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CircCEP85 upregulates IGF1 expression to promote breast cancer progression via sponging miR-1193

Abstract

Background

Increasing evidence has suggested that circular RNAs (circRNAs) play critical roles in breast cancer (BC) progression. However, the expression level and potential functional role of circRNA centrosomal protein 85 (circCEP85) in BC remains largely unknown. Here, we aimed to explore the role of circCEP85 in BC.

Methods

The levels of circCEP85, insuline-like growth factor I (IGF1) mRNA and microRNA-1193 (miR-1193) were examined by quantitative real-time polymerase chain reaction. The protein level was measured by Western blot. Cell proliferation, migration, apoptosis, angiogenesis and stemness were assessed by cell counting kit-8, 5-ethynyl-2’-deoxyuridine assay, transwell assay, flow cytometry, tube formation and sphere formation assays. Xenograft mouse models were conducted to evaluate the effect of circCEP85 in BC in vivo. Moreover, dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays were preformed to confirm the interaction between miR-1193 and circCEP85 or IGF1.

Results

CircCEP85 was upregulated in BC tissues and cells. Silencing of circCEP85 inhibited proliferation, invasion, angiogenesis and stemness, but promoted apoptosis in BC cells in vitro. In addition, circCEP85 silencing inhibited tumor growth in vivo. Mechanistically, circCEP85 elevated IGF1 expression via sponging miR-1193 to promote breast cancer progression.

Conclusion

The circCEP85-miR-1193-IGF1 axis regulated BC progression via the competitive endogenous RNA (ceRNA) mechanism. CircCEP85 might be a prognostic biomarker and therapeutic target for BC.

Highlights

  1. 1.

    CircCEP85 is up-regulated in breast cancer

  2. 2.

    CircCEP85 functions as a sponge for miR-1193 to regulate its target IGF1 in breast cancer cells

  3. 3.

    CircCEP85 contributes to breast cancer progression via the miR-1193/IGF1 axis

Introduction

Breast cancer (BC) is a serious malignant tumor, with the highest incidence rate among women [1]. More than 1.3 million women worldwide are diagnosed with BC each year, and more than half a million of them die from BC [2, 3]. With the deterioration of lifestyle and ecological environment, the proportion of new cases of BC in China is still growing [4, 5]. Therefore, clarifying the pathogenesis, controlling the incidence rate and improving the therapeutic efficiency of BC have become the major problems to be solved.

Circular RNA (circRNA) is one of non-coding RNA possessing a closed-loop structure without 5' and 3' ends, which is reversely spliced by exons, introns, or both, [6]. CircRNA has a stable structure and is not easily degraded by various factors, and has tissue and disease specificity [7]. Growing evidence has demonstrated that circRNAs ate involved in the progression many of cancers [8,9,10], including breast cancer [11]. For instance, circRNA circWHSC1 was highly expressed and promoted the growth, metastasis and glycolysis of BC cells through regulating miR-212-5p and AKT3 [12]. CircPRMT5 was increased in BC and accelerated the proliferation and migration of BC cells [13]. Circ_0005046 and circ_0001791 expression levels were all upregulated in BC, and they might function as early detection biomarkers for BC [14]. CircRNA centrosomal protein 85 (circCEP85, circbase ID: hsa_circ_0000033, derived from CEP85 gene on chr1:26584087-26586293) has been reported to be markedly increased in BC tissues by high-throughput sequencing [15]. However, the function and underlying molecular mechanism of circCEP85 in BC are still unclear.

In our study, we found circCEP85 was increased in BC tissues and cells, and BC patients with highly expressed circCEP85 had poor prognosis. Silencing of circCEP85 obviously inhibited BC malignancy by regulating miR-1193 and IGF1 expression. Our findings revealed that circCEP85 might function as a tumor promoter in BC.

Materials and methods

Tissue sample

The BC tissues and adjacent non-cancer tissues (n = 66) were obtained from The Third Hospital of Mianyang. Tissue specimens were immediately stored and kept at − 80 ℃. None of the patients had received radiotherapy or chemotherapy before operation. The Ethics Committee of The Third Hospital of Mianyang approved this study.

