Background
Breast cancer (BC) is the most commonly diagnosed malignancy and the leading cause of cancer-related deaths in women [
1]. Despite robust advances in surgical and medical management in BC, the morbidity and mortality are not decreasing [
2]. Higher rates of metastasis, recurrence and drug resistance are the significant reasons of cancer-associated deaths among breast cancer patients. Therefore, further investigating the molecular mechanism of BC remains an urgent and difficult task.
Circular RNAs (circRNAs), a novel class of endogenous non-coding RNAs (ncRNAs), are formed from exons or introns through special selective shearing [
3]. Unlike linear RNAs that are terminated with 5’caps and 3’tails, circRNAs are single-stranded covalently closed circular transcripts [
4,
5], providing them higher ability to resist to environmental degradation [
6]. Recent years, circRNAs have been proved as diagnostic biomarkers of hepatoblastoma [
7] and bladder cancer [
8] in blood or plasma according to their stability and tissue specificity [
9,
10]. Aberrant expression of circRNAs are correlated with the progression and prognosis of various cancers [
11]. Emerging evidence illustrated that circRNAs play key oncogenic or anti-cancer roles in multiple cancers, including BC. For example, circRNA_0025202 regulates tamoxifen sensitivity and tumor progression via regulating FOXO3a [
12]. CircFBXW7 inhibits the progression of TNBC through encoding a 185-aa Protein [
13]. Therefore, the potential function and molecular mechanisms of circRNAs in BC need further investigation.
Hippo signaling pathway, a highly conserved pathway, performs an important role in governing cell proliferation and apoptosis of various cancer types, especially in BC. The dysregulation and inactivation of Hippo pathway always result in cancer initiation and progression [
14]. YAP1 (yes-associated protein), a downstream gene of Hippo signaling pathway, is a significant oncogene in BC [
15,
16]. Inhibiting the activity of YAP1 could restrain vascular invasiveness of BC cells [
17]. Since the mutation, activation and overexpression of YAP1 are related to the occurrence and progression of BC, finding a new molecule that could suppress YAP1 is imperative.
In this research, we identified hsa_circ_0005273, a circRNA of Protein Tyrosine Kinase 2 (PTK2), was significantly high-expressed in BC tissues and cell lines. Importantly, hsa_circ_0005273 exerted its oncogenic role in BC via acting as a sponge of tumor suppressor miR-200a-3p to inactivate YAP1-Hippo signaling pathway in vivo and in vitro. Our findings provided new insights into the diagnosis and treatment of BC.
Materials and methods
CircRNA expression profiling analysis
Tissue samples
Tumor tissues and their adjacent normal tissues of 120 BC patients were collected from the Department of Breast and Thyroid Surgery of Shanghai Tenth People’s Hospital of Tongji University (Shanghai, China). None of the patients received any local or systemic treatment before surgery and all tissue specimens were immediately snap-frozen in liquid nitrogen until further use. Our study protocols were approved by Institutional Ethics Committees of Shanghai Tenth People’s Hospital and informed consent was obtained from all patients or their relatives. The methodology of this study adhered to the standards outlined in the Declaration of Helsinki.
Cell culture and transfection
The human BC cell lines MDA-MB-231, MCF-7, HCC-1937, SKBR3 and normal breast epithelial cell line MCF-10A were purchased from Chinese Academy of Sciences (Shanghai, China). MDA-MB-231, MCF-7, HCC-1937 and SKBR3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) with 10% Fetal Bovine Serum (FBS) (Gibco, USA), penicillin (100 units/ml) and streptomycin (100 μg/ml) (Enpromise, China). MCF-10A cells were cultured in Mammary Epithelial Basal Medium (MEBM) (Cambrex, USA). All these cells were cultured at 37 °C with 5% CO2. Small, interfering, specifically targeting human hsa_circ_0005273 (si-circ_0005273), non-specific negative control oligos (si-NC) and hsa_circ_0005273 lentiviral plasmid (lv-circ_0005273) were purchased from IBSBio (Shanghai, China). Human miR-200a-3p-mimics, non-specific negative control (miR-200a-3p-NC) and miR-200a-3p inhibitor were purchased from RiboBio (Guangzhou, China). MDA-MB-231, MCF-7 and SKBR3 cells were cultured and transfected with reagents above using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. We used DNA Midiprep Kits (Qiagen, Hilden, Germany) to prepare plasmid vectors. A lentivirus carrying si-circ_0005273 was constructed by ZORIN (Shanghai, China) and transfection procedures were performed according to the manufacturer’s instructions.
