Introduction
Breast cancer (BC) is the most commonly diagnosed cancer among females, and it is the leading cause of cancer death [
1]. In 2018, about 2.1 million new cases of BC were diagnosed, accounting for a quarter of all cancer cases among women [
2]. Although many treatments for BC, including radical surgery and adjuvant therapy, the prognosis for BC remains poor [
3]. Hence, it is significant to better understand the molecular mechanisms of BC and develop more effective therapeutic strategies for treatment BC.
Circular RNAs (circRNAs) are a special type of non-coding RNAs (ncRNAs) that are widely expressed in mammals [
4]. CircRNAs are characterized by covalently closed-loop structures with neither 5’ caps nor 3’ poly (A) tails, and circRNAs are conserved across species because of their resistance to RNase R (unlike lncRNA) [
5]. Many reports have shown that circRNAs are involved in the regulation of gene transcription, suggesting that circRNAs play essential roles in multiple diseases, including cancer [
6,
7]. Some circRNAs have been shown to play vital roles in regulation of BC progression [
8‐
10]. Besides, a previous study indicated that circRNA polo-like kinase-1 (circPLK1; hsa_circ_0038632, chr16:23691404-23701688) was upregulated in triple-negative breast cancer (TNBC) and tightly related to poor survivals [
11]. However, more roles and molecular mechanisms of circPLK1 in BC still need to be further explored.
Previous studies have indicated that circRNAs participate in many physiological and pathological processes by acting as competitive endogenous RNAs (ceRNAs) or microRNA (miRNA) sponges to regulate target genes and protein expression [
12]. Hence, the circRNA-miRNA-mRNA axis may play a crucial role in the regulation of BC progression. A previous study showed that high level of insulin-like growth factor 1 (IGF1) was positively associated with bad prognosis in BC patients [
13]. Moreover, miR-4500 has been identified to serve as an anti-oncogene in BC [
14]. Bioinformatics tool predicted the putative binding sites between miR-4500 and circPLK1 or IGF1. Therefore, we supposed that circPLK1 might regulate BC progression through functioning as a sponge for miR-4500 to affect IGF1 expression.
In this work, we examined the expression of circPLK1, miR-4500 and IGF1 in BC tissues and cells, and explored their effects on BC cell growth, migration and invasion. Additionally, the potential regulatory mechanism among them in BC progression was also explored. The purpose of this research was to identify promising therapeutic targets for BC treatment.
Materials and methods
Tissue collection
In this research, BC tissues (n = 35) and adjacent non-tumor tissues (n = 35) were provided by the patients who had undergone surgery at Liaoning Provincial Cancer Hospital. All samples were timely frozen in liquid nitrogen and then maintained in −80 ℃. All enrolled patients signed the informed consents. The present study was approved by the Research Ethics Committee of Liaoning Provincial Cancer Hospital.
Cell culture and transfection
Two BC cells (BT549 and HCC38) and breast epithelial cells (MCF-10A) were purchased from COBIOER (Nanjing, China). These cells were maintained in Dulbecco’s modified eagle medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Invitrogen). Then these cells were cultivated at 37 ℃ under a humidified atmosphere with 5% CO2.
For transfection, short-hairpin RNA (shRNA) against circPLK1 (sh-circPLK1), circPLK1 overexpression vector (circPLK1), pcDNA3.0-IGF1 overexpression plasmid (pcDNA3.0-IGF1), miR-4500 mimic (miR-4500), miR-4500 inhibitor (anti-miR-4500), and negative controls (sh-NC, Vector, pcDNA3.0-NC, miR-NC, and anti-NC) were obtained from RiboBio (Guangzhou, China). Transient transfection was performed using the Lipofectamine 3000 reagent (Invitrogen).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA from tissues (tumor and normal) and cells (BT549, HCC38 and MCF-10A) was extracted using TRIzol reagent (Invitrogen). High-Capacity cDNA Reverse Transcription Kit and TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) were applied to synthesize complementary DNA (cDNA). Next, qRT-PCR was conducted in a 7500 Real-Time PCR System (Thermo Fisher Scientific) with PrimeScript RT Reagent Kit (Takara, Shiga, Japan). The sequences of primers used in this research were listed as followed: circPLK1 (Forward, 5′-ACTATGGTGGACAAGCTGCT-3′; Reverse, 5′-GGAGGGCAGCTATTAGGAGG-3′); PLK1 (Forward, 5′-GGCAACCTTTTCCTGAATGA-3′; Reverse, 5′-AATGGACCACACATCCACCT-3′); miR-4500 (Forward, 5′-TGAGGTAGTAGTTTCTTGCGCC-3′; Reverse, 5′-CTCTACAGCTATATTGCCAGCCAC-3′); IGF1 (Forward, 5′-GCTCTTCAGTTCGTGTGTGGA-3′; Reverse, 5′-CGACTGCTGGAGCCATACC-3′); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Forward, 5′-GGCTGAGAACGGGAAGCTTGTCA-3′; Reverse, 5′-CGGCCATCACGCCACAGTTTC-3′); U6 (Forward, 5′-ATTGGAACGATACAGAGAAGATT-3′; Reverse, 5′-GGAACGCTTCACGAATTTG-3′). The expression of genes was normalized to GAPDH (for circPLK1, PLK1 and IGF1) or U6 (for miR-4500), and calculated with 2
−ΔΔCt method [
15].