Cell culture and transient transfection

Five BC cells (BT-549, MDA-MB-231, MDA-MB-453, and MDA-MB-468) and normal breast epithelial MCF-10A cell were bought from ATCC (Manassas, VA, USA). All cells were incubated in DMEM medium (Gibco, Carlsbad, CA, USA), containing 10% FBS and 1% penicillin/streptomycin at 37℃ in an incubator with 5% CO2.

Short hairpin RNAs targeting circCEP85 (sh-circCEP85#1, sh-circCEP85#2, sh-circCEP85#3) were constructed by Invitrogen (Carlsbad, CA, USA), miR-1193 mimic or inhibitor (miR-1193 or anti-miR-1193) and corresponding controls were all bought from RiboBio (Guangzhou, China). IGF1 overexpression plasmids (IGF1) and vectors were obtained from RiboBio. BC cells were treated with above oligonucleotides or vectors through lipofectamine 3000 reagent (Invitrogen).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNAs were dissociated from BC cells or samples with RNA Extraction Kit (TaKaRa, Japan), and the concentration was quantitatively analyzed by NanoDROP 2000 (Thermo Fisher). Then, RNAs were subjected to reverse transcription through PrimeScript RT reagent kit (Exiqon, Aarhus, Denmark). The SYBR Premix Ex Taq II kit (Takara) was used to perform qRT-PCR. CircCEP85, miR-1193, and IGF1 expression levels were analyzed by using 2−∆∆Ct method. Primer sequences were shown in Table 1.

RNase R assay

About 1 µg of RNA was digested with 1 U of RNase R at 37 ℃ for 15 min. Then, they were reversely transcribed into cDNAs, and circCEP85 expression and GAPDH mRNA expression were all assessed by qRT-PCR.

Cell counting kit 8 (CCK8) assay

CCK8 assay (Beyotime, Shanghai, China) was performed to assess cell viability. Transfected BC cells were seeded into 96-well plates, and incubated for 48 h. 10 µL CCK8 reagent was added into cells and incubated for 4 h. The absorbance at 450 nm was examined.

5-ethynyl-2’-deoxyuridine (EDU) assay

Transfected BC cells were plated into 96 well plates. BeyoClick™ EdU-647 kit (beyotime, shanghai, China) was used to perform EDU assay. Cells incubated with EDU buffer. Then, 4% formaldehyde was performed to fix the cells. After that, DAPI staining was conducted for 30 min in the dark, and EDU-positive rate was assessed by a fluorescence microscopy.

Apoptosis analysis

Annexin V-FITC Apoptosis Detection Kit (beyotime, shanghai, China) was used to detect cell apoptosis. Annexin V-FITC and PI were used to stain the transfected BC cells. The apoptosis of cells was assessed by a flow cytometer (Becton, USA).

Transwell invasion assay

Transfected BC cells suspended with serum-free medium were seeded into the Matrigel-coated transwell chambers (Bechman Coulter, Brea, CA). After incubation for 24 h, the invaded cells were fixed with 4% paraformaldehyde for 30 min, and then stained with 0.1% crystal violet for 15 min. The pictures were obtained by using a light microscope.

Tube formation assay

A 96-well plate was coated with 50 μL Matrigel (356235, Corning, Tewksbury, MA, USA). HUVECs were resuspended in 100 μL condition medium of assigned BC cells, and then reseeded (3 × 104 cells/well) onto Matrigel-coated wells. After 12 h, a microscope (Olympus, Japan) was used to observe the cells.

Sphere formation assay

Transfected BC cells were cultivated in DMEM medium containing basic fibroblast growth factor (10 ng/ml), insulin (4 ng/ml), B27 (2%), and epidermal growth factor (100 ng/ml) in the Ultra-low attachment 6-well plate (Costar, Corning) for 10 days. All above relative factors were bought from Sigma-Aldrich. The fresh medium was replaced after 2–3 days. The microscope was used to observe and photograph the sphere formation.

