BC tissue specimens and adjacent non-tumor tissue specimens (5 cm from tumor tissues) were collected from 50 female patients (median age, 53 years; age range, 29-86 years) with BC at The First Hospital of Jilin University (Changchun, China) between March 2016 and November 2017. The exclusion criteria were as follows: i) Patients who received preoperative treatment, including chemotherapy or radiotherapy; and ii) patients who had other types of tumor in other organs. The inclusion criteria were as follows: i) All clinicopathological diagnoses were confirmed by two pathologists; ii) none of the patients received any treatments before surgery; iii) no history of other synchronous malignancies; and iv) no death in the perioperative period. All samples were immediately frozen in liquid nitrogen. All participants provided written informed consent before surgical resection. The present study was approved by the Ethics Committee of The First Hospital of Jilin University.

A panel of human BC cell lines (MCF-7, SK-BR-3, MDA-MB-231 and MDA-MB-468) and a human breast epithelial cell line (MCF-10A) were purchased from BeNa Culture Collection. BC cell lines and MCF-10A cell line were cultured with RPMI-1640 (MCF-10A cell line) or DMEM (BC cell lines) (both from Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin (100 units/ml)/streptomycin (100 μg/ml) mixture (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C with 5% CO₂.

RNA samples were isolated from tissues and cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RT was conducted using the Bio-Rad iScript kit (Bio-Rad Laboratories, Inc.) and the TaqMan RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, to obtain cDNA, which was later used as the template for qPCR. The amplification of circ_0000511, ribonuclease P RNA component H1 (RPPH1) and TAZ was performed using iQSYBR-Green SuperMix (Bio-Rad Laboratories, Inc.), while the PCR reaction of miR-326 was conducted using the TaqMan MicroRNA assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR started at 95°C for 5 min, followed by 40 cycles at 95°C for 30 sec, 60°C for 40 sec and 72°C for 30 sec. GAPDH (for circ_0000511, RPPH1 and TAZ) and U6 (for miR-326) served as the internal references. The quantification was performed with the 2^−ΔΔCq method (23). The specific primers are displayed in Table I.

RNase R digestion was used to test the stability and circular structure of circ_0000511 and RPPH1 mRNA. RNA was isolated from MCF-7 and MDA-MB-468 cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), and 10 μg RNA was digested with 40 U RNase R (Epicentre; Illumina, Inc.). The enrichment of circ_0000511 and RPPH1 mRNA was detected by RTq-PCR, as aforementioned.

Small interfering (si)RNA of circ_0000511 (si-circ_0000511), scrambled siRNA negative control (si-NC), short hairpin (sh)RNA of circ_0000511 using pGIPZ vector (sh-circ_0000511), pGIPZ empty vector (sh-NC), miR-326 mimics (miR-326), miR-NC, miR-326 inhibitor (anti-miR-326), anti-miR-NC, TAZ overexpression plasmid (TAZ) and pcDNA empty vector (pcDNA) were purchased from Shanghai GenePharma Co., Ltd. The sequences were as follows: si-circ_0000511 sense, 5′-AAU GGC UGA GGU GAG UUC CCA-3′ and antisense, 5′-GGA ACU CAC CUC AGC CAU UGG-3′; si-NC sense, 5′-AAC AGG CAC ACG UCC CAG CGU-3′ and antisense, 5′-ACG CUG GGA CGU GUG CCU GUU-3′; miR-326, 5′-AGG UCU GGA CCG UAA GUC AG-3′; miR-NC, 5′-UGG ACG GAC CAC UGA CAU G-3′; anti-miR-326, 5′-CGU GGA UCG AAC GUU GGA C-3′; and anti-miR-NC, 5′-ACG GAC GUU GAG ACA CGG G-3′. A total of 1 μg plasmid or 0.5 μM oligonucleotides was transfected into MCF-7 or MDA-MB-468 cells using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). After transfection for 7 h at 37°C, the culture medium was replaced by fresh complete culture medium. BC cells were utilized for further analysis after 24 h of transfection.

A total of 150 MCF-7 or MDA-MB-468 cells in the control group and experimental groups were seeded into 6-well plates and allowed to grow for 2 weeks at 37°C. The culture medium was replenished every 6 days. The visible colonies were immobilized using 4% poly methanol for 15 min at room temperature, and stained using 0.5% crystal violet for 10 min at room temperature. Colonies containing >50 cells were counted under a light microscope.

