CircZFR functions as a sponge of miR-578 to promote breast cancer progression by regulating HIF1A expression

In this study, we enrolled 70 BC patients admitted to The First Affiliated Hospital of Zhengzhou University from April 2013 to June 2014. The clinicopathological features of these patients were provided in Table 1. Seventy pairs of primary tumor tissues and matched healthy breast tissues were collected and stored at − 80 °C to detect the expression levels of circZFR, miR-578 and HIF1A (Accession: NM_181054.3). These patients were followed-up for at least 60 months and the follow-up information was obtained by telephone calls every 3 months. The study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University, and written informed consent was provided by all participants.

BC cell lines MCF7 (ATCC^®HTB-22), BT-549 (ATCC^®HTB-122), MDA-MB-231 (ATCC^®HTB-26) and MDA-MB-453 (ATCC^®HTB-131, American Type Collection Culture, ATCC, Manassas, VA, USA) were cultured in RPMI-1640 medium, supplemented with 10% fetal calf serum (FCS) and 1% antibiotic solution (all from HyClone, Logan, UT, USA). The immortalized MCF10A cell line (ATCC^®CRL-10317, ATCC) was maintained in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) with 10% FCS, 20 ng/mL epidermal growth factor, 0.5 μg/mL hydrocortisone and 5 μg/mL insulin (all from HyClone). All cells were cultured in a 5% CO₂ incubator at 37 °C.

The expression levels of circZFR, HIF1A and miRNAs were gauged by qRT-PCR. Complementary DNA (cDNA) synthesis was done using total RNA (100 ng) isolated by Isogen (Nippon Gene, Tokyo, Japan) from tissues and cell lines. The levels of circZFR and HIF1A were quantified using the TaqMan Gene Expression Assays with the indicated primers, and mature miRNAs were assayed using the TaqMan MicroRNA Assays with TaqMan-specific primer probes as recommended by the manufacturers (Applied Biosystems, Rotkreuz, Switzerland). qRT-PCR was run in triplicate on the iCycler iQ5 device (Bio-Rad, Munich, Germany) using the PCR conditions as previously reported [17]. Results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 (internal control) using the 2^−ΔΔCt method [17]. Primer sequences for circZFR were: forward, 5′-ATGGTCTGCAGTCCTGTGTG-3′ and reverse, 5′-TGGTGGCATGTTTTGTCATT-3′; for HIF1A were: forward, 5′-TTCCCGACTAGGCCCATTC-3′ and reverse, 5′-CAGGTATTCAAGGTCCCATTTCA-3′; for miR-578 were: forward, 5′-GTGCAGGGTGTTAGGA-3′ and reverse, 5′-GAAGAACACGTCTGGT-3′; for miR-944 were: forward, 5′-GAGTAGGCTACATGTTATTAAA-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; for miR-532-3p were: forward, 5′-ATCCTCCCACACCCAAGG-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; for GAPDH and U6 were described previously [18].

CircZFR knockdown in MCF7 and BT-549 cell lines was achieved by the transduction of corresponding lentiviruses expressing three different sequence shRNAs specific to circZFR (sh-circZFR#1 (sh-circZFR), sh-circZFR#2 and sh-circZFR#3, Fulengen, Guangzhou, China), and nontarget shRNA lentiviruses (sh-NC) were used as the negative control. Vector-transduced cells were selected by puromycin (Solarbio, Beijing, China) at a final concentration of 2.5 μg/mL at least 72 h. MiR-578 overexpression and knockdown cell lines were generated using the synthetic miR-578 mimic (30 nM, Ribobio, Guangzhou, China) and inhibitor (anti-miR-578, 30 nM, Ribobio), respectively, with a corresponding nontarget oligonucleotide (miR-NC mimic or anti-NC, Ribobio) as the negative control. To elevate HIF1A expression in BC cell lines, a recombinant overexpressing plasmid for HIF1A (HIF1A, 100 ng, Ribobio) or negative control plasmid (vector, Ribobio) was transiently transfected into cell lines using Lipofectamine 3000 reagent (Invitrogen, Breda, The Netherlands) as per the manufacturers’ protocols. Each experiment was performed in triplicate.