Western blot analysis

The proteins were isolated from collected samples and transfected cells by RIPA buffer (Sigma-Aldrich). The proteins were separated by SDS-PAGE and transferred onto the PVDF membranes (Merck, Darmstadt, Hesse, Germany), and the PVDF membranes were then incubated with the primary antibodies, including anti-IGF1 (1:1,000, ab182408, Abcam), anti-PCNA (1:1,000, #71395, CST), anti-cleaved caspase 3 (1:1,000, #9661, CST), anti-VEGFA (1:1,000, ab52917, Abcam), anti-Naong (1:1,000, ab109250, Abcam), or anti-β-actin (1:4,000, ab7817, Abcam). Then, the secondary antibody was hatched with the membrane, and ECL kit was perform to observe the protein bands.

RNA pull-down assay

Cells were transfected with biotinylated-labelled circCEP85 (circCEP85 probe) or (oligo probe). 48 h later, the lysis buffer was used to lyse the cells, and the lysate was incubated with magnetic beads for 24 h. Finally, the bound RNA was isolated, and circCEP85 enrichment was assessed by qRT-PCR.

Dual-luciferase reporter assay

CircCEP85 and IGF1 sequences contained the wild type (WT) or mutant type (MUT) miR-1193 complementary binding sites were cloned and inserted into the pmirGLO dual luciferase reporter vectors by RiboBio, and named as circCEP85-WT, circCEP85-MUT, IGF1-WT or IGF1-MUT. BC cells were transfected with these luciferase reporter vectors and miR-1193 mimic using Lipofectamine™ 3000 kit. After 24 h, the luciferase activities were detected.

RNA immunoprecipitation (RIP) assay

According to the instructions of the Magna RIP Kit (Abcam, Cambridge, UK). BC cells were lysed by RIP lysis buffers, and cell lysates were cultured with the magnetic bead with anti-Ago2 (1:500, ab186733, Abcam) or anti-IgG. The level of miR-1193 and circCEP85 was evaluated by qPCR.

Xenograft mice model

BT549 cells transfected with sh-circCEP85#1 or sh-NC were resuspended with PBS (4 × 106 cells/200 µL PBS), and then the cell suspensions were subcutaneously injected into the mice (n = 5/group). Tumor volume was recorded every week and the mice were sacrificed for tumor weight measurement after 35 days. The tumor samples were analyzed by qRT-PCR and western blot. IGF1 and Ki-67 Immunohistochemistry (IHC) staining was conducted using SP Kit (Invitrogen) with anti-IGF1 (1:500, ab263903, Abcam) and anti-Ki-67 (1:500, ab1550, Abcam). Our research was permitted by The Third Hospital of Mianyang.

Statistical analysis

All experiments at least repeated for three times. Graphpad Prism 7.0 software was used to analyze data. The differences between two groups were analyzed by Student’s t-test, and differences among three or more group were analyzed by one-way analysis of variance. P < 0.05 was considered as statistically significant.

Results

CircCEP85 was highly expressed in BC

The expression of circCEP85 in BC tissues and adjacent non-cancer tissues was detected by qRT-PCR. The results showed that circCEP85 was increased in BC tissues compared with adjacent non-cancer tissues (Fig. 1A). According to the median expression of circCEP85, the patients were grouped into high and low expression groups. Kaplan–Meier analysis results showed that patients with highly expressed circCEP85 had a low overall survival rate (Fig. 1B). Besides, the expression of circCEP85 in BC cells (BT-549, MDA-MB-231, MDA-MB-453, and MDA-MB-468) was increased compared with normal breast epithelial MCF-10A cell (Fig. 1C). Next, the stability of circCEP85 was assessed using RNase R in BC cells, and GAPDH mRNA was used as a control. The results suggested the expression of circCEP85 had no obvious change after RNase R treatment (Fig. 1D). These results indicated that circCEP85 was a circular RNA and was upregulated in BC tissues and cells.