RNA extraction and RT-qPCR
Total RNA was extracted from frozen tissues and cultured cells by Trizol reagent (Invitrogen, Carlsbad, CA, USA) and the concentration and purity of RNA samples was assessed with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). cDNA was synthesized by a commercial cDNA synthesis kit (Takara Biotechnology, Dalian, China). We conducted quantitative real-time polymerase chain reaction (RT-qPCR) by using the SYBR Green PCR Kit (Takara Biotechnology, Dalian, China) and primer sequences were designed and synthesized by RiboBio (Guangzhou, China). Expression of circRNA, miRNA and mRNA were assessed by threshold cycle (CT) values and analyzed using the 2
-ΔΔCt method. Primers and siRNAs designed in this study are shown in supplemental Table
S1.
Confirming specificity for hsa_circ_0005273
PCR products amplified by primers were separated on 1% agarose gel to verify the specificity of the hsa_circ_0005273 PCR products. Sanger sequencing was performed to validate the sequence of hsa_circ_0005273.
RNase R resistance analysis of circRNAs
MDA-MB-231, MCF-7 and SKBR3 cell lines were treated with RNase R (4 U/mg, Epicenter) and incubated for 30 min at 37 °C. Then, the treated RNAs were reverse transcribed with specific primers and detected by RT-qPCR assay.
MTT assay
A density of 2000 cells per well were placed into 96-well plates. The cells were detected in accordance with the manufacturer’s instructions using MTT assay kit (Sigma, Santa Clara, CA, USA). The 490 nm optical density was detected by a microplate reader respectively at 24, 48, 72 and 96 h.
A density of 1000 cells per well were transferred into six-well plates. Cell colonies were washed twice by using cold phosphate buffered saline (PBS), fixed with 75% ethanol and stained with 0.1% crystalline purple until the colonies were visible. Then colonies were counted and photographed.
Wound healing assay
MDA-MB-231, MCF-7 and SKBR3 cells were transfected with a range of constructs as indicated in 6-well plates. When the treated cells reached about 80% confluent, a scratch was produced in the cell monolayer by drawing a 200-μl-pipette tip over the surface of each well, holding the tip perpendicular to the plate. The monolayers were washed twice with 1x PBS and cultured with DMEM medium with 2%FBS. Wound healing was observed under a light microscope and pictures were taken at 0 h, 24 h and 48 h at the same position to observe cell movement.
Migration assays
Transwell chambers (Corning, Inc., Lowell, MA, USA) were used to measure the migration ability of the cells in 24-well plates. Cells were transferred into the upper chamber with 200 μl serum-free medium and medium with 10% FBS was added to the lower chamber. 24 h later, cells in the upper chamber were carefully removed by a cotton swab. Then, the cells on the opposite side of the filter were fixed with 70% ethanol for 30 min and stained with 0.1% crystal violet for 10 min. Representative pictures were taken with a microscope (Leica Microsystems, Mannheim, Germany) and migrated cells were counted in five random fields.
Cell cycle assay
MDA-MB-231, MCF-7 and SKBR3 cells were transfected with a range of constructs in 12-well plates. Cells were collected and fixed in ice-cold ethanol for more than 4 h. Then each sample was added with 0.5 ml 0.05 mg/ml propidium iodide (PI) staining solution and incubated for 30 min at 37 °C. Flow cytometer (FACSCantoTM II, BD Biosciences) was used to analyze the cell cycle .
Dual-luciferase reporter assay
To confirm that miR-200a-3p directly targets hsa_circ_0005273 and YAP1 3′-UTR, wild and mutant reporter plasmids of hsa_circ_0005273 and YAP1 were individually designed and synthesized by IBSBio (Shanghai, China). 293 T cells were co-transected with the constructed reporter plasmids, together with miR-200a-3p mimics or miR-200a-3p-NC using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, USA). 24 h later, luciferase activities were measured by the Dual-Luciferase® Reporter Assay kit (Promega, Madison, USA). Then firefly to Renilla luciferase ratios were calculated.
