells to knockdown the expression of circKIF4A. Knockdown efficiency was detected by qRT-PCR, and we found that circKIF4A was conspicuously decreased in si-circKIF4A transfected MCF-7 and MDA-MB-231 cells compared with si-NC group (Fig. 2a). Furthermore, transwell assay indicated that cell migration and invasion were markedly decreased in si-circKIF4A transfected group in reference to control group in MCF-7 and MDA-MB-231 cells (Fig. 2b and c). Flow-cytometric analysis showed that circKIF4A silencing led to a marked enhancement of cell apoptosis in MCF-7 and MDA-MB-231 cells compared to NC group (Fig. 2d). Then the level of caspase-3 was determined by qRT-PCR, we found that si-circKIF4A transfection caused an obvious elevation of caspase-3 in MCF-7 and MDA-MB-231 cells (Fig. 2e). These data implicated that circKIF4A knockdown suppressed BC cell progression.

It is widely accepted that circRNAs can modulate gene expression by sponging miRNAs [24]. By searching online website starBase v2.0 (http://​starbase.​sysu.​edu.​cn/​), circKIF4A was found to contain the binding sequences of miR-152 (Fig. 3a). To confirm this prediction, dual-luciferase reporter assay was firstly conducted. The outcomes suggested that the luciferase activity was obviously reduced in circKIF4A-WT and miR-152 co-transfected cells compared to circKIF4A-WT and miR-NC co-transfected groups, whereas the luciferase activity was not changed in circKIF4A-MUT group (Fig. 3b and c). Then, RIP assay was further carried to verify the interaction between circKIF4A and miR-152. The data showed that the enrichment of circKIF4A was greatly increased in BC cells transfected with miR-152 (Fig. 3d and e), which further confirmed our prediction. As we expected, miR-152 was drastically downregulated in BC tissues and cells relative to normal tissues and cells (Fig. 3f and g). Furthermore, we determined the level of miR-152 in MCF-7 and MDA-MB-231 cells transfected with pcDNA-circKIF4A or si-circKIF4A. The results indicated that miR-152 was markedly downregulated by circKIF4A overexpression, whereas miR-152 expression was significantly upregulated after si-circKIF4A transfection in both MCF-7 and MDA-MB-231 cells (Fig. 3h and i). Collectively, circKIF4A directly interacted with miR-152 and suppressed miR-152 expression in BC cells.

By searching online software DINAN tool (http://​diana.​imis.​athena-innovation.​gr/​DianaTools/​index.​php), the potential target of miR-152 was predicted. It showed that 3′ UTR of ZEB1 might contain the complementary sequences of miR-152 (Fig. 4a). To confirm it, dual-luciferase reporter assay was performed. Our results presented that the luciferase activity was markedly inhibited in both MCF-7 and MDA-MB-231 cells co-transfected with ZEB1-WT and miR-152, while there was no significant difference in ZEB1-MUT groups (Fig. 4b and c). RIP assay presented that the enrichment of ZEB1 was distinctly increased in MCF-7 and MDA-MB-231 cells treated with miR-152 (Fig. 4d and e). Moreover, we observed that ZEB1 mRNA was elevated in BC tissues and cells in reference to that in normal tissues and cells (Fig. 4f and g). Subsequently, the protein level of ZEB1 in MCF-7 and MDA-MB-231 cells treated with miR-152, anti-miR-152, circKIF4A, si-circKIF4A or their corresponding controls was measured using western blot assay. The results implicated that miR-152 obviously suppressed ZEB1 expression and miR-152 inhibition significantly increased ZEB1 expression in both MCF-7 and MDA-MB-231 cells (Fig. 4h and i). Overexpression of circKIF4A resulted in a remarked increase of ZEB1 and knockdown of circKIF4A resulted in a significant decrease of ZEB1 in MCF-7 and MDA-MB-231 cells (Fig. 4j and k). To sum up, circKIF4A positively regulated ZEB1 expression through targeting miR-152 in BC cells.

To further illustrate the relationship between circKIF4A and miR-152 in BC, MCF-7 and MDA-MB-231 cells transfected with si-NC, si-circKIF4A, si-circKIF4A + anti-miR-152 or si-circKIF4A + anti-miR-NC. As presented in Fig. 5a, the upregulation of miR-152 caused by si-circKIF4A transfection was partially restored by miR-152 inhibition in MDA-MB-231 and MCF-7 cells. Transwell assay indicated that the migration and invasion of MCF-7 and MDA-MB-231 cells were effectively suppressed by si-circKIF4A transfection, while the effects could be abolished by anti-miR-152 transfection (Fig. 5b and c). Flow-cytometric analysis indicated that circKIF4A knockdown significantly induced the apoptosis of MCF-7 and MDA-MB-231 cells, whereas depletion of miR-152 could partially abolish the effect (Fig. 5d). Moreover, the increased level of caspase-3 caused by circKIF4A knockdown was partly attenuated by miR-152 inhibition in MCF-7 and MDA-MB-231 cells (Fig. 5e). Thus, we concluded that circKIF4A silencing could decelerate BC cell progression by interacting with miR-152.

To further investigate the relationship between circKIF4A and ZEB1, MCF-7 and MDA-MB-231 cells were assigned to si-NC, si-circKIF4A, si-circKIF4A + pcDNA and si-circKIF4A + ZEB1. As displayed in Fig. 6a, si-circKIF4A led to a reduction of ZEB1 in MCF-7 and MDA-MB-231 cells, while the elevation of ZEB1 overturned the impact. Then we evaluated the migration, invasion and apoptosis of MCF-7 and MDA-MB-231 cells by transwell assay or flow-cytometric analysis The results displayed that the suppressive roles in cell migration and invasion and the promotional role in apoptosis in MCF-7 and MDA-MB-231 cells mediated by circKIF4A silencing were also partially rescued following the treatment of ZEB1 (Fig. 6b, c and d). Additionally, the elevation of caspase-3 caused by si-circKIF4A transfection was decreased by ZEB1 overexpression in both MCF-7 and MDA-MB-231 cells (Fig. 6e). These data illustrated that circKIF4A affected BC progression by regulating ZEB1 expression.