Fig. 1
figure 1

CircCEP85 was highly expressed in BC tissues and cells. A The expression of circCEP85 in BC tissues (n = 66) and adjacent non-cancer tissues (Normal) (n = 66) was detected; B Kaplan–Meier survival curves of the correlation between circCEP85 expression and overall survival of BC patients; C Relative expression of circCEP85 was detected in BC cells and MCF10A cells; D The levels of circCEP85 and GAPDH mRNA were detected by qRT-PCR after the treatment of RNase R. ***P < 0.001

Knockdown of circCEP85 inhibited proliferation, invasion, angiogenesis, stemness and induced apoptosis of BC cells in vitro

To explore the roles of circCEP85 in BC progression, circCEP85 expression was knockdown by shRNA of circCEP85 in BC cells (Fig. 2A). In three shRNA of circCEP85, sh-circCEP85#1 with the most significant difference was selected for subsequent research, which was then transfected into BT-549 and MDA-MB-231 cells. CCK8 assay and EDU assay results revealed that circCEP85 inhibition evidently inhibited the proliferation of BC cells (Fig. 2B, C). Flow cytometry analysis showed that knockdown of circCEP85 obviously elevated cell apoptosis rate (Fig. 2D). The invasion of BC cells transfected with sh-circCEP85#1 was also greatly hindered (Fig. 2E). This indicated that silencing of circCEP85 could inhibit the proliferation and invasion of BC cells. In addition, we also found that circCEP85 inhibition decreased the angiogenesis capability and stemness in BC cells (Fig. 2F, G). Moreover, we found the levels of proliferation-related (PCNA), angiogenesis-related (VEGFA) and stemness-related (Naong) protein were decreased, but the level of apoptosis-related (cleaved caspase-3) protein was increased in BC cells with circCEP85 silencing (Fig. 2H). These results demonstrated that knockdown of circCEP85 suppressed the proliferation, invasion, angiogenesis, stemness, and induced apoptosis of BC cells in vitro.

Fig. 2
figure 2

Knockdown of circCEP85 inhibited proliferation, invasion, angiogenesis, stemness and induced apoptosis of BC cells in vitro. A Relative expression of circCEP85 was detected by qRT-PCR in BT-549 and MDA-MB-231 cells transfected with sh-NC, sh-circCEP85#1, sh-circCEP85#2, or sh-circCEP85#3; BH BT-549 and MDA-MB-231 cells were transfected with sh-NC or sh-circCEP85#1. B, C CCK-8 assay and EDU assay were performed to measure cell proliferation; D Flow cytometry analysis was conducted to evaluate cell apoptosis rate; E Transwell assay was used to detect cell invasion ability; F Angiogenesis capability was assessed by tube formation assay; G Sphere formation assay was used to detect cell stemness; H Western blot assay was employed to detect the levels of proliferation-related (PCNA), apoptosis-related (cleaved caspase-3), angiogenesis-related (VEGFA) and stemness-related (Naong) protein. **P < 0.01, ***P < 0.001

CircCEP85 functioned as a sponge for miR-1193

To investigate the underlying molecular mechanisms of circCEP85 in BC progression, we first analyzed the location of circCEP85 in BC cells. As showed in Fig. 3A, circCEP85 was mainly located in the cytoplasm. CircRNAs in the cytoplasm can act as miRNA sponges to play a regulatory role in tumors[16]. Then, Circatlas and Starbase were used to predict the potential miRNAs of circCEP85, and we found miR-1193 might bind to circCEP85 according to the Venn diagram (Fig. 3B). MiR-1193 expression was decreased in BC tissues and cells, and correlation analysis suggested that miR-1193 had a negative correlation with circCEP85 (Fig. 3C–E). The overexpression efficiency of miR-1193 mimic was demonstrated in Fig. 3F. The complementary binding sites between circCEP85 and miR-1193 were displayed in Fig. 3G. To confirm the relationship between circCEP85 and miR-1193 in BC cells. Dual-luciferase reporter assay was performed, and the results showed that co-transfection of miR-1193 and WT-circCEP85 obviously decreased the relative luciferase activity in BC cells, but there was no significant change in the luciferase activity of BC cells co-transfected with miR-1193 and MUT-circCEP85 (Fig. 3H, I). Then, RNA pull down assay also demonstrated that circCEP85 directly bound to miR-1193 (Fig. 3J). Furthermore, RIP assay revealed both circCEP85 and miR-1193 were enriched by Ago2 antibodies compared with IgG antibodies (Fig. 3K). Overall, the present results indicated that circCEP85 could serve as a competing endogenous RNA (ceRNA) to sponge miR-1193 in BC cells.

