Background
Thyroid cancer (TC) is the most prevalent cancer in the endocrine system whose incidence and morbidity are steadily growing around the world [
1]. About 83% patients with TC belong to papillary thyroid carcinoma (PTC) [
2]. The risk factors of PTC include genetic mutation and environmental exposure [
3]. Most PTC patients at the early stage show a favorable prognosis after treatment with thyroidectomy and radioactive iodine [
4]. However, the recurrence is greatly increased if metastasis exists [
5]. Hence, it is still important to understand the molecular mechanism of PTC progression. Furthermore, developing novel therapeutic approach is urgently required.
Circular RNA (circRNA), together with microRNA (miRNA) and long noncoding RNA (lncRNA) are most explored noncoding RNAs (ncRNAs) in the past years [
6]. CircRNA is characterized by a covalently closed loop and has no capacity to code protein [
7]. Recent researches have proven that circRNA participates in various physiological and pathological processes [
8,
9]. Especially, the roles of circRNA in tumorigenesis are widely investigated recently. For example, Chen et al. predicted that hsa_circ_0032462, hsa_circ_0028173 or hsa_circ_0005909 promotes osteosarcoma progression through regulating CADM1 expression [
10]. Zeng et al. showed that circ-VANGL1 promotes bladder cancer cell proliferation, migration and invasion via miR-605-3p/VANGL1 axis [
11]. Song et al. reported that hsa_circ_0007534 silencing inhibits breast cancer growth and metastasis through modulating miR-593/MUC19 pathway [
12]. In PTC, the study about circRNAs has just begun. Only a few circRNAs have been investigated, such as circZFR [
13] and circ-ITCH [
14]. Thus, it is essential to reveal the association between circRNA expression and PTC development.
CircRNA_102171, with a length of 309 nucleotides, is derived from back-splicing of SMURF2 mRNA. The function of circRNA_102171 is unknown. In this study, we aimed to reveal its function and molecular mechanism in PTC progression. We showed that circRNA_102171 expression was elevated in PTC tissues. Functionally, circRNA_102171 promotes PTC cell proliferation, migration and invasion while suppressing apoptosis. Mechanistically, circRNA_102171 interacts with CTNNBIP1 and blocks its association with the β-catenin/TCF complex, leading to activation of Wnt/β-catenin pathway. Our study provides a novel signaling pathway involved in PTC progression.
Methods
Patients and tissue specimens
Forty-seven pairs of PTC tissues and normal tissues were obtained from The Third Affiliated Hospital of Harbin Medical University. This study was approved by the ethics committee of The Third Affiliated Hospital of Harbin Medical University. All written informed consents were acquired from patients. All tissues received no radiotherapy or chemotherapy before surgery and immediately stored in liquid nitrogen after surgery.
Cell culture and transfection
PTC cell lines (TPC-1, NPA87 and KAT-5) and normal cell line Nthy-ori3–1 were from the Chinese Academy of Sciences (Shanghai, China). Cell lines were cultured in DMEM (Gibco, Carlsbad, CA, USA) containing 10% FBS (Gibco) and maintained in a humidified incubator at an atmosphere with 5% CO2.
Small interfering RNA (siRNA) against circRNA_102171 (si-circ; 5′-AGAGGACAGATAGTAGGACTT-3′) was designed using the circinteractome tool (
https://circinteractome.nia.nih.gov/bin/) and bought from RiboBio Co., Ltd. (Guangzhou, China). siRNAs (50 nM) were transfected into TPC-1 and KAT-5 cells using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR)
Total RNAs were isolated from PTC tissues and cell lines using TRIzol reagent following the manufacturer’s instructions. qRT-PCR were carried out according to a previous study [
15]. All primers acquired from Sangon Biotech (Shanghai, China). GAPDH or U6 works as internal control.
Cell viability assay
Cell viability was measured using the cell counting kit-8 (CCK-8, Sigma-Aldrich) following the manufacturer’s instructions. In brief, 2000 cells per well were seeded in 96-well plates and cultured at described days. Then CCK8 solution were added and absorbance at 450 nm were determined through a SpectraMax microtiter plate reader (Molecular Devices, Carlsbad, CA, USA).
One thousand PTC cells per well were seeded into 6-well plates and cultured for 14 days. Then cells were fixed with methanol and stained with 0.5% crystal violet. Colonies were then counted using a Nikon Eclipse E600 microscope (Nikon Instruments, Melville, NY).
Cell apoptosis assay
Cell apoptosis were measured using an Annexin V-FITC Apoptosis Staining/Detection kit (Cambridge, MA) according to the manufacturer’s instructions. The samples were analyzed with a FACScan flow cytometer (BD Biosciences, San Jose, CA).
Transwell assay
Cell migration and invasion was analyzed using the transwell chamber (8 μ pore size; Corning Incorporated, Corning, NY, USA). In brief, 2 × 104 PTC cells in 200 μl serum-free DMEM were seeded into the upper chamber pre-coated with Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA) for invasion analysis. The lower chamber was filled with 600 μm complete DMEM medium. After incubation for 48 h, the cells in the upper chamber were removed. And cells in the down chamber were fixed and stained with 0.5% crystal violet (Beyotime Institute of Biotechnology, Jiangsu, China). Cell number was counted using a light microscope.