RNA antisense purification (RAP)
Approximately 6 × 107 MDA-MB-231 cells were washed with ice-cold 1 × PBS, lysed in lysis buffer and incubated for 10 min at 37 °C with DNase salt stock. 100 ul high-affinity biotin RNA probe was added into RAP samples, hybridized at 37 °C for 30 min, incubated at 50 °C for 5 min, then hybridized at 37 °C for 2 h. Streptavidin beads and RAP samples were incubated for 30 min at room temperature and washed for 5 times with 500 μl washing buffer. After elution and purification, the RNA was restored by 30 μl RNase-free water analyzed by qRT-PCR. Hsa_circ_0005273 probe was designed by IBSBio (Shanghai, China) and RNA Antisense Purification (RAP) Kit was purchased from BersinBioTM (Guangzhou, China).
RNA Immunoprecipitation assay (RIP)
RIP assay was conducted in MDA-MB-231 cells using the BersinBioTM RNA-Binding Protein Immunoprecipitation Kit (Guangzhou, China) according to manufacturer’s instructions. Anti-AGO2(Abclonal, China) and anti-IgG(Abclonal, China) were used. The extracted RNAs were analyzed by RT-qPCR.
Western blotting analysis
Proteins were extracted by using RIPA lysis buffer (Beyotime, Jiangsu, China) and the concentrations were detected by using the protein assay kit (Beyotime, Jiangsu, China). Protein lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to nitrocellulose membrane (Beyotime, Jiangsu, China), which was incubated 1 h with 5% non-fat milk and immunoblotted overnight at 4 °C with primary antibodies: anti-PCNA(Proteintech, USA), anti-YAP1(Abclonal, China), anti-p-YAP1(Abclonal, China), anti-MST1(Abclonal, China), anti-p-MST1(Sigma, USA), anti-LATS1/2(Abclonal, China), anti-p-LATS1/2(Abclonal, China), anti-PTK2(Abclonal, China), anti-β-actin(Abclonal, China), and anti-LaminA(Proteintech, USA). The next day, the membranes were incubated in secondary antibodies for 1 h at room temperature. Dilutions of all antibodies used in this study were 1/1000. Signals of protein bands were scanned by Odyssey Infrared scanning system (Li-Cor, Lincoln, NE, USA).
FISH assay
Ribo™ Fluorescent In Situ Hybridization Kit (Ribo, China) was used in FISH assay. Specific probes for the hsa_circ_0005273 were designed and synthesized by RiboBio (Guangzhou, China) and specific probes for the miR-200a-3p were designed and synthesized by IBSBio (Shanghai, China). 4′,6-Diamidino-2-Phenylindole (DAPI) was used to stain cell nuclei. Fluorescence microscope (Olympus BX53 Biological Microscope) was used to capture the images of cells.
Xenograft tumor assay
Athymic nude mice (age, 4–6 weeks; weight, 18–22 g) were ordered from the laboratory animal center of Shanghai. Approximately 1 × 106 MDA-MB-231 cells with stable expression of si-circ_0005273 or si-NC were injected into the second mammary fat of the mice (n = 4, each group). Then, tumor size was measured and calculated every week using the following formula: Volume (mm3) = 0.5 * width2 * length. After 5 weeks, the mice were killed by cervical dislocation and the tumors were collected. The animal protocols complied with the rule of the ethics committee of Tongji University.
Immunohistochemistry (IHC)
Fresh tumor tissue samples from the nude mice were fixed in 4% paraformaldehyde, hydrated through ethanol solution and embedded in paraffin. The paraffin-embedded tissue was sectioned into 4 μm slides, then the sections were incubated with anti-YAP1, anti-MST1 and anti-LATS1/2 antibodies to measure YAP1, MST1 and LATS1/2 expression. Images were captured under a microscope (Leica Microsystems, Mannheim, Germany) at the appropriate magnification.