Fig. 3
figure 3

CircCEP85 functioned as a sponge for miR-1193. A The location of circCEP85 was tested by cytoplasmic and nuclear RNA analysis assay; B Prediction of downstream target genes for circCEP85 binding using bioinformatics websites; C Relative expression of miR-1193 in BC tissues and adjacent non-cancer tissues was detected by qRT-PCR; D The correlation between circCEP85 and miR-1193 in BC tissues was analyzed by Pearson correlation analysis; E Relative expression of miR-1193 in BC cells was detected; F The overexpression efficiency of miR-1193 mimic was detected by qRT-PCR; G Schematic diagram of binding sites between circCEP85 and miR-1193; H, I Dual-luciferase reporter assay was performed to analyze the target binding relationship between circCEP85 and miR-1193; J RNA pull down assay was performed to assess the relationship between circCEP85 and miR-1193; K Enrichment levels of circCEP85 and miR-1193 were measured by RIP assay. **P < 0.01, ***P < 0.001

MiR-1193 suppressed proliferation, invasion, angiogenesis and stemness, but induced apoptosis in BC cells

The potential roles of miR-1193 in BC cells were further revealed. Overexpression of miR-1193 markedly decreased the proliferation of BC cells (Fig. 4A, B). Upregulation of miR-1193 significantly enhanced cell apoptosis rate in BC cells (Fig. 4C). In addition, the invasion ability was repressed in BC cells after treating with miR-1193 mimics (Fig. 4D). Furthermore, miR-1193 mimics restrained the angiogenesis capability and stemness of BC cells (Fig. 4E, F). Meanwhile, the protein levels of PCNA, VEGFA, and Nanog were downregulated, and cleaved caspase-3 protein level was increased in miR-1193-overexpressed BC cells (Fig. 4G). All results suggested that miR-1193 could suppress the tumorigenesis of BC cells in vitro.

Fig. 4
figure 4

MiR-1193 suppressed proliferation, invasion, angiogenesis, stemness and induced apoptosis of BC cells. A, B Effect of miR-1193-mimics on proliferation in BC cells was assessed by CCK8 and EDU assays; C Effect of miR-1193-mimics on apoptosis in BC cells was assessed by flow cytometry analysis; D Effect of miR-1193-mimics on invasion in BC cells was assessed by transwell assay; E Tube formation assay was conducted to detect the effects of miR-1193-mimics on HUVEC angiogenesis capability; F Sphere formation assay was performed to assess cell stemness in BC cells treated with miR-1193-mimics; G Western blot analysis was used to determine the protein levels of PCNA, cleaved caspase 3, VEGFA and Naong in BT-549 and MDA-MB-231 cells transfected with miR-NC or miR-1193. ***P < 0.001

IGF1 was a direct target of miR-1193

The downstream target genes of miR-1193 were then explored, and we screened out eight mRNA molecules by using miRDB and Targetscan (Fig. 5A), among them, only the expression of IGF1 was decreased in BC cells after miR-1193 mimics transfection (Fig. 5B). Their binding sites were displayed in Fig. 5C. Then, dual-luciferase reporter assay was conducted to demonstrate the binding relationship between miR-1193 and IGF1. The luciferase activity was significantly reduced after the transfection of WT-3’UTR IGF1 and miR-1193 mimics in BC cells (Fig. 5D). In addition, overexpressed miR-1193 evidently repressed IGF1 protein level (Fig. 5E). Furthermore, the expression of IGF1 was upregulated in BC tissues as well as BC cells when compared with adjacent non-cancer tissues and MCF-10A cells, respectively (Fig. 5F–I). These results suggested that miR-1193 targetedly suppressed IGF1 expression in BC cells.

