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Circ_0049447 acts as a tumor suppressor in gastric cancer through reducing proliferation, migration, invasion, and epithelial-mesenchymal transition

Tang, Kai-Wen; Guo, Zhe-Xu; Wu, Zhong-Hua; Zhou, Cen; Sun, Jie; Wang, Xin; Song, Yong-Xi; Wang, Zhen-Ning

Editor(s): Guo, Li-Shao

Author Information
doi: 10.1097/CM9.0000000000001494
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Abstract

Introduction

Gastric cancer (GC) is one of the most common malignant neoplasms. Although its morbidity has declined over time, GC is still the fifth most commonly diagnosed cancer and ranks third as a cause of cancer-associated deaths.[1,2] There were over 1,000,000 new cases of GC and an estimated 783,000 deaths caused by GC worldwide in 2018.[2] Since there are no obvious symptoms for early stage GC, patients are often diagnosed at an advanced stage when many are already metastatic, rendering them inoperable or necessitating palliative chemotherapy.[3] Therefore, there is an urgent need to find novel targets that can promote GC diagnosis and therapy.

Circular RNAs (circRNAs) are characterized as covalently closed loops of RNA without 5′ caps or 3′ tails.[4] As a result of this structure, circRNAs are more stable and not easily degraded by endonucleases.[5] With the development of high-throughput sequencing, many circRNAs have been recognized. Accumulating recent evidences have indicated that circRNAs are abnormally expressed in lung cancer, breast cancer, and other cancers.[6,7] CircRNAs have been reported to regulate gene expression, to sponge microRNAs (miRNAs) and proteins, and to act as scaffolds for circRNA-protein complexes.[8] It has been shown that circRNAs can regulate proliferation, invasion, metastasis, and stemness in cancer cells via sponging miRNAs. For example, upregulated circFGFR1 acts as a sponge for miR-381-3p and enhances the expression of CXCR4 to promote lung cancer progression and drug resistance.[9] In addition, Du et al[10] found that circSKA3 acts as a binding partner to Tks5 and integrin β1 in the formation of invadopodia to promote tumor invasiveness. Although abnormally increased expression of circRNAs has been observed in various cancers, there are a small number of researches on circRNAs in GC has emerged, suggesting that circRNAs are promising therapeutic targets for GC. However, research on circRNAs in GC is still in the initial stage. Therefore, the function and mechanism of circRNAs in GC still needs further exploration.

In this study, we explored the expression and function of a novel circRNA, circ_0049447, in GC. Circ_0049447 is located at chr19:11446123–11448136 and has a spliced length of 533 nt. We show that circ_0049447 is downregulated in GC and that overexpression of circ_0049447 could reduce proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in GC cells.

Methods

Ethical approval

The present study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University (No. AF-SOP-07-1.0-01). Patients signed an informed written consent form.

Specimens and clinicopathological information

GC tissue specimens were obtained from 80 patients undergoing radical gastrectomy in the First Affiliated Hospital of China Medical University from 2016 to 2017, including 61 male patients and 19 female patients. All GC specimens were selected from the central mucosa of the tumor, and normal adjacent tissues were selected from the location >5 cm from the tumor edge. To ensure specimen integrity and prevent degradation, all fresh specimens were frozen in liquid nitrogen tanks before storage at −80°C.

The clinicopathological information of all specimens was collected. The diagnosis of GC patients was confirmed by post-operative pathology. All patients in this study did not receive radiotherapy or chemotherapy before the operation. Clinicopathological information was used to analyze the significance and prognostic value of circ_0049447.