RNase R treatment
RNase R (Epicentre Technologies, Madison, WI, USA) treatment was used to degrade linear RNA. In short, total RNA (2 µg) was incubated by RNase R (3 U/µg) for 0.5 h at 37 ℃. Next, the treated cells were harvested, and the corresponding levels were measured by qRT-PCR.
Cell viability assay
Cell Counting Kit-8 (CCK-8; Boster, Wuhan, China) was employed to examine BC cell viability [
16]. In brief, BT549 and HCC38 cells were seeded in 96-well plates and subjected to different treatments. After 48 h, CCK-8 solution (10 µL) was added to per well at 37 ℃ for 2–3 h. A microplate reader was utilized to examine the absorbance at 450 nm wavelength.
Cell cycle assay
BT549 and HCC38 cells were seeded in 6-well plates and subjected to different treatments. After 48 h, BT549 and HCC38 cells were collected and fixed with ethanol (75%) overnight. Afterwards, cells were incubated with propidium iodide (PI; 25 µg/mL, Keygen, Nanjing, China), Triton X-100 (0.2%) and DNase-free RNase (20 µg/mL). After incubation for 15 min in the dark, flow cytometry (Partec AG, Arlesheim, Switzerland) was used for detecting cell cycle distribution.
Western blot assay
Total protein was extracted using RIPA lysis buffer (Solarbio, Beijing, China). The protein concentration was evaluated by BCA protein assay kit (Solarbio), and then extracted protein samples (about 40 µg/lane) were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto polyvinylidene fluoride membranes, and then blocked with 5% nonfat milk for 1–2 h. These membranes were incubated by the primary antibodies (Abcam, Cambridge, UK), including cyclin-dependent kinase (CDK) 4 (1:2000, ab137675), CDK6 (1:2000, ab151247), IGF1 (1:1000, ab9572) and GAPDH (1:1000, ab37168). After that, membranes were then continuously incubated with secondary antibody (1:4000, ab205718, Abcam). After incubation for 2 h, immunoreactive bands were visualized by enhanced chemiluminescence reagent (Solarbio). ImageJ software was employed to assess the bands density [
17].
Transwell assay
For cell invasion assay, the upper chambers (Costar, Corning, NY, USA) were pre-coated by Matrigel (BD Biosciences, San Jose, CA, USA). Transfected cells (BT549 and HCC38) in 200 µL of serum-free medium (DMEM) were added to the top compartment of the chamber. At the same time, the bottom chambers were filled with complete medium (600 µL). Cells which had invaded into the lower chamber were then stained and photographed under a microscope at 100 × magnification. For cell migration, the steps were similar to cell invasion method except that the top chambers were not pre-coated with Matrigel.
Dual-luciferase reporter assay
The potential complementary sequence of miR-4500 and circPLK1 or IGF1 was predicted using starBase v3.0. The wild-type sequences of circPLK1 or IGF1 containing the binding sites of miR-4500 were amplified and individually inserted into the pmirGlO luciferase reporter vector (Promega, Madison, WI, USA), thereby generating wild-type plasmids (wt-circPLK1 and wt-IGF1). Similarly, the mutant sequences were also designed and inserted into the pmirGLO vector to generate mutant-type plasmids (mut-circPLK1 and mut-IGF1). Briefly, the above reporters were co-transfected with miR-NC or miR-4500 into BT549 and HCC38 cells for 48 h. After that, dual-Luciferase Reporter Assay System (Promega) was employed for measuring the luciferase activity.
RNA pull-down assay
RNA pull-down assay was carried out in BT549 and HCC38 cells using RNA-Protein Pull-Down Kit (Thermo Fisher Scientific). Briefly, miR-4500 and miR-NC were labeled with biotin and transfected into BT549 and HCC38 cells, respectively. Subsequently, streptavidin agarose beads were used to incubate cell lysates for 1 h. At last, qRT-PCR was employed for examining the levels of circPLK1 and IGF1.
Xenograft tumor model
Female BALB/c nude mice (5–6 weeks old) were obtained from Huafukang (Beijing, China). Stably transfected HCC38 cells (sh-NC or sh-circPLK1) were injected subcutaneously into nude mice (n = 6/group). Tumor volume was detected at the indicated times and calculated as follows: volume = (length × width2)/2. After five weeks, mice were killed, and the formed tumors were excised, weighed and collected for further study. This in vivo experiments were approved by the Animal Care and Use Committee of Liaoning Provincial Cancer Hospital.