Fig. 5
figure 5

IGF1 was a direct target of miR-1193. A The downstream target genes of miR-1193 were predicted using bioinformatics websites; B The expression of downstream target genes in BC cells treated with miR-1193 mimics was detected by qRT-PCR; C Schematic diagram of binding sites between IGF1 and miR-1193; D The relative luciferase activities of the reporter were detected in BC cells; E Western blot was conducted to detect the protein level of IGF1 in BC cells after the transfection with miR-1193 mimics; F Relative expression of IGF1 mRNA in BC tissues and adjacent non-cancer tissues was detected by qRT-PCR; G, H IHC and western blot assays were used to detect IGF1 expression in BC tissues and adjacent non-cancer tissues; I The protein level of IGF1 in BC cells was assessed by western blot. ***P < 0.001

CircCEP85 promoted BC progression via regulating miR-1193/IGF1 axis

Next, we aimed to explore the roles of circCEP85/miR-1193/IGF1 axis in BC cells. MiR-1193 expression was markedly downregulated in BC cells after the transfection of miR-1193 inhibitor (anti-miR-1193) (Fig. 6A). IGF1 protein level was obviously increased in BC cells by overexpressing IGF1 (Fig. 6B). Subsequently, rescue experiments were performed in BC cells. The repressive impacts of circCEP85 silencing on cell proliferation were reversed by miR-1193 inhibition or SLC7A5 overexpression (Fig. 6C, D). Flow cytometry analysis showed that knockdown circCEP85 increased cell apoptotic rate, but the enhanced apoptosis by silencing circCEP85 was recuperated by the co-transfection of anti-miR-1193 or IGF1 in BC cells (Fig. 6E). Additionally, sh-circCEP85-induced suppression of cell invasion ability was reversed after anti-miR-1193 or IGF1 transfection (Fig. 6F). The inhibitory influences of circCEP85 inhibition on angiogenesis capability and stemness were also relieved by the co-transfection of anti-miR-1193 or IGF1 (Fig. 6G, H). The decreased protein expression of PCNA, VEGFA, and Nanog, and the increased protein expression of cleaved caspase 3 caused by circCEP85 inhibition were also rescued by miR-1193 inhibitors or IGF1 overexpression (Fig. 6I). Besides that, the synergistic effects of IGF1 and miR-1193 were also evaluated. As shown Additional file 1: Fig. S1A, miR-1193 mimic transfection significantly led to a reduction of IGF1 expression level in BC cells, while this reduction was rescued after IGF1 vector introduction, indicating the successful and stable transfection. Thereafter, functional experiments showed that miR-1193 overexpression in BC cells suppressed cell proliferation (Additional file 1: Fig. S1B-C), induced apoptosis in cells (Additional file 1: Fig. S1D), repressed cell invasion (Additional file 1: Fig. S1E) and angiogenesis capabilities (Additional file 1: Fig. S1F) and impeded stemness property (Additional file 1: Fig. S1G), and IGF1 up-regulation could attenuate these effects mediated by miR-1193 overexpression in BC cells (Additional file 1: Fig. S1B-G). The results verified miR-1193 acted as a tumor suppressor to inhibit BC tumorigenesis via repressing IGF1. In all, circCEP85 promoted BC cell progression by regulating miR-1193/IGF1 axis.

Fig. 6
figure 6

CircCEP85 promoted BC progression via regulating miR-1193/IGF1 axis. A The knockdown efficiency of miR-1193 was detected by qRT-PCR; B The overexpression efficiency of IGF1 was assessed by western blot analysis; CI BC cells were treated with sh-NC, sh-circCEP85#1, sh-circCEP85#1 + anti-NC, sh-circCEP85#1 + anti-miR-1193, sh-circCEP85#1 + vector and sh-circCEP85#1 + IGF1, respectively; Cell proliferation was detected by CCK8 assay (C) and EDU assay (D); Cell apoptotic rate was assessed by flow cytometry analysis (E); Cell invasion ability was measured by transwell assay (F); Angiogenesis capability was evaluated by tube formation assay (G); Cell stemness was estimated by sphere formation assay (H); I The levels of PCNA, cleaved caspase 3, VEGFA and Naong proteins were detected by western blot analysis. **P < 0.01, ***P < 0.001