MCF-7 and MDA-MB-468 cells were seeded into 96-well plates to settle down at 37°C overnight. The next day, BC cells were transfected with plasmids or RNAs, as aforementioned. After transfection for specific intervals (0, 1, 2 or 3 days), BC cells were incubated with 10 μl MTT (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 4 h. A total of 100 μl DMSO (Sangon Biotech Co., Ltd.) was added to the wells to dissolve the formazan product. The absorbance was detected at 570 nm.

The apoptotic rate of transfected MCF-7 and MDA-MB-468 cells was assessed by flow cytometry. BC cells in the early and late stage of apoptosis were differentiated from normal or necrotic BC cells through double staining with Annexin V-FITC and PI at 37°C for 15 min in the dark (both Beijing Solarbio Science & Technology Co., Ltd.). The apoptotic rate was evaluated using FlowJo software (v7.6; FlowJo LLC) on a FC-500 flow cytometer (Beckman Coulter, Inc.).

After plasmid or RNA transfection, MCF-7 and MDA-MB-468 cells were lysed using cell lysis buffer (Abcam). The BCA method was used to examine the concentrations of different protein samples. Protein samples (25 μg/lane) were separated via 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). After incubating with 5% skimmed milk for 1 h at room temperature, the PVDF membranes were incubated overnight at 4°C with specific primary antibodies, including anti-B cell leukemia/lymphoma 2 (anti-Bcl-2; 1:5,000; cat. no. ab185002; Abcam), anti-Bcl-2 associated X apoptosis regulator (anti-Bax; 1:8,000; cat. no. ab32503; Abcam), anti-cleaved caspase-3 (1:10,000; cat. no. ab2302; Abcam), anti-TAZ (1:5,000; cat. no. ab242313; Abcam) and anti-GAPDH (1:20,000; cat. no. ab181602; Abcam). The next day, an HRP-conjugated secondary antibody (1:5,000; cat. no. ab205718; Abcam) was incubated with the PVDF membranes for 2 h at room temperature. The immunoreactive protein bands were measured using an enhanced chemiluminescent visualization (ECL) kit (Pierce; Thermo Fisher Scientific, Inc.). Image Lab analysis software (v4.0; Bio-Rad Laboratories, Inc.) was used to analyze the intensities of protein bands.

Transwell chambers (Costar; Corning, Inc.) were used in the present study. For Transwell migration assays, 200 μl cell suspension (1×10⁴ MCF-7 and MDA-MB-468 cells in serum-free DMEM) was added to the upper chambers. The lower chambers contained culture medium with 10% FBS. After incubation at 37°C for 24 h, BC cells on the upper surface of the membrane were discarded using a cotton swab, and the migrated BC cells on the lower surface of the membrane were stained using 0.5% crystal violet for 10 min at room temperature. Five random fields at a magnification of ×100 were selected to count the number of migrating cells under a light microscope.

For Transwell invasion assays, the upper chambers were pre-coated with 40 μl Matrigel (BD Biosciences) at the dilution of 1:8 at 37°C for 30 min. The remaining steps were similar as for the Transwell migration assays.

The circ_0000511-miRNAs interactions and miR-326-mRNAs interactions were explored using the StarBase database (http://starbase.sysu.edu.cn).

To test the direct interaction between miR-326 and circ_0000511 or TAZ mRNA, the wild-type (wt) partial sequences in circ_0000511 and TAZ mRNA 3′-untranslated region (UTR), containing the putative binding sequence with miR-326, were amplified and constructed in the psiCHECK-2 luciferase vector (Promega Corporation), generating circ_0000511-wt and TAZ-wt. Meanwhile, circ_0000511-mutant (mut) and TAZ-mut luciferase plasmids containing the mutant binding sites with miR-326 were generated using GeneArtä Site-Directed Mutagenesis System (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the constructed luciferase plasmids were co-transfected into BC cells with miR-NC or miR-326 using Lipofectamine 3000, as aforementioned. Luciferase activities were measured after 24 h of transfection using the Dual Luciferase Reporter Assay kit (Promega Corporation). The firefly luciferase activity was normalized to Renilla luciferase activity.

