Introduction
Gastric cancer is known as the third most common malignancy worldwide with an estimated over 782,000 deaths in 2018 [
1]. Owing to the lack of obvious symptoms and screening programs, most patients were found with advanced gastric cancer during the first diagnosis [
2]. Although the therapeutic effects of GC have been improved so far, the prognosis of patients with GC remained largely unsatisfactory [
3]. Therefore, further exploration of the molecular mechanisms underlying tumorigenesis and progression of GC is in urgent need to develop more efficient therapeutic strategies.
CircRNAs are a rediscovered class of endogenous non-coding RNAs within eukaryotes, which are typically comprised of exons and characterized by a covalently closed continuous loop and a canonical splicing junction site [
4,
5]. Traditionally, they were regarded as meaningless by-products of processes in gene rearrangements and splicing [
6]. Nevertheless, due to advances in high-throughput sequencing technologies and bioinformatics analysis, emerging studies have revealed circRNAs are generally stable, conserved and low-expressed and varies with different cell types and tissue types in expression level. Also, circRNAs are of multiple functions, including acting as miRNA sponges, binding to RNA-binding protein (RBP) and involving in protein translation [
7‐
10]. In addition, circRNAs could participate in the initiation and development of multiple cancers and exert various influences on biological processes, including proliferation, invasion and metastasis [
11,
12]. For example, circNHSL1 served as an oncogenic circRNA in progression and metastasis of gastric cancer through the circNHSL1/miR-1306-3p/SIX1/Vimentin regulatory gene network [
11]; circDONSON promoted progression and development of GC cells via the NURF complex-dependent activation of SOX4 signaling [
12]. However, the roles and functions of circRNAs in GC have not been fully elucidated.
MicroRNAs (miRNAs) are a class of pervasive, conserved small non-coding RNAs that act as negative gene regulators to repress the expression of target genes [
13]. They involve a wide range of biological processes in cancers, such as proliferation, invasion, differentiation and apoptosis [
14,
15]. Recent studies have reported that circRNAs could act as miRNA sponges by competitively binding with miRNA response elements (MREs) to inhibit their expression and function [
16,
17]. For example, circMTO1 acts as a sponge of miR-9 to suppress the progression of hepatocellular cancer [
18]. However, little is known about the circRNA-miRNA regulatory network in the progression of GC.
Caveolin-1 (CAV1) acts as a major scaffold protein of caveolae, which is omega-shaped vesicular invaginations of cell membrane [
19]. Various classes of signaling molecules localized in the caveolae are negatively regulated by CAV1 through its “caveolin-scaffolding domain” (CSD) [
20]. Previous studies demonstrated that CAV1 could recruit β-catenin to caveolae membranes. Then, recruited β-catenin could combine with E-cadherin to form cadherin-catenin complexes, which could promote cell adhesion and inhibit tumor metastasis [
21‐
23]. Meanwhile, CAV1 could block the EGF-induced β-catenin-mediated transactivation and repress the transcription of cyclinD1, thus suppressing the cell cycle progression and tumor proliferation [
24‐
26]. Moreover, results from our early studies revealed that CAV1 acted as a critical role in the progression and metastasis of pancreatic cancer [
27,
28] and gastric cancer [
29,
30]. However, the upstream regulation mechanism of CAV1 is still vague.
Here, based on the results of RNA-seq, we first identified a novel GC-related circRNA termed circCCDC9 derived from exons 6 and 7 of the CCDC9 gene with a circBase ID of hsa_circ_0000944. Subsequently, to obtain insights into the function and underlying molecular mechanism of circCCDC9 in the development and progression of GC, the clinical significance of circCCDC9 was explored in GC tissues. CircCCDC9 was down-regulated in GC tissues and acted as a sponge of miR-6792-3p to affect the expression of CAV1, and eventually modulated the tumorigenesis and progression of GC cells. Collectively, our findings have provided insights into the prognosis prediction and therapeutic target of GC.