CircCEP85 promoted BC tumor growth in vivo

To study the function of circCEP85 on tumor growth in vivo. BT549 cells stably transfected with sh-circCEP85 or sh-NC were injected into the right flank of the mice to establish xenograft tumor models. As expected, circCEP85 inhibition effectively decreased the volume and weight of tumors (Fig. 7A, B). IHC assay showed that ki-67 and IGF1 positive cells were decreased in xenograft tumors of circCEP85 silencing group (Fig. 7C). The qRT-PCR results indicated that miR-1193 in xenograft tumors was obviously increased, while circCEP85 and IGF1 were decreased markedly (Fig. 7D). Next, western blot analysis showed that sh-circCEP85 significantly reduced IGF1, PCNA, VEGFA, and Nanog expression, while increased cleaved caspase 3 expression in xenograft tumors (Fig. 7D). These data explained that circCEP85 promoted the tumor growth of BC in vivo.

Fig. 7
figure 7

CircCEP85 promoted BC tumor growth in vivo. A Tumor growth curves in nude mice within 5 weeks; B After 5 weeks, the mice were executed and the tumors were weighed; C Relative expression of ki-67 and IGF1 in xenograft tumor tissues were measured by IHC assay; D The expression levels of circCEP85, miR-1193 and IGF1 in xenograft tumor tissues were detected by qRT-PCR; E The protein levels of IGF1, PCNA, cleaved caspase 3, VEGFA and Naong were evaluated by western blot analysis. ***P < 0.001

Discussion

BC is the leading cause of cancer-related death among women worldwide [17]. Therefore, it is very important to clarify the mechanisms related to the occurrence and development of BC and find promising therapeutic targets for BC treatment. Mounting evidence has proved that circRNAs were involved in BC progression. For example, CircFOXK2 could increase IGF2BP3 expression by sponging miR-370, thus accelerating the metastasis of BC cells [18]. Circ-MMP11 promoted lapatinib resistance of BC cells by changing ANLN expression via targeting miR-153-3p [19]. Gong et al., found that circ_0084927 was elevated in BC, and circ_0084927 inhibition could notably repress the growth and invasion of BC cells [20]. In our study, we also proved that circCEP85 expression was upregulated in BC tissues and cells, and knockdown of circCEP85 could apparently repress the proliferation, invasion, angiogenesis and stemness of BC cells and could promote the apoptosis of BC cells. Meanwhile, circCEP85 inhibition suppressed the tumor growth in vivo, and survival analysis suggested highly expressed circCEP85 was correlated with a poor survival rate in BC patients. These findings suggested that circCEP85 played a key role in BC progression.

Recent studies have shown that cytoplasmic circRNAs could bind miRNAs to regulate the expression of downstream target of miRNAs in cancers [16, 21, 22]. Our studies suggested that circCEP85 is mainly located in the cytoplasm, suggesting that circCEP85 might act as a sponge for miRNAs in BC. We predicted that miR-1193 might be a target miRNA of circCEP85. Zhang et al., reported miR-1193 was decreased in cervical cancer (CC), and overexpression of miR-1193 decelerated tumor growth and invasion of CC cells by downregulating CLDN7 expression [23]. Shen et al., found that miR-1193 was obviously downregulated and inhibited the malignancy of Jurkat human T-cell leukemia cells by regulating TM9SF3 [24]. Especially, the role of miR-1193 in BC has been reported to be lowly expressed and could act as a tumor suppressor in BC progression. Similarly, this study found the expression of miR-1193 was notably reduced in BC tissues and cells, and miR-1193 could markedly inhibit the proliferation, invasion, angiogenesis and stemness of BC cells, and elevated cell apoptotic rate. Recuse assay results showed that the inhibitory effects of circCEP85 inhibition on BC cells were restored by the co-transfection of miR-1193 inhibitor. Those findings implied that circCEP85 promoted BC progression by binding miR-1193.