RNA IP
RNA IP was utilized to determine endogenous interaction between circRNA_102171 and CTNNBIP1. In brief, PTC cell lysates were incubated with anti-CTNNBIP1 for 4 h at 4 °C. Then beads were added and incubated for another 2 h. The precipitated RNAs were eluted and analyzed by qRT-PCR.
Animal study
Xenograft assay was used to analyze the role of circRNA_102171 in vivo. Animal assay was approved by the ethics committee of The Third Affiliated Hospital of Harbin Medical University. In brief, 1 × 107 shcircRNA_102171 or control TPC-1 cells were subcutaneously injected into the flanks of nude mice from Charles River (Beijing, China). At described times, tumor volumes were measured. 25 days later, tumor weights were analyzed.
RNA electrophoretic mobility shift assays (RNA-EMSA)
RNA-EMSA was performed using the LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific) according to a previous study [
16].
Statistical analysis
Statistical analysis was completed using SPSS software (version 22.0, SPSS Inc., Armonk, NY, USA). Statistical significance was measured using Student’s t-test or the ANOVA test. Each result from at least three independent experiments was displayed as mean ± standard deviation (SD). p < 0.05 was considered as statistical significance.
Discussion
As the most common cancer in endocrine system, PTC has developed into a challenge for public health. Sequencing studies have revealed many differentially expressed circRNAs in PTC tissues compared to paired normal tissues [
18]. However, the roles of circRNAs in PTC remain largely unknown. Therefore, our study investigated the role of circRNA_102171. We demonstrated circRNA_102171 promotes PTC cell proliferation, migration and invasion while inhibiting apoptosis.
CircRNAs are often aberrantly expressed in cancer tissues and have been reported to regulate various human cancers, including breast cancer [
12], bladder cancer [
19], liver cancer [
20], non-small cell lung cancer [
8], oral squamous cell carcinoma [
21] and cervical cancer [
22]. For instance, Chen et al. reported that circ-ANAPC7 overexpression promotes acute myeloid leukemia development through targeting miR-181 family [
23]. Yuan et al. showed that circ_0026344 upregulation predicts favorable prognosis and inhibits colorectal cancer development by targeting miR-21 and miR-31 [
24]. Importantly, Wei et al. showed that circZFR promotes proliferation and metastasis of PTC cells through miR-1261/C8orf4 axis [
13]. Wang et al. found that circ-ITCH inhibits PTC development by regulating miR-22-3p/CBL/β-catenin cascade [
14]. Nevertheless, how circRNA_102171 functions in PTC remains unclear. Our study showed that circRNA_102171 level was elevated in PTC tissues. Through CCK8, colony formation and EdU incorporation assays, we demonstrated that circRNA_102171 promotes PTC cell proliferation, migration and invasion. Our study demonstrated that circRNA_102171 works as an oncogenic role in PTC, suggesting circRNA_102171 might be a potential therapeutic target.
Wnt/β-catenin signaling is a classical pathway involved in tumorigenesis. Aberrantly activation of Wnt/β-catenin pathway usually contributes to development of various cancers, such as gastric cancer [
25], ovarian carcinoma [
26], bladder cancer [
27] and colorectal cancer [
28]. Wnt/β-catenin is also reported to be important in PTC progression [
14]. Wnt/β-catenin pathway is elaborately regulated by multiple factors in cancer. After association with TCF protein, β-catenin/TCF complex recognizes specific elements and initiates transcription [
17]. Many molecular mechanisms are implicated in the regulatory process of Wnt/β-catenin pathway. For instance, APC degradation complex could promote the degradation of β-catenin, leading to inactivation of Wnt/β-catenin pathway [
29]. lnc-β-Catm enhances the stability of β-catenin through promoting β-catenin methylation [
30]. In our study, we reported a novel regulatory manner of Wnt/β-catenin activation. Through unbiased RNA-pulldown and MS identification, we screened that circRNA_102171 might interact with CTNNBIP1. According to RNA IP and pulldown assays, we demonstrated their interaction. Moreover, RNA-EMSA assay further demonstrated their direct interaction in PTC cells. CTNNBIP1 is β-catenin interacting protein. Evidences showed that CTNNBIP1 interacts with β-catenin to negatively regulate Wnt/β-catenin pathway by preventing formation of β-catenin/TCF complex [
17]. The mechanism regulating the interaction between CTNNBIP1 and β-catenin remains vague. Our results showed that circRNA_102171 interacts with CTNNBIP1 and impaired the formation of CTNNBIP1/ β-catenin complex. Consequently, circRNA_102171 promotes the association of β-catenin with TCF proteins and activation of Wnt/β-catenin pathway.
Previous researches showed that CTNNBIP1 negatively regulates cancer progression [
17,
31]. For example, CTNNBIP1 downregulation promotes progression of lung adenocarcinomas [
32]. CTNNBIP1 upregulation inhibits glioma cell proliferation [
33]. Whether CTNNBIP1 possess a similar role in PTC remains unknown. Through TCGA database and qRT-PCR analysis, we showed that CTNNBIP1 level was significantly downregulated in PTC tissues, suggesting a potential anti-tumor role. Furthermore, gain-of-function assays demonstrated that CTNNBIP1 overexpression suppressed the proliferation, migration and invasion of PTC cells. Our study reveals CTNNBIP1 is a tumor suppressor in PTC.
Acknowledgements
The authors thank all patients involved in this study.