Methods
Patient tissue samples and cell lines
There were two groups of gastric cancer and paired adjacent normal tissues in our study. One group contained fresh-frozen GC tissues and corresponding adjacent normal gastric epithelial tissues, which were obtained from 48 patients who underwent gastrectomy at Shanghai General Hospital between 2015 and 2018 in accordance with the Helsinki Declaration. After surgical resection, all specimens were frozen in liquid nitrogen and stably stored at − 80 °C until RNA extraction. The other group included 57 paired gastric cancer and matched normal tissues from 2013 to 2014, which were collected from patients who had underwent primary surgical treatment for GC in Shanghai General Hospital and immediately fixed with formalin. Then, these samples were embedded in paraffin to construct tissue microarray (TMA) and the final TMA contained 54 paired gastric cancer samples. All clinicopathological diagnoses were confirmed by two pathologists according to the 8th edition of the American Joint Commission on Cancer (AJCC) and the Union for International Cancer Control (UICC). The study was approved by the Ethics Committee of Shanghai General Hospital and informed consents were obtained from all patients.
The normal human gastric epithelial mucosa cell line (GES-1), and human GC cell lines (AGS, BGC-823, HGC-27, MGC-803, MKN-28, MKN-45, SGC-7901) were all purchased from the Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cells were maintained and stored following the instructions obtained from their providers. Briefly, AGS, BGC-823, HGC-27, MGC-803, MKN-28, MKN-45 and SGC-7901 were maintained in RPMI-1640 medium (Gibco, USA) and HEK-293 T cell was maintained in DMEM medium (Gibco, USA), respectively. Both medium were supplemented with 10% fetal bovine serum (Gibco, USA), 1% penicillin and streptomycin (Gibco, USA). All cell lines were cultured in a humidified incubator containing 5% CO2 at 37 °C.
RNA extraction and quantitative real-time polymerase reaction (qRT-PCR)
The total RNAs of the GC tissues/cell lines and corresponding normal tissues/cell lines were extracted using TRIzol Reagent (Takara, Japan) and genomic DNAs (gDNA) of those were isolated with FastPure DNA Isolation (Vazyme, China), according to the manufacturer’s instructions. The concentration and purity of RNA samples were measured by Nanodrop 2000 (Thermo Fisher Scientific, USA). For circRNA and mRNA, reverse transcriptions were performed using the PrimeScript RT Master Mix (Takara, Japan) with random primers. For miRNA, reverse transcriptions were performed using the PrimeScript RT Reagent Kit (Takara, Japan) with specific stem-loop primers. And cDNA amplification was performed using TB Green Premix Ex Taq II (Takara, Japan) with an ABI Prism 7500 sequence detection system (Applied Biosystems, USA). GAPDH and U6 were used as internal control, and each sample was repeated three times. Relative quantification of circRNA, miRNA and mRNA expression was compared to internal control and analyzed using the 2
-ΔΔCT method. Divergent primers were used to detect backsplice junction of circRNA and convergent primers were used to detect linear mRNA. The primers were listed in Additional file
1: Table S1.
Nucleic acid electrophoresis
The cDNA and gDNA PCR products were investigated using 2% agarose gel electrophoresis with TAE running buffer. DNA was separated by electrophoresis at 100 V for 30 min. The DNA marker was Marker L (50-500 bp) (Sango Biotech, China). The bands were examined by UV irradiation.
RNase R treatment
The RNAs (10 μg) from AGS and MKN-45 cells were treated with RNase R (3 U/μg, Epicenter) and incubated for 30 min at 37 °C. Then, the treated RNAs were reverse transcribed with divergent primer or convergent primer and detected by qRT-PCR assay and PCR assay followed by nucleic acid electrophoresis.
Oligonucleotide transfection
MKN-45 and AGS cells were seeded in 6-well plates and cultured to 60–70% confluence before transfection. Si-circCCDC9, miR-6792-3p mimics, miR-6792-3p inhibitor and negative control oligonucleotides were designed and synthesized by GenePharma (Shanghai, China). Lipofectamine™2000 reagent (Invitrogen, USA) was used as transfection medium according to the manufacturer’s protocol. Oligonucleotide sequences were listed in Additional file
1: Table S2.