A total of 10 female 4-week-old BALB/c nude mice (weight, 16-20 g) from Orient Bio, Inc., were randomly divided into two groups with 5 mice in each group. Mice were maintained under pathogen-free conditions at 22±2°C and 60% humidity with a 12-h light/dark cycle, and were supplied with food and water ad libitum. The weight of mice was measured twice a week to monitor the health of mice. Mice weight in the two groups exhibited no significant difference during the experiment. The maximum volume of xenograft tumor was <1,000 mm³. The mice were euthanized by carbon dioxide suffocation. The CO₂ displacement rate used for mouse euthanasia was 30% chamber volume/min. The MCF-7 cell line stably transfected with sh-NC or sh-circ_0000511 was collected and re-suspended in PBS buffer. A total of 200 μl cell suspension containing 2×10⁶ MCF-7 cells was subcutaneously injected into the right flank of nude mice. The length and width of tumor masses were detected every week until 4 weeks after injection when the mice were euthanized. The tumor volume was analyzed with the following formula: Volume = width² × length × 0.5. The tumors were weighed after removing the tumors from the mice. The abundance of circ_0000511, miR-326 and TAZ mRNA and protein was examined by RT-qPCR and western blot assay, respectively. Animal manipulation was approved by the Research Ethics Committee of The First Hospital of Jilin University.

The results were analyzed using GraphPad Prism software (v7.0; GraphPad Software, Inc.) and the data are displayed as the mean ± SD. All in vitro experiments were repeated three times. Differences between BC tissues and adjacent normal tissues were analyzed using paired Student's t-test, while unpaired Student's t-test was utilized to assess the differences between two groups of BC cells. One-way ANOVA followed by Tukey's test was used to analyze the differences in multiple groups. Linear correlation analysis was performed using Spearman's correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.

Circ_0000511 expression was significantly elevated in BC tissues compared with in matching adjacent normal tissues (Fig. 1A). The expression levels of circ_0000511 in the normal breast epithelial MCF-10A cell line and four BC cell lines (MCF-7, SK-BR-3, MDA-MB-231 and MDA-MB-468) were detected by RT-qPCR. As shown in Fig. 1B, circ_0000511 expression was significantly enhanced in BC cell lines compared with in the MCF-10A cell line. Moreover, to test the stability and loop structure of circ_0000511, exonuclease RNase R was used in subsequent experiments to digest linear RNA but not circular RNA, and the linear form of circ_0000511 (RPPH1 mRNA) was used as the control. RNA samples isolated from BC cells were equally divided into two parts, and these two parts were exposed to RNase R or not. As shown in Fig. 1C and D, RNase R addition significantly downregulated the expression levels of RPPH1, while the abundance of circ_0000511 remained almost unchanged in the RNase R treatment and no treatment groups, suggesting the loop structure of circ_0000511. Overall, circ_0000511 was identified as a circular RNA, and its expression was upregulated in BC tissues and cell lines.

si-circ_0000511 was designed to silence circ_0000511 in BC cell lines to test the biological effects of circ_0000511. Circ_0000511 abundance was significantly downregulated in MCF-7 and MDA-MB-468 cells transfected with si-circ_0000511 compared with the si-NC-transfected group (Fig. 2A). MCF-7 and MDA-MB-468 cell lines were chosen for loss-of-function experiments due to their higher expression of circ_0000511 compared with the other two BC cell lines (SK-BR-3 and MDA-MB-231). The proliferation of BC cells was analyzed using colony formation and MTT assays. As shown in Fig. 2B, circ_0000511 silencing significantly decreased the number of colonies, suggesting that circ_0000511 interference suppressed the proliferative ability of BC cells. Additionally, the results of MTT assay revealed that circ_0000511-knockdown impaired the proliferation of BC cells (Fig. 2C and D). Furthermore, circ_0000511 interference triggered the apoptosis of BC cells (Fig. 2E). To further clarify the influence of circ_0000511 interference on the apoptosis of BC cells, western blot assay was conducted to detect the protein expression levels of Bcl-2, Bax and Cleaved caspase-3 in BC cells. As shown in Fig. 2F and G, the expression levels of the pro-apoptotic proteins (Bax and Cleaved caspase-3) were significantly upregulated in BC cells with the silencing of circ_0000511, while circ_0000511 interference significantly downregulated the expression levels of the anti-apoptotic protein Bcl-2, demonstrating that circ_0000511-knockdown promoted the apoptosis of BC cells. The migratory and invasive abilities were both significantly suppressed with the silencing of circ_0000511 compared with in the si-NC group (Fig. 2H and I), suggesting that circ_0000511-knockdown restrained the migration and invasion of BC cells. Overall, high circ_0000511 expression increased the malignant behaviors of BC cells.