Statistical analysis
All data from at least 3 independent experiments were presented as mean ± standard deviation (SD). GraphPad Prism was used for statistical analysis. Student’s t-test or a one-way analysis of variance (ANOVA) was applied to analyze significant differences between different groups. P value < 0.05 was considered as significant difference. ***P < 0.001.
Discussion
BC is one of the most common and aggressive cancers among women, causing a large number of deaths [
2]. Recent reports have demonstrated that circRNAs participate in a variety of cellular physiobiological processes and play vital roles in regulation of gene expression [
19]. In the current research, we found that circPLK1 silence suppressed cell growth, migration and invasion by regulation of miR-4500/IGF1 pathway in BC cells.
Many reports have shown that circRNAs are considered as promising biomarkers for the diagnosis and prognosis of various cancers due to their abundance and stability in plasma and tissues [
20]. Recent studies have confirmed that dysregulation of circRNAs is strongly linked to the occurrence and development of cancers, including BC. For example, Liang et al. demonstrated that circ-ABCB10 was upregulated in BC tissues, and its knockdown repressed the proliferation and accelerated cell apoptosis of BC cells [
21]. Besides, Liu et al. revealed that hsa_circ_0008039 downregulation inhibited BC cell proliferation and migration via sponging miR-432-5p and inhibiting E2F3 expression [
22]. More importantly, Kong et al. reported that circPLK1 was overexpressed in TNBC, and circPLK1 knockdown inhibited TNBC cell growth and invasion by regulating miR-296-5p/PLK1 axis [
11]. In our research, high expression of circPLK1 was also observed in BC tissues and cells (Fig.
1), and its deficiency inhibited BC cell viability, arrested cell cycle progression, and repressed metastasis (Fig.
2). Likewise, circPLK1 knockdown led to the inhibition of tumor growth
in vivo (Fig.
8). These findings disclosed that circPLK1 played a pivotal role in BC progression, and circPLK1 might be a promising prognostic biomarker and therapeutic target for BC.
IGF1, a tyrosine kinase receptor, has a significant influence on the control of cell and body size [
23]. IGF1 has been acknowledged to be involved in diverse pathological processes, including cancer [
24]. IGF1 was considered to be a key factor in the carcinogenesis of some tumors, including colon cancer [
25], esophageal cancer [
26], and lung cancer [
27]. Notably, Walsh and his colleague pointed out that IGF1 increased invasive potential of MCF 7 breast cancer cells [
28]. Nevertheless, the roles of IGF1 in BC cell growth and metastasis remain largely unknown. In this work, we uncovered that the expression of IGF1 was enhanced in BC tissues and cells (Fig.
3). Next, we explored whether IGF1 participated in circPLK1-mediated functions in BC cells. Rescue experiments showed that overexpression of IGF1 abated the suppressing effects of circPLK1 deficiency on cell growth, migration and invasion (Fig.
4). Our data proved that circPLK1 exerted its biological functions by regulating IGF1 expression.
An increasing number of studies have suggested that circRNA can serve as miRNA sponge to affect target gene expression [
29,
30]. To explore the functional mechanism of circPLK1, the potential target miRNAs of circPLK1 were predicted by starBase v3.0. The data showed that miR-4500 might interact with circPLK1. Through dual-luciferase reporter and RNA pull-down assays, we validated the association between miR-4500 and circPLK1. Increasing evidence has confirmed that miRNAs exert their functional effects through binding to 3’UTR of target mRNAs [
31]. So, potential target of miR-4500 was predicted by starBase v3.0. Interestingly, IGF1 was predicted as target for miR-4500. This prediction was confirmed by dual-luciferase reporter and RNA pull-down assays. Previous studies indicated that miR-4500 could inhibit the progression of many caners, including lung cancer [
32], colorectal cancer [
33], and papillary thyroid cancer [
34]. Similarly, Li et al. proved that miR-4500 expression was declined in BC cells, and miR-4500 inhibited BC progression by downregulating RRM2 and inhibiting MAPK signaling pathway [
14]. Consistent with this research, we also observed that miR-4500 was lowly expressed in BC (Fig.
5), and miR-4500 exerted anti-tumor roles via targeting IGF1 (Fig.
6). Besides, our study indicated that IGF1 expression was regulated by circPLK1/miR-4500 axis in BC (Fig.
7).
In conclusion, circPLK1 and IGF1 were highly expressed and miR-4500 was lowly expressed in BC. Moreover, our study for the first time proved that circPLK1 downregulation suppressed BC cell growth, migration and invasion via regulating miR-4500/IGF1 axis, which provided a new mechanism for better understanding the pathogenesis of BC.
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