RNA preparation and real-time polymerase chain reaction (PCR)

Total RNA was extracted from specimens and cultured cells using the TRIzol reagent (Invitrogen, Waltham, MA, USA), then a nanophotometer (Implen, München, Germany) was used to measure RNA concentration and purity by ultraviolet (UV) spectrophotometry (A260/A280 > 1.8). The first strand of complementary DNA (cDNA) was synthesized using the PrimeScript™ RT reagent Kit (Takara, Japan) and real-time PCR was operated by using TB Green® Premix Ex Taq™ II (Takara) as described.[11] The specificity of the PCR amplification product was validated by the melting curve for each experiment. Additionally, the product from the circRNA primers was validated by sequencing. Relative expression of circ_0049447 was normalized to a housekeeping gene (ACTB) and calculated using the 2−ΔΔCt method.[12] Primers used are as follow: ACTB forward: 5′-GAGGCGTACAGGGATAGCAC-3′ and reverse: 5′-GTCACCAACTGGGACGACAT-3′; circ_0049447 forward: 5′-GTATGACATCGCCAATCAGG-3′ and reverse: 5′-AGAGGAGACGGGCGTCCAAG-3′.

Cell culture

Human GC cell lines AGS (American type culture collection [ATCC]), HGC-27 (Institute of Biochemistry, Cell Biology [IBCB], Chinese Academy of Sciences [CAS]), and MGC-803 (IBCB, CAS) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen) with 10% fetal bovine serum (FBS) and incubated at 37°C and 5% CO2.

Plasmids and transfection

To construct overexpression vectors, full-length circ_0049447 cDNA was cloned into the pcDNA3.1 plasmid (circ_0049447 group). A pcDNA3.1 negative control plasmid with no functional cDNA was also constructed (pcDNA3.1 group). Transfections were performed using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's protocol. The cells were passaged at a 1:3 dilution with 400 μg/mL of G418 to screen the cells stably overexpressing circ_0049447. Transfection efficiency was confirmed by real-time PCR.

Cell counting kit-8 (CCK-8) proliferation assay

To check cellular proliferation capacity, 3 × 103 transfected cells were placed in a 96-well plate with 100 μL of cell suspension. Absorbance values were measured at 0, 24, 48, 72, or 96 h.

Colony formation assay

Transfected cells (1 × 103) were placed in a six-well plate and incubated, with culture media changed every 2 days. After 1 week, colonies were stained with crystal violet for 10 min. After washed with phosphate buffer saline, colonies were counted by the naked eye.

Migration and invasion assay

Cell migration and invasion activity were determined by using transwell (Corning, Corning, NY, USA) and matrigel (BD Biosciences, San Jose, CA, USA). Transfected GC cells (approximately 5 × 104) in 200 μL FBS-free medium were placed into the top chamber. For the invasion assay, 50 μL diluted matrigel was first added to the top chamber and incubated for 4 h at 37°C with 5% CO2 before plating the cells. After 48 h of culture, the number of cells adhering to the bottom side was counted by using a microscope.

Western blotting

Cellular protein was collected by total protein extraction kit (KeyGen Biotech, Nanjing, China). Western blotting was performed using precast gels (KeyGen Biotech) according to the manufacturer's protocol. The proteins were incubated with primary antibodies: E-cadherin (SAB, #48355), N-cadherin (CST, 13116S), twist (Santa, sc-81417), vimentin (SAB, #41532), β-catenin (Santa, sc-7963), and β-actin (SAB, #21800).

Luciferase reporter assay

The MGC-803 cells were seeded in a 24-well plate overnight and then co-transfected with pmirGLO-circ_0049447 wide-type (circ_0049447 WT) or mutant (MUT) and miR-324-5p mimics or negative control. Forty-eight hours later, the supernatant was obtained after lysis of cells. Luciferase activities were measured with the dual-luciferase reporter assay system (Promega, Madison, WI, USA).

Xenograft mouse model

Six-week-old male BALB/C nude mice were randomly divided into two groups (n = 5/group) and 2 × 106 GC cells were subcutaneously injected into the right axillary area. The volume of the tumor was measured once 2 days (volume = 0.5 × length × width2) and weight of tumor was recorded when the mice were sacrificed on day 14. Tumors were isolated and were further processed for immunohistochemistry.