Considering the miRNA sponge role of circRNAs (13), the present study investigated whether circ_0000511 accelerated the progression of BC through targeting miRNAs. The complementary binding sites between circ_0000511 and miR-326 predicted using the StarBase database are displayed in Fig. 3A. To test if circ_0000511 interacted with miR-326 through the predicted binding sites, these sites in circ_0000511 were mutated to 'AAACUCU' (Fig. 3A). The transfection efficiencies of miR-326 and anti-miR-326 were both high in BC cells (Fig. 3B). The partial sequence in circ_0000511, including the wt or mut binding sites with miR-326, was cloned into luciferase vectors, named as circ_0000511-wt or circ_0000511-mut. The luciferase reporter vector was co-transfected with miR-NC or miR-326 into BC cells. As shown in Fig. 3C and D, miR-326 overexpression significantly decreased the luciferase activity in the circ_0000511-wt group compared with in the miR-NC and circ_0000511-wt co-transfected group. However, miR-326 transfection had no influence in luciferase activity in the circ_0000511-mut group compared with in the miR-NC and circ_0000511-mut group (Fig. 3C and D). The results of the dual-luciferase reporter assay demonstrated that miR-326 was a direct target of circ_0000511 in BC cells. Additionally, circ_0000511 silencing significantly upregulated the expression levels of miR-326 in BC cells (Fig. 3E), suggesting a negative regulatory association between miR-326 and circ_0000511 in BC cells. Among three cell lines, including MCF-10A and two BC cell lines, miR-326 expression was significantly decreased in BC cell lines compared with in MCF-10A cells (Fig. 3F). Furthermore, miR-326 expression was significantly downregulated in BC tissues compared with that in normal tissues (Fig. 3G). The analysis of Spearman's correlation coefficient suggested that miR-326 expression was negatively correlated with circ_0000511 expression in BC tissues (Fig. 3H). Overall, the present results indicated that miR-326 was a direct target of circ_0000511 in BC cells.

Circ_0000511-knockdown significantly upregulated miR-326 expression, which was then significantly decreased by the addition of anti-miR-326 in BC cells (Fig. 4A). Therefore, further rescue experiments were conducted via transfecting si-circ_0000511 alone or together with anti-miR-326 into BC cells. The introduction of anti-miR-326 partly recovered the number of colonies (Figs. 4B and S1A) and the number of viable BC cells after 3 days of transfection (Fig. 4C and D), demonstrating that anti-miR-326 addition partly recovered the proliferative ability of BC cells. Meanwhile, the apoptosis of these transfected BC cells was assessed by flow cytometry. Circ_0000511 silencing-induced apoptosis in BC cells was largely counteracted by the introduction of anti-miR-326 (Figs. 4E and S1B). The protein expression levels of Bax and Cleaved caspase-3 were significantly upregulated by the silencing of circ_0000511, while the addition of anti-miR-326 significantly downregulated the expression levels of these two proteins (Fig. 4F and G). The abundance of the anti-apoptotic protein Bcl-2 was significantly downregulated in the si-circ_0000511 group, and the addition of anti-miR-326 largely recovered Bcl-2 expression (Fig. 4F and G). The present findings further demonstrated that circ_0000511 restrained the apoptosis of BC cells via targeting miR-326. Furthermore, migration and invasion were suppressed with the silencing of circ_0000511, and the introduction of anti-miR-326 largely rescued the migratory and invasive abilities of BC cells (Figs. 4H and I, and S1C and D). Overall, circ_0000511 accelerated the proliferation, migration and invasion, and inhibited the apoptosis of BC cells via sponging miR-326.

miRNAs are implicated in the pathogenesis of cancer partly through downregulating the expression levels of target mRNAs associated with cellular proliferation, apoptosis, migration and invasion (16,17). miR-326-mRNA interactions were searched using the StarBase 3.0 database. The complementary sequence between TAZ mRNA and miR-326 is shown in Fig. 5A. The site-directed mutation in TAZ is also shown in Fig. 5A. MCF-7 and MDA-MB-468 cells were co-transfected with miR-NC or miR-326 and TAZ-wt or TAZ-mut, and the luciferase activities in these four transfected groups were detected using the dual-luciferase reporter assay. Compared with the miR-NC and TAZ-wt co-transfected group, miR-326 transfection significantly decreased the relative luciferase activity in the TAZ-wt group (Fig. 5B and C). However, miR-NC or miR-326 transfection did not affect the luciferase activity in the TAZ-mut groups (Fig. 5B and C), suggesting a direct association between miR-326 and TAZ mRNA in BC cells. The present study further explored the regulatory association between miR-326 and TAZ by detecting the mRNA and protein expression levels of TAZ in BC cells transfected with miR-NC, miR-326, anti-miR-NC or anti-miR-326. miR-326 overexpression significantly downregulated the mRNA and protein expression levels of TAZ, while the inhibition of miR-326 significantly upregulated TAZ mRNA and protein expression (Fig. 5D and E). The current findings suggested that TAZ was negatively regulated by miR-326 in BC cells. The expression feature of TAZ and the expression correlation between TAZ and miR-326 were tested in BC. As shown in Fig. 5F and G, TAZ mRNA and protein expression was significantly upregulated in BC tissues compared with in adjacent normal tissues. The results of Spearman's correlation coefficient analysis revealed that there was a negative correlation between miR-326 and TAZ mRNA expression in BC tissues (Fig. 5H). Additionally, TAZ mRNA and protein expression was significantly upregulated in BC cell lines compared with in the MCF-10A cell line (Fig. 5I and J). Overall, miR-326 negatively regulated the abundance of TAZ by directly targeting it in BC cells.