The downstream gene of miR-1193 was predicted by miRDB and Targetscan, and we found that insuline-like growth factor I (IGF1) was a target gene of miR-1193. IGF-1 is a polypeptide hormone belonging to the growth factor hormone family that helps control the growth and development of organs, muscles, and tissues in the body, besides, it is also involved in regulating glucose metabolism and brain function, IGF-1 assists with natural bodily development, while abnormal accumulation of IGF-1 can negatively affect health [25]. Currently, IGF1 was found to be involved in many cancers, such as lung cancer [26], cervical cancer [27], pancreatic cancer [28], and esophageal carcinoma [29]. In breast cancer, IGF-1 gene polymorphism was associated with an increased risk for this cancer [30]. IGF-1 binding to the insulin-like growth factor receptor-type 1 (IGF-1R) can modulate cell metabolism, stimulate proliferation, and promote metastasis in BC [31,32,33,34,35]. In present study, we also demonstrated that IGF1 expression was upregulated in BC tissues and cells, and forced expression of miR-1193 could evidently repress the level of IGF1. In addition, circCEP85 could modulate IGF1 expression through sponging miR-1193 in BC cells. Meanwhile, upregulation of IGF1 could reverse the effect of circCEP85 silencing on BC cell progression. Moreover, IGF overexpression also abolished the anticancer effects of miR-1193 mimic on BC cells. These findings showed that circCEP85 accelerated BC progression largely by miR-1193/IGF1 axis, implying their synergistic effects on the breast cancer progression therapy.

Taken together, this study found that circCEP85 might act as an oncogene in BC, and promote BC progression by sponging miR-1193 to elevate IGF1 abundance. These findings indicated that circCEP85 siRNA might be a potential molecular for the targeted therapy in BC. Currently, nanocomposite materials based on functionalized metal and semiconductor nanoparticles in targeted drug delivery have received increased attention due to their abilities in enhancing cell adhesion and internalization, controlling targeted release, reinforcing biodegradation, convenient detection in the body, etc. [36, 37]. Previous researches have reported the use of nanoparticles for cancer-related applications [37,38,39,40,41]. Therefore, circCEP85 siRNA combined with nanoparticles might be a promising therapeutic strategy for BC therapy.

Table 1 Primers sequences used for PCR

Availability of data and materials

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

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Conceptualization and Methodology: JH and LJ; Formal analysis and Data curation: JH, YW and MC; Validation and Investigation: FG and JH; Writing—original draft preparation and Writing—review and editing: FG, JH, and LJ; Approval of final manuscript: all authors. All authors read and approved the final manuscript.

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Correspondence to Jianjun Han.

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The present study was approved by the ethical review committee of The Third Hospital of Mianyang (Sichuan Mental Health Center). Written informed consent was obtained from all enrolled patients.

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Supplementary Information

Additional file 1: Figure S1.

MiR-1193 suppressed proliferation, invasion, angiogenesis, stemness and induced apoptosis of BC cells by targeting IGF1. (A-G) BT-549 and MDA-MB-231 cells were transfected with miR-NC, miR-1193, miR-1193 + vector, or miR-1193 + IGF1. (A) Relative expression of IGF1 was detected by western blot in BT-549 and MDA-MB-231 cells. (B, C) The proliferation of BC cells was assessed by CCK8 and EDU assays; (D) BC cell apoptosis was assessed by flow cytometry analysis; (E) BC cell invasion was assessed by transwell assay; (F) Tube formation assay was conducted to detect HUVEC angiogenesis capability; (G) Sphere formation assay was performed to assess BC cell stemness; **P < 0.01, ***P< 0.001.

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Gao, F., Han, J., Jia, L. et al. CircCEP85 upregulates IGF1 expression to promote breast cancer progression via sponging miR-1193. Appl Biol Chem 65, 44 (2022). https://doi.org/10.1186/s13765-022-00709-7

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Keywords

  • circCEP85
  • miR-1193
  • IGF1
  • Breast cancer