Immunohistochemistry staining

After the in vivo tumor is prepared into the slide, immunohistochemistry staining was performed using immunohistochemical kit (ZSGB-BIO, Beijing, China) according to the manufacturer's protocol. And primary antibodies against β-catenin (Abcam, ab32572, Cambridge, England) and wnt1 (Abcam, ab15251) were used.

Statistical analysis

Data are presented as mean ± standard deviation. All statistical analyses in this study were performed using SPSS 22.0 software (IBM Corp, Armonk, NY, USA) and GraphPad Prism 8. Student's t test, Mann-Whitney U test, and Kruskal-Wallis H test were used to evaluate the significance of variance. The Kaplan-Meier method with Wilcoxon test was used to evaluate survival.

Results

Expression of circ_0049447 in GC and its relationship with clinicopathological characteristics

To detect circ_0049447 expression in GC, we designed primers according to its reverse spliced sequence. To confirm the primers’ specificity, we verified the sequence of the real-time PCR product by Sanger sequencing and found that the primers have a high specificity [Figure 1A]. Next, we examined the circ_0049447 expression in 80 pairs of GC specimens using real-time PCR. The results indicated that circ_0049447 was significantly downregulated in GC (P < 0.001) [Figure 1B]. The mean −ΔCT value of circ_0049447 in GC tissues and non-tumorous tissues were −15.25 ± 1.05 and −13.50 ± 1.47, respectively [Figure 1C]. Since circ_0049447 was significantly downregulated in GC, we further explored the relationship between circ_0049447 expression and clinicopathological characteristics of GC patients. We found a significant correlation between circ_0049447 expression and pN stage (P = 0.029), but no significant correlations were observed between its expression and other characteristics such as age (P = 0.136), location (P = 0.751), tumor size (P = 0.15), histological grade (P = 0.869), or tumor-node-metastasis stage (P = 0.275) [Table 1]. To evaluate the diagnostic value of circ_0049447 in GC, we constructed a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) reached 0.838, while sensitivity was 82.3% and specificity was 77.2% [Figure 1D]. The results suggest that circ_0049447 has good diagnostic efficiency and that it may be useful as a potential biomarker. We next used the Kaplan-Meier analysis to estimate the correlation between circ_0049447 expression and overall survival (OS) for GC patients, but we found no significant difference in OS (P = 0.125) between the two groups [Figure 1E]. Overall, we found that circ_0049447 is downregulated in GC and it is a promising biomarker for GC diagnosis.

Figure 1
Figure 1:
Expression of circ_0049447 in GC and its clinical value. (A) Simulated diagram and reverse splicing sequence of circ_0049447. (B) The percentage of low expression of circ_0049447 in tissue accounts for 83.75% (67/80). (C) Relative expression level of circ_0049447 in 80 GC tissues and in paired adjacent normal tissues (n = 80, P < 0.001). (D) The ROC curve of circ_0049447. The area under the curve (AUC) was up to 0.838, while sensitivity was 82.3% and specificity was 77.2%. (E) Kaplan-Meier survival analysis for OS of 80 patients with GC in the relatively low and high expression groups showed no significant difference between the two groups. P < 0.001. GC: Gastric cancer; OS: Overall survival; ROC: Receiver operating characteristic.
Table 1 - Association between the expression of circRNA circ_0049447 and the clinicopathological features in 80 patients with GC.
Characteristics n Expression level P values
Age 0.136
 ≤61 years 40 0.22 (0.12–0.44)
 >61 years 40 0.37 (0.11–0.96)
Gender 0.655
 Male 61 0.27 (0.10–0.72)
 Female 19 0.23 (0.14–0.89)
Location 0.751
 Upper 8 0.39 (0.23–0.72)
 Middle 33 0.31 (0.09–0.92)
 Lower 39 0.23 (0.12–0.63)
Tumor size 0.150
 ≤4.3 cm 39 0.22 (0.10–0.61)
 >4.3 cm 41 0.33 (0.14–0.92)
Macroscopic type 0.776
 Borrmann I + II 5 0.28 (0.17–1.51)
 Borrmann III + IV 68 0.27 (0.128–0.86)
Histological grade 0.850
 Good 20 0.24 (0.11–0.72)
 Poor 60 0.27 (0.12–0.74)
pT stage 0.197
 T1 6 0.10 (0.08–0.29)
 T2 9 0.27 (0.10–0.49)
 T3 18 0.24 (0.08–0.81)
 T4 47 0.33 (0.15–0.92)
pN stage 0.029
 N0 24 0.39 (0.09–1.02)
 N1 8 0.08 (0.06–0.20)
 N2 22 0.26 (0.11–0.70)
 N3 26 0.29 (0.15–0.70)
pTNM stage 0.275
 I 10 0.10 (0.08–0.70)
 II 20 0.38 (0.13–1.02)
 III 50 0.27 (0.13–0.65)
Invasion into lymphatic vessels 0.696
 Negative 19 0.21 (0.09–0.91)
 Positive 61 0.28 (0.13–0.72)
Median relative expression (25th–75th percentile). Seven patients were early GC. circRNA: Circular RNA; GC: Gastric cancer; p: Pathological; TNM: Tumor-node-metastasis.