Vector construction and stable transfection
To construct stable overexpression vector of circCCDC9, the full-length circCCDC9 cDNA was synthesized and cloned into overexpression vector pLCDH-ciR, which contained a front and back circular frame to promote RNA circularization. The empty vector with no circCCDC9 sequence was used as negative control. Stable transfection in AGS cells was performed as previously shown [
31].
Luciferase reporter assay
The sequences of circCCDC9 and CAV1–3’UTR and their corresponding mutation were designed, synthesized and inserted into luciferase reporter vector GP-miRGLO (Genepharma, China), termed circCCDC9-WT, circCCDC9-MUT, CAV1–3’UTR-WT and CAV1–3’UTR-MUT, respectively. All these plasmids were co-transfected with miR-6792-3p mimics or inhibitor to MKN-45 or AGS cell, respectively. Then, the relative luciferase activity was examined by Dual Luciferase Assay Kit (Promega, USA) in line with the manufacturer’s protocol.
Biotin-coupled probe RNA pull down assay
Biotinylated circCCDC9 and miR-6792-3p (GenePharma, China) pull down assays were performed as described earlier [
9]. In brief, 1 × 10
7 GC cells were harvested, lysed, and sonicated. The probe was incubated with probes-M280 streptavidin dynabeads (Invitrogen, USA) at 25 °C for 2 h to generate probe-coated beads. The cell lysates were incubated with the probe-coated beads mixture at 4 °C overnight. After washing with the wash buffer, the RNA complexes bound to the beads were eluted and purified with Trizol Reagent (Takara, Japan) for further analysis. The sequences were listed in Additional file
1: Table S2.
RNA immunoprecipitation
RNA immunoprecipitation (RIP) assay was performed using Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, USA) following the manufacturer’s protocol. MKN-45 cells were lysed in complete RNA immunoprecipitation lysis buffer after transfected with miR-6792-3p mimics or negative control. Then, the cell extract was incubated with magnetic beads conjugated with anti-Argonaute 2 (AGO2) or anti-IgG antibody (Millipore, USA) for 6 h at 4 °C. The beads were washed and incubated with Proteinase K to remove proteins. Finally, isolated RNA was extracted using TRIzol Reagent (Takara, Japan), then, the purified RNA was subjected to agarose gel electrophoresis and qRT-PCR analysis.
Fluorescence in situ hybridization (FISH)
FISH assay was executed to observe the location of circCCDC9 and miR-6792-3p in GC cells. Cy3-labeled circCCDC9 probes and FAM-labeled miR-6792-3p were designed and synthesized by GenePharma (Shanghai, China). Hybridization was performed overnight with circCCDC9 and miR-6792-3p probes according to the manufacturer’s instructions. The images were acquired on Zeiss LSM710 Laser Scanning Confocal Microscope (Zeiss Instrument Inc., Germany). Similarly, FISH assay was also performed in TMA, which contained 54 paired gastric cancer samples by Cy3-labeled miR-6792-3p (Boster, Shanghai). The intensity of staining was scored and termed as expression level by multiplying the intensity score (0 = negative, 1 = low, 2 = high) and the percentage of the total cell population (0 = none stained, 1 = 1 ~ 24% stained, 2 = 25 ~ 49% stained, 3 = 50 ~ 74% stained, 4 = 75 ~ 100% stained). The sequences of circCCDC9 and miR-6792-3p probe for FISH were listed in Additional file
1: Table S2.
In situ hybridization (ISH)
The relative expression of circCCDC9 in GC tissues was detected by in situ hybridization with a specific digocin-labeled circCCDC9 probe (Digoxin-5′-CGCTCCTCCCACTCCTGCCGCTCGT-3′- Digoxin) (Geneseed, China) on TMAs containing 54 paired paraffin-embedded GC tissues. Briefly, after dewaxing and rehydration, the samples were digested with proteinase K, fixed in 4% paraformaldehyde, and hybridized with the digoxin-labeled circCCDC9 probe overnight, then incubated with anti-Digoxin mAb (Roche, Switzerland) at 4 °C overnight. The tissues were stained with NBT/BCIP and observed. The staining scores were scored and termed as expression level by multiplying the intensity score (0 = negative, 1 = low, 2 = high) and the percentage of the positively stained cells (0 = none stained, 1 = 1 ~ 24% stained, 2 = 25 ~ 49% stained, 3 = 50 ~ 74% stained, 4 = 75 ~ 100% stained).