Statistical analysis
The significance of differences between groups was assessed by GraphPad Prism V8.0 (GraphPad, CA, USA) and SPSS 20.0 (IBM, SPSS, IL, USA). Comparisons between groups were analyzed with the Student’s t test. Data were obtained from three independent experiments which are presented as the means ± standard deviation (SD) and a P-value < 0.05 was considered significant.
Discussion
The past few decades have witnessed the rapid research progress of circRNAs and a number of circRNAs have been confirmed to participate in the tumorigenesis of multiple cancers, especially in BC. CircRNAs have covalently closed ring structure with no 5′ caps and 3′ ploy-A tails, thus they are stable and insensitive to RNase R [
19]. Considerable quantities of circRNAs are located in the cytoplasm and few are found in the nucleus [
20]. Considering the stability, long half-life period of circRNAs as well as tissue-specific expression, circRNAs have distinct advantages to act as potential biomarkers of cancer diagnosis and prognosis [
21].
Through analyzing GEO database and our experiment results, we identified a novel circRNA, hsa_circ_0005273, was remarkably upregulated in BC tissues. More importantly, high expression hsa_circ_0005273 was positively associated with tumor size, TNM stage, lymph node metastasis and distant metastasis in BC patients. Knocking down hsa_circ_0005273 could inhibit the proliferation, migration and cell cycle of BC cells as well as the growth of tumors in vivo, indicating its promotive role in BC tumorigenesis.
Previous studies have demonstrated various physiological functions of circRNAs, such as circRNAs serving as competing endogenous RNAs (ceRNAs) or natural miRNA sponges [
22,
23], interacting with proteins [
24‐
26], regulating gene transcription [
20,
27] and translation [
28‐
31]. Normally, the function of circRNAs are related to their subcellular localization. CircRNAs enriched in cytoplasm contain a large number of miRNA response elements MER (miRNA response element), thus allows them to act as miRNA sponges and reverse the inhibitory effect of miRNA on its target genes [
3,
32]. Through FISH assay and subcellular fractionation, we confirmed that hsa_circ_0005273 was mainly located in cytoplasm of BC cells. Therefore, we effort to explore the molecular mechanism of hsa_circ_0005273 as a miRNA sponge. RIP assay was first performed to verify that hsa_circ_0005273 could target miRNAs in an AGO2 manner. Then, dual luciferase reporter assays, RNA pull-down analysis and FISH study were conducted to demonstrated the interaction between hsa_circ_0005273, miR-200a-3p and YAP1. Additionally, we observed YAP1 was positively correlated with hsa_circ_0005273 expression in both mRNA and protein levels. Eventually, rescue assays further verified hsa_circ_0005273 could regulate YAP1 via targeting miR-200a-3p.
As a tumor suppressor pathway, inactivation of Hippo signaling pathway could culminate epithelial-to-mesenchymal transition (EMT), cancer stem cell generation and therapeutic resistance [
18]. YAP1, primarily as the downstream gene of Hippo signaling pathway, was demonstrated as an important oncogene in a considerable number of cancer types. Normally, upstream factors of the Hippo pathway, such as MST1/2 and LATS1/2, operate an inhibitory phosphorylation of YAP1, thereby inhibiting it nuclear translocation and YAP-mediated transcription [
33,
34]. When the Hippo signaling pathway is inactivated, YAP1 translocate to the nucleus where they interact with TEADs, inducing target gene expression [
35‐
37]. Considering YAP1 as a critical factor in Hippo pathway, we hypothesized that hsa_circ_0005273 could inactivate Hippo signaling pathway via targeting YAP1. Thereafter, we verified that LATS1/2 and MST1expression were negative related to hsa_circ_0005273. In addition, the protein and mRNA levels of LATS1/2, MST1 and their phosphorylation status were negatively regulated by hsa_circ_0005273. More importantly, hsa_circ_0005273 promoted YAP1 translocate to the nucleus, which is a key evidence of Hippo pathway inactivation [
18].
This study is first to suggest that the hsa_circ_0005273/miR-200a-3p/YAP1/Hippo pathway may play a key role in the progression of BC and provide a novel strategy for inhibiting YAP1.
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