Overexpression of circ_0049447 inhibits GC cell proliferation

To explore the function of circ_0049447 in GC cells, we performed gain-of-function assays by transfecting pcDNA3.1-circ_0049447 (circ_0049447 group) or negative control (pcDNA3.1 group) into AGS, HGC-27, and MGC-803 human GC cell lines. The CCK-8 assay indicated that circ_0049447 overexpression significantly inhibited the proliferative capacity of AGS, HGC-27, and MGC-803 cells compared with negative controls (P < 0.05). The colony formation assay also showed decreased numbers of colonies in the circ_0049447 group compared with the pcDNA3.1 group (P < 0.05). Thus, we demonstrated that increased expression of circ_0049447 can decrease proliferation in GC cells [Figure 2].

Figure 2
Figure 2:
Upregulation of circ_0049447 inhibits proliferation in GC. CCK-8 assay (A) and colony formation assay (B, C) in AGS, HGC-27, and MGC-803 cell lines showed upregulating the expression of circ_0049447 can inhibit proliferation in GC. P < 0.05. CCK-8: Cell counting kit-8; GC: Gastric cancer.

Overexpression of circ_0049447 impedes GC cell migration, invasion, and EMT

We next examined GC cell motility after upregulating circ_0049447 expression using transwell assays. Transwell migration assay showed fewer cells migrated from the transwell chambers in the circ_0049447 group compared with the pcDNA3.1 group in AGS, HGC-27, and MGC-803 cells, suggesting that increased expression of circ_0049447 could inhibit migration capacity in GC cells (P < 0.05) [Figure 3A and 3B]. Similarly, overexpression of circ_0049447 also significantly impeded invasion ability in GC cells (P < 0.05) [Figure 3C and 3D].

Figure 3
Figure 3:
Overexpression of circ_0049447 impedes migration, invasion, and EMT in GC. (A, B) Transwell migration assay showed overexpressed circ_0049447 can inhibit migration in GC. (C, D) Transwell invasion assay showed upregulated circ_0049447 can impede invasion in GC. (E) Western blotting showed high expression of circ_0049447 downregulated the mesenchymal markers N-cadherin, twist, vimentin, and β-catenin whereas upregulated E-cadherin. P < 0.05. GC: Gastric cancer; EMT: Epithelial-mesenchymal transition.

As EMT can lead to cancer cell migration and invasion,[13] we detected the expression of EMT-associated markers via Western blotting. After overexpression of circ_0049447, the mesenchymal markers N-cadherin, twist, vimentin, and β-catenin were downregulated, whereas the epithelial marker E-cadherin was upregulated [Figure 3E]. Taken together, these insinuate that overexpression of circ_0049447 can reduce GC migration, invasion, and EMT.