Cell counting kit-8 proliferation assay
A total of 3000 cells were plated in each well of a 96-well plate. Then, on the indicated time (24 h, 48 h, 72 h, 96 h), 10 μL of CCK-8 reagent (Dojindo Crop, Japan) was added directly into the culture medium. Then, the cells were incubated for 2 h at 37 °C, and the optional density (OD) was measured at 450 nm using a microplate reader (BioTek Instruments, USA). These experiments were repeated for three times.
Transfected cells were seeded in 6-well plates at a density of 800 cells per well and then cultured at 37 °C in 5% CO2 for 2 weeks. Then, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min and stained with a 0.5% crystal violet solution for another 20 min. The colonies were then counted and analyzed. These experiments were repeated for three times.
5-Ethynyl-20-deoxyuridine (EdU) incorporation assay
The EdU assay was performed to assess the proliferation of cells by using a Cell-Light EdU DNA Cell Proliferation Kit (RiboBio, Guangzhou, China) following the manufacturer’s protocol. GC cells were cultured for 24 h in 24-well plates. These two cell lines were fixed using 4% paraformaldehyde after incubation with 50 mM EdU solution for 2 h. Then, cell lines were sealed with Apollo Dye Solution and Hoechst 33342 in sequence. The EdU cell lines were photographed and counted under an Olympus FSX100 microscope (Olympus, Tokyo, Japan). These experiments were repeated for three times.
Wound healing assays
GC cells were cultured in six-well plates, and cell monolayer was subsequently scratched with a 200ul pipette tip. Representative images of cell migration were captured by photographing 10 high-power fields at 0 h and 24 h after injury. Remodeling was measured as the diminishing distance across the induced injury area normalized to the 0 h control and expressed as a relative migration rate. For each one, the experiments were repeated for at least three times with three replicates.
Transwell migration and invasion assays
For Transwell assays, according to the manufacturer’s protocol, GC cells were seeded in upper chambers with 200ul of serum-free medium. The transwell chamber (Corning, USA) was paved with matrigel mix (BD Biosciences, USA) for invasion assays and paved without matrigel mix for migration assays. The bottom chamber was filled with medium and 10% FBS as a gastric cancer cell chemoattractant. After incubation for 24 h, the upper chambers were fixed and then stained by crystal violet (Kaigen, China) for 15 min. For visualization, the cell lines were photographed and counted in different five fields. These experiments were repeated for three times.
Western blot
Cells were lysed in Radio Immunoprecipitation Assay lysis buffer (RIPA, Beyotime, China). The protein was prepared and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). The same amounts of protein were extracted by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The blocked protein with 5% skim milk powder was incubated with primary antibody anti-CAV1 (1:10000, abcam, MA, USA), anti-β-catenin (1:1000, Cell Signaling Technology, MA, USA), anti-cyclin D1 (1:1000, Cell Signaling Technology, MA, USA), anti-E-cadherin (1:1000, Cell Signaling Technology, MA, USA) and anti-GAPDH (1:5000, Cell Signaling Technology, MA, USA) at 4 °C overnight. Then the prepared membranes were incubated with secondary antibody (1:5000, Cell Signaling Technology, MA, USA) for 2 h. Finally, the blots were visualized by ECL chemiluminescent reagent (Millipore, Germany) and related data was analysed by Image Lab Softwave.
IHC examination
Tissue samples were fixed in 4% paraformaldehyde embedded in paraffin, and sectioned. The tissue sections were incubated with anti-CAV1, anti-β-catenin, anti-E-cadherin, anti-cyclin D1 and anti-Ki-67 primary antibodies at 4 °C overnight and then incubated with an HRP-conjugated secondary antibody.