sh-circZFR-infected or sh-NC-transduced cell lines were transfected with or without anti-NC or anti-miR-578, and MCF7 and BT-549 cell lines were carried out the indicated transfections, followed by the incubation for 24 h at 37 °C. Cell viability assay was done using the Cell Counting Kit-8 (CCK-8, Abcam, Cambridge, UK) assay as per the manufacturer’s guidance. Cell colony formation was tested using standard colony formation protocols as previously reported [19]. Cell apoptosis measurement was performed by flow cytometry using double-staining with fluorescein isothiocyanate (FITC)-labeled Annexin V and propidium iodide (PI) based on the directions of manufacturers (Invitrogen). 10,000 events were analyzed by a flow cytometer, and the apoptotic cells were determined by calculating the sum of early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic cells. All experiments were done in triplicate.

Cell migration and invasion were detected by the transwell assay using modified Boyden chambers in 24-transwell plates (8 μm pores, Corning, Amsterdam, The Netherlands). Chambers of cell invasion assays consisted of Matrigel-precoated membrane inserts (Corning). After transfection, BC cell lines were seeded into the top chamber of a 24-transwell insert, and medium containing 15% FCS was used as a chemoattractant in the lower chamber. 24 h later, the well was stained with 1% crystal violet (Solarbio), and the number of cells that had migrated or invaded to the basal side of the membrane was counted under a microscope (Nikon, Shinagawa, Tokyo, Japan) at 100× magnification. Each experiment was performed in triplicate.

The Colorimetric Glucose Uptake Assay Kit, L-Lactate Assay Kit and ATP Assay Kit (all from Abcam) were used to determine the levels of glucose uptake, lactate product and ATP, according to the recommendations of manufacturers. Briefly, the lysates of transfected cells were prepared using the Assay buffer and incubated with standard protocols for the indicated time point, followed by the measurement of the absorbance with a microplate reader (Invitrogen) at OD 412 nm for glucose uptake, 450 nm for lactate product and 570 nm for ATP level. All assays were done in triplicate.

The xenograft models were constructed to assess the role of circZFR on tumor growth in vivo. Animal studies were implemented in accordance with a protocol approved by the Ethics Committee on Animal Use and Care of the First Affiliated Hospital of Zhengzhou University. Twelve 6–8 weeks BALB/c female mice (Shanghai Animal Laboratory Center, Shanghai, China) were used and randomly divided into two groups (n = 6 per group): sh-NC and sh-circZFR. Approximately 5 × 10⁶ sh-NC-infected or sh-circZFR-transduced BT-549 cell line was subcutaneously injected into the dorsal flank of the mice. One week later, tumor size was measured every week with callipers and tumor volume was calculated using the following formula: volume = 0.5 × length × (width)². All mice were sacrificed at 35 days after implantation, and tumor tissues were collected for weight.

Analysis for the targeted miRNAs of circZFR was performed using the online web CircInteractome (https://​circinteractome.​nia.​nih.​gov/​index.​html) and CircBANK (http://​www.​circbank.​cn/​searchCirc.​html). The putative targets of miR-578 were predicted by TargetScan v.7 online software (http://​www.​targetscan.​org/​vert_​71/​).

Targeted relationships among circZFR, miR-578 and HIF1A were confirmed by dual-luciferase reporter and RNA pulldown assays. In dual-luciferase assays, the fragment of circZFR containing the miR-578-binding sites and HIF1A 3′UTR were individually cloned into pmirGLO vector (Promega, Madison, WI, USA) to construct corresponding wild-type reporters (circZFR-WT and HIF1A-3′UTR-WT). The TaKaRa MutanBest Kit was used to construct the corresponding mutations (circZFR-MUT and HIF1A-3′UTR-MUT) as per the insturctions of manufacturers (TaKaRa, Dalian, China). Each reporter construct (200 ng) and 30 nM of miR-578 mimic or miR-NC mimic were cotransfected into BC cell lines. Cell line extracts were prepared with RIPA buffer (TaKaRa) 24 h post-transfection, and the ratio of Renilla to firefly luciferase was detected using the Promega Dual-luciferase Assay. In RNA pulldown assays, cell lysates were incubated with the biotin-labeled miR-578 mimic (Bio-miR-578, Ribobio) or nontarget control sequence (Bio-NC, Ribobio) for 4 h at 4 °C before adding to the streptavidin beads (Sigma-Aldrich) for 2 h. The beads were collected, and total RNA was extracted for circZFR quantification. Each experiment was performed in triplicate.