Prediction of miRNAs bound by circ_0049447

Current evidence indicates that circRNAs can act as competitive endogenous (ce) RNAs that bind with miRNAs to modulate the expression of their downstream gene targets.[14] This is especially seen in circRNAs derived from exons.[15] circ_0049447 is composed of RNA derived from exons of RAB3D. Given that circ_0049447 may affect GC progression by acting as a ceRNA to sponge miRNAs and regulate their downstream targets, it is important to identify the signaling axis in the ceRNA network. To explore whether certain miRNAs can bind to circ_0049447 to potentially mediate GC processes, we used Circinteractome, circBank, and RegRNA to predict potential miRNA-binding partners.[16–18] Six miRNAs were co-predicted by all three databases [Figure 4A and 4B]. According to these results, circ_0049447 may act as a sponge for certain miRNAs whose validated functions include potential roles in GC development[19–37] [Table 2]. To further explore the underlying mechanisms, we detected the expression of these predicted miRNAs in MGC-803 cells overexpressed circ_0049447. miR-324-5p, the most downregulated after overexpressing circ_0049447, was chosen for further analysis [Figure 4C]. Next, we used luciferase reporter assays to check whether miR-324-5p directly targets circ_0049447. A significant reduction of luciferase activity was detected in MGC-803 cells co-transfected with miR-324-5p mimics and circ_0049447 WT, but not with the MUT vector [Figure 4D]. Then, we detected the expression of miR-324-5p in 30 pairs of GC tissues. The results indicated that the expression of miR-324-5p in GC tissues was upregulated [Figure 4E]. Then, we searched the target genes directly regulated by miR-324-5p that have been reported in tumors and detected the expression of them in MGC-803 cells. So far, only FBXO11 and PTPRD are directly negatively regulated by miR-324-5p in lung cancer and thyroid cancer, respectively.[23,24] We found PTPRD was upregulated in MGC-803 cells overexpressed circ_0049447, while FBXO11 had no differences [Figure 4F]. It has been shown that upregulated miR-324-5p can directly target PTPRD and promote migration and invasion in thyroid cancer.[24] And these results were consistent with our prediction that circ_0049447 inhibits GC by sponging certain miRNA and implied a mechanism by which circ_0049447 may regulate migration and invasion in GC [Figure 4G].