Xenografts in mice
All animal care and experiments were performed according to the guidelines of the National Institutes of Health and approved by the Animal Care Committee of Shanghai General Hospital. We chose 4-week-old male BALB/c nude mice for tumor xenografts experiments to study the effect of circCCDC9 on tumor growth and metastasis. In tumor growth assay in vivo, AGS cells stably transfected with circCCDC9-overexpressing or control vector were subcutaneously injected into the upper back of the nude mice (1 × 10
7, 150 μl). Volumes of tumors were measured weekly with a caliper. The tumor volume was calculated as (length×width
2)/2. Finally, mice were sacrificed and subcutaneous tumor tissues were detected for tumor weight, WB and IHC staining. In tumor metastasis in vivo, circCCDC9-overexpressing or control AGS cells (5 × 10
6) were intravenously injected into ileocolic vein of nude mice [
32]. After 30 days, the livers were removed, paraffin-embedded and finally validated by hematoxylin and eosin (H&E) staining.
Statistical analysis
Statistical analyses were performed using SPSS 22.0 (IBM, SPSS, Chicago, USA) and GraphPad Prism 7. Student’s t-test and one-way ANOVA were performed to analyze whether two or more groups had statistical significance. The Pearson correlation coefficient was used to analyze the correlations. The results were presented as the mean ± SD. OS curves and DFS curves were calculated with the Kaplan-Meier method and analyzed with the log-rank test. For all analyses, group differences were considered statistically significant for P < 0.05 between groups.
Discussion
CircRNAs are a type of non-coding RNAs that have been discovered more than four decades ago [
41]. CircRNAs are pervasive but heterogeneous among multiple tissues and cell lines and the generation of circRNAs is regulated by specific
cis-regulatory elements and
trans-acting factors [
42,
43]. Previous studies considered circRNAs as transcriptional noise and aberrant splicing by-products [
5,
6]. However, accumulating evidence about the biogenesis and functions of circRNAs has given rise to a new perspective on understanding the pathogenesis of diverse diseases, including cancer, cardiovascular disease, autoimmune disease and so on [
44‐
46]. Some circRNAs were reported to function as oncogenes or tumor suppressors in diverse human cancers, such as colon cancer, lung cancer, hepatocellular carcinoma (HCC) and gastric cancer [
47]. So far, only a few GC-related circRNAs have been well recognized [
48]. In the present study, it is the first research shows that circCCDC9/miR-6792-3p/CAV1 pathway participates in GC progression.
In this study, we first revealed a novel circular RNA through circRNA microarray analysis termed circCCDC9 that was obviously downregulated in GC and significantly correlated with tumor size and lymph node invasion as well as overall survival of GC patients. Second, from functional study in vitro and in vivo, we identified that circCCDC9 acted as a tumor suppressor to inhibit the proliferation, metastasis and invasion of GC cells. Third, miR-6792-3p, predicted by bioinformatics analyses and validated by dual luciferase reporter assays, was significantly upregulated in GC cells and tissues and was correlated with patients’ OS and proliferation and metastasis of GC cells. Fourth, mechanistic experiments demonstrated that circCCDC9 functioned as a ceRNA through harboring miR-6792-3p to counteract its suppressive effect on the target gene CAV1 in GC progression. Given the above evidence, our results suggested the importance of circCCDC9 as a novel biomarker and therapeutic target in human GC.