Western blot was used to determine the expression of HIF1A using standard protocols. The preparation of cell line extracts was done using RIPA buffer with proteinase inhibitors (Roche, Charente, France). Proteins (50 μg) were resolved on a 10% SDS–polyacrylamide gel, electrophoretically blotted onto nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and probed with antibody against HIF1A (ab51608, 1 μg/mL, Abcam) or GAPDH (MA5-15738, 0.5 μg/mL, Invitrogen). Following the incubation with horseradish peroxidase-coupled IgG secondary antibody (ab97051, 0.1 μg/mL, Abcam), the signals were visualized by Cheniluminescence (GE Healthcare) as recommended by the manufacturers. All experiments were done in triplicate.

Data were shown as the mean ± standard deviation from at least three independent assays. Pairwise comparisons were done using a two-tailed Student’s t test, Mann–Whitney U test or analysis of variance (ANOVA) with SPSS version 19.0 software (SPSS, Chicago, IL, USA). For survival analysis, the Kaplan–Meier survival curve and log-rank test were used. Correlations among circZFR, miR-578 and HIF1A expression levels in BC tissues were determined by the Spearman correlation test. All tests were considered statistically significant at p-value < 0.05.

As demonstrated by qRT-PCR, circZFR was significantly up-regulated in BC tissues and cell lines compared with their counterparts (Fig. 1a, b). To determine its clinical relevance, we preliminarily examined the link between circZFR level and the prognosis of BC patients. Kaplan–Meier survival curves showed that the patients in low circZFR level group had a longer survival time than those in high circZFR group (Fig. 1c). Additionally, circZFR expression was remarkably associated with the distant metastasis and TNM stage of these patients (Table 1).

To study the biological role of circZFR in BC progression, the loss-of-function experiments were carried out using shRNAs against circZFR (sh-circZFR#1, sh-circZFR#2 and sh-circZFR#3). In contrast to the negative control, the transfection of the three shRNAs prominently reduced circZFR expression in both MCF7 and BT-549 cell lines (Fig. 2a, b). Notably, sh-circZFR#1 (also named sh-circZFR) caused the most significant down-regulation in circZFR expression, so we used it for further analyses. Functional experiments data revealed that the silencing of circZFR led to a striking inhibition in cell viability (Fig. 2c), colony formation (Fig. 2d, e), as well as a strong promotion in cell apoptosis (Fig. 2f, g).

To determine whether circZFR regulated BC tumor development in vivo, we performed the xenograft model assays. When we infected BT-549 cell line with sh-circZFR, tumor growth was remarkably blocked compared with the sh-NC control (Fig. 2h, i).

We also asked whether circZFR regulated BC cell migration, invasion and glycolysis in vitro. Transwell assays showed that in comparison to the control group, cell migration (Fig. 3a) and invasion (Fig. 3b) were significantly repressed by circZFR knockdown. Moreover, in both cell lines, circZFR silencing resulted in decreased glucose uptake (Fig. 3c), lactate product (Fig. 3d) and ATP level (Fig. 3e), demonstrating the suppression of circZFR knockdown on cell glycolysis.