Figure 4
Figure 4:
Prediction of miRNAs binding to circ_0049447 and mechanism simulated diagram of circ_0049447. (A, B) Predicting potential miRNAs by Circinteractome, circBank, and RegRNA and binding sites with circ_0049447. (C) Relative expression of predicted miRNAs in MGC-803 cell overexpressed circ_0049447, miR-324-5p is the most downregulated, P < 0.05. (D) Schematic illustration of circ_0049447 luciferase reporter vectors and luciferase activity of circ_0049447 WT or MUT after transfection with miR-324-5p mimics or negative control in MGC-803 cells, P < 0.01. (E) Relative expression of miR-324-5p in 30 pairs of GC tissues, P < 0.001. (F) Relative expression of the validated target gene of miR-324-5p in MGC-803 cells overexpressed circ_0049447, P < 0.05; ns: No significant difference. (G) Mechanism simulated diagram of circ_0049447 in GC. GC: Gastric cancer; miRNAs: MicroRNAs.
Table 2 - Prediction of miRNAs binding to circ_0049447 and their validated functions in cancer.
Potential miRNAs Target genes Function Tumor types Reference
miR-1204 ZNF418 Promote proliferation, inhibit apoptosis Hepatocellular carcinoma [19]
NR3C2 Aggravate proliferation, suppress apoptosis Glioblastoma [20]
PITX1 Promote proliferation Non-small cell lung cancer [21]
VDR Promote proliferation, invasion, and EMT Breast cancer [22]
miR-324-5p FBXO11 Chemotherapy resistance Non-small cell lung cancer [23]
PTPRD Promote migration and invasion Papillary thyroid carcinoma [24]
miR-661 RB1 Promote proliferation and metastasis Non-small cell lung cancer [25]
RUNX3 Promote proliferation and invasion [26]
INPP5J Promote proliferation Ovarian cancer [27]
Nectin-1, StarD10 Promote invasion Breast cancer [28]
miR-663b APC2 Promote proliferation, migration, and invasion Colorectal cancer [29]
TNK1 Promote proliferation and stemness [30]
ERF Promote proliferation and EMT Bladder cancer [31]
SMAD7 Promote proliferation and EMT Nasopharyngeal carcinoma [32]
TP73 Promote proliferation, inhibit apoptosis Osteosarcoma [33]
miR-671-5p TRIM67 Promote proliferation, migration, and invasion Colon cancer [34]
APC Promoted migration and invasion Clear cell renal cell carcinoma [35]
CDR1 Promote proliferation, migration, and invasion Glioblastoma [36]
miR-431 DAB2IP Promote migration and invasion Pancreatic neuroendocrine cancer [37]
APC: Adenomatous polyposis coli; APC2: Adenomatous polyposis coli 2; CDR1: Cerebellar degeneration-related protein 1; DAB2IP: DAB2-interacting protein; EMT: Epithelial-mesenchymal transition; ERF: Ets2-repressor factor; FBXO11: F-box protein 11; INPP5J: Inositol polyphosphate-5-phosphatase J; miRNAs: MicroRNAs; NR3C2: Nuclear receptor subfamily 3 group C member 2; PITX1: Paired like homeodomain 1; PTPRD: Protein tyrosine phosphatase receptor delta; RB1: RB transcriptional corepressor 1; RUNX3: Runt-related transcription factor 3; SMAD7: SMAD family member 7; TNK1: Tyrosine kinase non-receptor 1; TP73: Tumor protein p73; TRIM67: Tripartite motif-containing 67; VDR: Vitamin D receptor; ZNF418: Zinc-finger protein 418.

Upregulation of circ_0049447 impedes GC growth in vivo

To further explore whether circ_0049447 could influence the progression of GC in vivo, we used a subcutaneous xenograft model in nude mice. The volume of the tumor was measured once 2 days and the weight of the tumor was recorded when the mice were sacrificed. Compared with the negative control group, we discovered that mean tumor volume and weight in the circ_0049447 group were significantly smaller, indicating that circ_0049447 can impede GC proliferation in vivo [Figure 5A–D]. In addition, we used immunohistochemical analysis to detect the β-catenin and wnt1 in the tumor from the xenograft model. The results indicated that β-catenin and wnt1 were significantly inhibited in MGC-803 cells overexpressed circ_0049447, which may affect the growth of the tumor [Figure 5E].

Figure 5
Figure 5:
Upregulation of circ_0049447 impedes GC growth in vivo. (A) Representative images of in vivo tumors of nude mice. (B) Images of the tumor of each group at the endpoint of the experiment. (C) The tumor growth curves of in vivo tumor volumes. P < 0.05. (D) The mean weight of tumor in each group. P < 0.05. (E) Immunohistochemical staining of β-catenin and wnt1 in vivo tumor. GC: Gastric cancer.