The overexpression of TAZ was tested by RT-qPCR assay and western blot assay. As shown in Fig. S2A and B, the mRNA and protein expression levels of TAZ were both significantly upregulated following the transfection of TAZ plasmid in BC cells, suggesting the high transfection efficiency of the TAZ plasmid. miR-326 over- expression significantly downregulated the mRNA and protein expression levels of TAZ in BC cells, and the addition of TAZ overexpression plasmid rescued the mRNA and protein abundance of TAZ in BC cells (Fig. 6A and B). Therefore, further rescue experiments were conducted by transfecting miR-NC, miR-326, miR-326 + pcDNA or miR-326 + TAZ into BC cells to test if miR-326 functioned via decreasing TAZ expression. miR-326 overexpression suppressed cellular proliferation, migration and invasion, while apoptosis was increased with the overexpression of miR-326 (Fig. 6C-J), which further demonstrated that miR-326 exerted an antitumor role in BC cells. The addition of TAZ overexpression plasmid partly recovered the proliferative ability of BC cells according to the results of colony formation assay (Figs. 6C and S3A) and MTT assay (Fig. 6D and E). The results of flow cytometry (Figs. 6F and S3B) and western blot assay (Fig. 6G and H) both suggested that miR-326-induced apoptosis in BC cells was partly counteracted by the overexpression of TAZ. Additionally, the migration and invasion of BC cells were largely regained in the miR-326 and TAZ co-transfected group (Figs. 6I and J, and S3C and D). The current findings demonstrated that miR-326 suppressed the progression of BC via downregulating TAZ expression.

After identifying the negative regulatory association between miR-326 and circ_0000511 or TAZ, the present study subsequently explored the association among circ_0000511, miR-326 and TAZ in BC cells. MCF-7 and MDA-MB-468 cells were transfected with si-NC, si-circ_0000511, si-circ_0000511 + anti-miR-NC or si-circ_0000511 + anti-miR-326. The mRNA and protein expression levels of TAZ were analyzed by RT-qPCR and western blot assay, respectively. As shown in Fig. 7A and B, circ_0000511 silencing significantly downregulated the mRNA and protein expression levels of TAZ, and the addition of anti-miR-326 partly recovered the mRNA and protein abundance of TAZ in BC cells, suggesting that circ_0000511 enhanced the mRNA and protein expression levels of TAZ via acting as a sponge of miR-326.

On the basis of the oncogenic role of circ_0000511 in vitro, the present study further assessed the influence of circ_0000511 silencing on the growth of BC tumors in vivo. An MCF-7 cell line stably transfected with sh-NC or sh-circ_0000511 was established. Tumor growth curves were generated by recording the tumor volume every week for 4 weeks. As shown in Fig. 8A, tumors in the sh-NC group were bigger than tumors in the sh-circ_0000511 group. Additionally, by measuring the tumor weight after resecting tumors 4 weeks after inoculation, it was revealed that circ_0000511 silencing suppressed tumor growth in vivo (Fig. 8B). The mRNA and protein expression levels of circ_0000511, miR-326 and TAZ were measured by RT-qPCR and western blot assay, respectively. As shown in Fig. 8C, circ_0000511 abundance was significantly downregulated in the sh-circ_0000511 group compared with that in the sh-NC group. Additionally, miR-326 expression was significantly increased in the sh-circ_0000511 group compared with that in the sh-NC group (Fig. 8D), and TAZ mRNA and protein expression was significantly decreased in the sh-circ_0000511 group compared with that in the sh-NC group (Fig. 8E and F). Overall, the present results suggested that circ_0000511 accelerated the tumor growth of BC in vivo.