To further understand the role of circZFR in BC pathogenesis, we performed a detailed analysis for its targeted miRNAs. The two online algorithms CircInteractome and CircBANK collectively revealed that circZFR harbored a putative complementary sequence for miR-578, miR-944 and miR-532-3p (Fig. 4a). The data of qRT-PCR showed that the silencing of circZFR led to a striking overexpression in miR-578 and miR-532-3p levels, but miR-944 expression was not affected by circZFR knockdown in the two BC cell lines (Fig. 4b, c). Previous work had reported that circZFR regulated colorectal cancer progression through acting as a sponge of miR-532-3p [20]. So, we aimed to identify whether miR-578 was a molecular mediator of circZFR in BC progression. By contrast, miR-578 was prominently up-regulated in the two BC cell lines transfected with miR-578 mimic (Fig. 4d). We then cloned circZFR fragment containing the miR-578-binding sites into a luciferase plasmid and mutated the miR-578-binding sites (Fig. 4e). The elevated miR-578 expression significantly reduced the activity of circZFR wild-type reporter (circZFR-WT) (Fig. 4f). However, the mutation of the target sites (circZFR-MUT) completely abrogated the effect of miR-578 on reporter gene expression (Fig. 4f), indicating the validity of the target sequence for interaction. Additionally, RNA pulldown assays revealed that the enrichment level of circZFR was remarkably elevated by Bio-miR-578 in both cell lines (Fig. 4g).

In BC tissues, miR-578 expression was significantly decreased compared to the normal control (Fig. 5a), and it was inversely correlated with circZFR level (Fig. 5b). Moreover, miR-578 level was lower in BC cell lines than that of control (Fig. 5c). Subsequently, we analyzed the biological effect of miR-578 on BC progression. In contrast to the control group, the increased expression of miR-578 induced a distinct repression in cell viability (Fig. 5d) and colony formation (Fig. 5e), and a strong promotion in cell apoptosis (Fig. 5f), as well as a striking reduction in cell migration (Fig. 5G), invasion (Fig. 5H) and glycolysis (Fig. 5i–k).

A crucial question was whether circZFR regulated BC progression by miR-578. To address this, we reduced miR-578 expression in sh-circZFR-transduced MCF7 and BT-549 cell lines. As expected, in comparison to the negative control, the reduced level of miR-578 significantly reversed circZFR knockdown-induced anti-viability (Fig. 6a), anti-colony formation (Fig. 6b), pro-apoptosis (Fig. 6c), anti-migration (Fig. 6d), anti-invasion (Fig. 6e) and anti-glycolysis (Fig. 6f–h).

Using the software TargetScan, a predicted miR-578-binding sequence (ACAAGAA) was identified within the 3′UTR of HIF1A (Fig. 6a). When we cloned the 3′UTR fragment containing the putative miR-578-binding sites downstream of a luciferase coding sequence, the cotransfection of the luciferase reporter (HIF1A-3′UTR-WT) and miR-578 mimic into the two BC cell lines produced lower luciferase activity than in cell lines cotransfected with the miR-NC control (Fig. 7b, c). However, the mutation of the target sequence (HIF1A-3′UTR-MUT) prominently abolished the suppression of miR-578 (Fig. 7b, c). By contrast, HIF1A mRNA and protein levels were significantly reduced by miR-578 overexpression in the two BC cell lines (Fig. 7d, e). These data together pointed that HIF1A in BC cell lines was directly targeted and inhibited by miR-578.

We then examine whether circZFR influenced HIF1A expression in BC cell lines. As expected, in comparison to their counterparts, HIF1A expression was remarkably down-regulated by circZFR silencing at both mRNA and protein levels in the two BC cell lines, and this effect was significantly abrogated by anti-miR-578 introduction (Fig. 7f, g). Additionally, qRT-PCR data showed a striking up-regulation of HIF1A mRNA level in BC tissues (Fig. 7h). Furthermore, in BC tissues, HIF1A mRNA expression was positively correlated with circZFR expression and negatively correlated with miR-578 level (Fig. 7i).

To provide further insight into the link between miR-578 and HIF1A in BC progression, we elevated HIF1A expression using a recombinant overexpressing plasmid in miR-578 mimic-transfected BC cell lines. As a result, HIF1A protein level was prominently increased by the overexpressing plasmid in the two cell lines (Fig. 8a). Functional experiments results revealed that the up-regulation of HIF1A dramatically abolished miR-578 overexpression-mediated anti-viability (Fig. 8b), pro-apoptosis (Fig. 8c), anti-migration (Fig. 8d), anti-invasion (Fig. 8e) and anti-glycolysis (Fig. 8f–h).