Discussion

circRNAs were initially considered to be splicing errors when they were discovered in the 1970s.[38] With the progress of RNA-sequencing technologies and bioinformatics over the past few decades, an increasing number of circRNAs have been identified. circRNAs have received additional focus due to their stability, conserved properties, and abundance.[39] Recently, more biological functions have been attributed to circRNAs, including important roles in development and physiological conditions. For example, Shen et al[40] discovered that three circRNAs were differentially expressed in rat lung tissues at various points of embryonic development, and they suggested that these circRNAs participate in lung development. circRNAs have also been implicated in many diseases including cancer,[41] diabetes,[42] Alzheimer disease,[43] and others. For example, upregulation of circRNA_102231 aggravated lung cancer, and it was found to be associated with poor prognosis.[44] For the past few years, a small number of researches on circRNAs in GC have emerged, suggesting that circRNAs are promising therapeutic targets for GC. For example, it was suggested that circ_103809 promotes GC cell migration and invasion.[45] However, research on circRNAs in GC is still in the initial stage, which means that further exploration is needed to fully define the effects of circRNAs in GC.

There are currently no published reports regarding circ_0049447 in any diseases or cancers, including GC. Here, we show for the first time that circ_0049447, a novel circRNA, is downregulated in GC. Moreover, the AUC of circ_0049447 for GC tissues reached 0.838, while sensitivity was 82.3% and specificity was 77.2%. These results indicate that circ_0049447 may play a promising role in GC diagnosis as a potential biomarker conducive to GC screening. Although the expression of circ_0049447 in GC tissues was correlated with the pN stage, its expression level was much lower in the N1 stage than that in N2 and N3 stage. Therefore, we further compared GC patients with and without N metastasis, and the results showed that there was no difference between the two groups. We thought that significantly low expression of circ_0049447 in patients with N1 stage may be due to the low proportion of GC patients in this subgroup.

We validated that overexpression of circ_0049447 can decrease the proliferation of GC cell lines using CCK-8 and colony formation assays. We also found that migration and invasion capacities were impeded in GC cell lines after upregulation of circ_0049447. While exploring potential mechanisms for how circ_0049447 affects cell motility, we found that circ_0049447 could modify the EMT pathway by inhibiting mesenchymal phenotypes and facilitating epithelial phenotypes. After transfection with the circ_0049447 overexpression plasmid, the mesenchymal markers N-cadherin, twist, vimentin, and β-catenin were downregulated, whereas E-cadherin was upregulated, suggesting that circ_0049447 regulates GC cell migration and invasion through modulating the EMT pathway. In addition, the subcutaneous xenograft model also indicated that circ_0049447 can impede GC growth in vivo. Therefore, circ_0049447 acts as a tumor suppressor gene in GC. Increasing evidence indicates that circRNAs can act as ceRNAs to mediate the expression of downstream genes.[46] It has been shown that circRNAs derived from exons are especially adept at acting like miRNA sponges, such as circ5615 which promotes colorectal cancer progression by effectively sponging miR-149-5p.[47] circ_0049447 is derived from three exons, which means that circ_0049447 may also exert its function through sponging miRNA. In the current study, we confirmed circ_0049447 could impede the proliferation, migration, and invasion in GC cells, and sponge miR-324-5p. Previous studies have reported miR-324-5p could promote migration, invasion and inhibit apoptosis to enhance chemotherapy resistance.[23,24] Based on our founding and previous studies, we hypothesized that circ_0049447 could influence some specific phenotypes like apoptosis to impede chemotherapy resistance in GC through sponging miR-324-5p. In the future, we will focus on these phenotypes to further explore the underlying mechanisms of circ_0049447 in GC. Considering the anti-tumor effects of circ_0049447, future research should test whether recover its expression in vivo may allow physicians to target oncogenic miRNAs via circRNA nanosponge to help treat tumors in the future.

Acknowledgements

The authors thank the Department of Surgical Oncology of the First Affiliated Hospital of China Medical University for providing human gastric tissue samples.

Funding

This work was supported by grants from the Natural Science Foundation of China (No. 81872031), the Major R&D Plan of Liaoning Province (No. 2019JH1/10300007), the Xingliao Talents Program in Liaoning Province (No. XLYC1807164), and the Scientific and technological innovation talents program of Shenyang (No. RC190202).

Conflicts of interest

None.

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Keywords:

Gastric cancer; CircRNAs; Biomarker

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