Up to now, circRNAs have been mainly reported on its active role in cytoplasm, where they acted as miRNA sponges, interacted with RBP and involved in protein translation [
8‐
10]. Given that most circRNAs contained miRNA response elements (MREs), accumulating researches shown that the ceRNA mechanism represented the primary approach through which circRNAs perform its biological functions [
8,
11,
16,
18,
49]. For example, overexpression of circPRMT5 in urothelial carcinoma of the bladder activated the SNAIL1-induced EMT pathway by blocking the expression of miR-30c, which resulted in tumor metastasis [
49]. Moreover, it was reported that circAGFG1, which was significantly upregulated in triple-negative breast cancer, promotes tumor growth and metastasis by upregulating the downstream oncogene CCNE1. Meanwhile, miR-195-5p could silence the expression of CCNE1 gene and inhibit proliferation and metastasis of tumors, whereas circAGFG1 could facilitate this process by inhibiting miR-96-5p [
50]. Herein, analyzed by circNET, RNAhybrid and miRanda, circCCDC9 was predicted to harbor miRNA-binding sites of miR-6792-3p. Thus, a series of experiments such as FISH, biotin-labeled probe pull-down assay, dual-luciferase reporter assay and RIP assay, confirmed that circCCDC9 and miR-6792-3p were co-located in the cytoplasm of GC cells and circCCDC9 directly bound to miR-6792-3p. Subsequent rescue experiments further confirmed that circCCDC9 reversed the oncogenic roles of miR-6792-3p. Furthermore, we discovered that upregulated expression of miR-6792-3p suppressed the activation of CAV1 in GC cells, suggesting that miR-6792-3p was a critical negative regulator of the CAV1. Therefore, our results provided sufficient evidence to suggest that circCCDC9 acted as a miR-6792-3p sponge and provided an exploitable biomarker and therapeutic target for patients with GC.
CAV1 has been proved to contribute to multiple malignancies. Our previous studies showed that the tumor suppressor CAV1 regulated the epithelial-mesenchymal transition (EMT) and played critical roles in GC progression [
29]. To date, whether and how circRNAs contributed to CAV1-induced progression in GC remains elusive. In the present study, our results indicated that circCCDC9 interacted with miR-6792-3p to promote the expression of CAV1, and inferred a novel mechanistic role of circCCDC9/miR-6792-3p/CAV1 axis in regulating the progression of GC. Strikingly, a recent study indicated CAV1 acted as a stage-specific growth modulator, which served as a suppressor in the early stage but shifted its function in the later stage of GC [
51]. However, our study indicated CAV1 served as a tumor suppressor in GC. It was speculated that the reason for this might be different tumor microenvironments in different stages or cell-types [
52]. Therefore, further studies are necessary to obtain a better understanding of the function of CAV1 in tumor progression.
A recent study also revealed CAV1 depletion could stabilize HER2 at the cell surface, further to increase the tumor avidity for trastuzumab on HER2-positive gastric cancer [
53,
54]. Thus, we explored the role of circCCDC9/miR-6792-3p/CAV1 axis on HER2 expression in GC. Our findings in this study had supported previous research that the negative correlation between CAV1 expression and HER2 expression (Fig.
S4). In addition, our results also shown the negative correlation between circCCDC9 expression and HER2 expression, suggested that circCCDC9 might affect HER2 expression by CAV1, at least partially. Hence, further studies are necessary to explore the underlying mechanism between circCCDC9 and HER2 in GC.
To our knowledge, this is the first study that thoroughly investigates the expression, regulation, function and clinical implication of circCCDC9 in GC. Moreover, this is also the first research to study the relationship between miR-6792-3p and CAV1. These findings may bring light to the treatment of GC. However, there are several limitations to the interpretation of our study results. Firstly, GC tissues used for RNA-seq in our study were taken from a homogenous population from one hospital, we could not rule out the possibility that there might be other critical downregulated circRNAs which are also involved in CAV1-induced progression in GC. Secondly, our preliminary study suggested that circCCDC9 was significantly downregulated in GC tissues. So, further investigation of circCCDC9 in body fluids is also needed, which makes it possible for circCCDC9 to be an ideal biomarker and therapeutic target in GC. Thirdly, our study demonstrated the ability of circCCDC9 to bind to miR-6792-3p. Whereas, there might be other miRNAs that were bound to circCCDC9 to regulate the progression and development of GC, which were not predicted by bioinformatics analyses. Fourth, whether circCCDC9 regulated the development of GC through other mechanisms such as interacting with RNA-binding proteins and sponging trans-acting elements required further investigation. Therefore, a deeper understanding of the therapeutic potential of circCCDC9 in GC is in need of further exploration.
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