Circular RNA circ_0000039 enhances gastric cancer progression through miR-1292-5p/DEK axis

Abstract

BACKGROUND:

Circular RNA (circRNA) is a class of non-coding RNA that is vital for regulating gene expression and biological functions. Mounting studies demonstrate that circRNA is crucial for human cancer development. However, the role of circ_0000039 in gastric cancer (GC) remains uncertain.

METHODS:

Normal human gastric tissues and GC tissue samples were collected, and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect the expression levels of circ_0000039, miR-1292-5p, and DEK. GC cell lines with overexpression and low expression of circ_0000039 were constructed. Cell counting kit-8 (CCK-8), scratch healing and Transwell experiments were used to assess the function of circ_0000039 on the proliferation, migration and invasion of GC cells. Bioinformatics analysis and dual-luciferase reporter assays were employed to detect the targeting relationship between circ_0000039 and miR-1292-5p.

RESULTS:

Circ_0000039 expression was up-regulated in GC tissues and cell lines, and it was significantly related with poor differentiation of tumor tissues. In addition, circ_0000039 overexpression enhanced the proliferation, migration and invasion of GC cells, while circ_0000039 depletion inhibited these malignant biological behaviors. In terms of mechanism, it was found that circ_0000039 promoted the proliferation and progression of GC cells by adsorbing miR-1292-5p and up-regulating the expression of DEK.

CONCLUSION:

Circ_0000039 is a new oncogenic circRNA in GC, which regulates the miR-1292-5p/DEK axis to modulate the malignant biological behaviors of GC.

Keywords

Gastric cancer circ_0000039 miR-1292-5p DEK proliferation metastasis

1. Introduction

Gastric cancer (GC) is a common malignancy of the human digestive system, which is the fourth most common malignancies, as well as the third leading cause of cancer-related death in the world [1, 2, 3, 4]. Although radical gastrectomy and chemotherapy are widely used, the prognosis of the patients in advanced stage is still disappointing [5, 6]. Therefore, it is particularly important to understand the molecular mechanism of GC progression.

Circular RNA (circRNA) is a non-coding RNA commonly found in the cytoplasm of various eukaryotic cells [7]. It is formed by back-splicing from immature RNA, lacking 5’ caps and 3’ poly (A) tail [8]. Moreover, it possesses higher stability and evolutionary conservation than linear RNA [9]. CircRNAs have been recognized as promising biomarkers for the diagnosis of diverse diseases in recent years, and they participate in regulating the biological behaviors of cancer cells [10, 11]. For instance, knockout of the circCCDC66 inhibits tumor growth and invasion in colonic cancer [12]. Circ_0000190 expression is down-regulated in GC tissues, and dysregulation of circ_0000190 is remarkably linked to tumor diameter, lymph node metastasis, and TNM staging [13]. Circ_0000745 expression in GC tissue is closely related to the degree of tumor tissue differentiation [14]. However, the role of circ_0000039 in GC has yet to be fully elucidated.

Accumulating studies show that circRNA functions as a miRNA sponge to modulate the expression of genes, and this mechanism is associated with tumorigenesis and cancer progression [15]. For instance, circ_101280 promotes the carcinogenesis of hepatocellular carcinoma via adsorbing miR-375 and up-regulating JAK2 expression [16]. Circ-BPTF facilitates bladder cancer progression through the miR-31-5p/RAB27A axis [17]. In addition, recent research indicates that abnormal regulation of the circRNA-miRNA interaction is implicated in the occurrence and progression of GC [18]. For example, the expression of circRNA_100269 is down-regulated in GC and circRNA_100269 inhibits tumor cell growth via targeting miR-630 [11].

DEK is a nuclear protein with a molecular weight of 42 KDa, and involved in many biological processes, including heterochromatin integrity, transcriptional regulation, and DNA damage repair and so on [19, 20, 21, 22]. Moreover, DEK is significantly associated with the tumorigenesis and metastasis of diverse tumors [23, 24]. In GC, it is reported that miR-1292-5p targets DEK to suppress cancer cell proliferation, migration and invasion [25].

In this study, we found that circ_0000039 and DEK expressions were up-regulated in GC tissues, and showed a positive correlation. Interestingly, bioinformatics database Circinteractome (https://circinteractome.nia.nih.gov/) predicted that circ_0000039 was a molecular sponge of miR-1292-5p, suggesting that it could probably participate in the GC progression via regulating miR-1292-5p/DEK axis. This study aimed to explore the expression and function of circ_0000039 in GC, and validate the scientific hypothesis mentioned above.

2. Materials and methods

2.1 Clinical samples

In this study, 27 GC tissues and matched adjacent non-tumor tissues were obtained from the Caoxian People’s Hospital, and none of the patients enrolled in this study received preoperative radiotherapy or chemotherapy. The samples were obtained during surgery and then stored in liquid nitrogen for further analysis. The adjacent tissue, 5 cm from the margin of the tumor, was evaluated by two pathologists and non-tumor tissue was observed. Written informed consent was obtained from all patients. The experiments were conducted with the approval and guidance of the Ethics Committee of Caoxian People’s Hospital (Approval Number: CXRMYY-2017075).

2.2 Cell lines and culture

Four human GC cell lines (MGC-803, SGC-7901, BGC-823, and AGS) and human normal gastric mucosal epithelial cell lines (GES-1) were available from the Chinese Academy of Sciences Biochemistry and Cell Biology Research (Shanghai, China). All cells were cultured in Dulbecco Modified Eagles Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA), 100 U/mL penicillin and 100 $\mu$ g/mL streptomycin (HyClone, Logan, UT, USA) at 37 ${}^{\circ}$ C in 5% CO ${}_{2}$ . The cells in logarithmic growth phase were selected for all of the following experiments. The medium was refreshed at an interval of 2–3 d. When the cells reached 80–90% confluence, the cells were treated with 0.25% trypsin (HyClone, Logan, UT, USA) and subcultured.

2.3 RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

In accordance with the instructions, total RNA in tissues and cells was extracted using TRIzol reagent (Invitrogen, Shanghai, China) and miRNA was extracted employing mirPremier ${}^{\text{\textregistered}}$ microRNA Isolation Kit (Sigma, St. Louis, MO, USA). RNA was reversely transcribed into cDNA using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Light Cycler Fast Start DNA MasterPlus SYBR Green I kit (Roche Diagnostics, Burgess Hill, UK) was used for qRT-PCR. The relative expressions of circ_0000039, miR-1292-5p, and DEK were calculated using the 2 ${}^{\rm-\Delta\Delta CT}$ method. Primer sequences were obtained from Genecopoeia (Guangzhou, China), and each reaction was repeated for three times. GAPDH was used as the standard internal reference for circ_0000039 and DEK, and U6 was used as the standard internal reference for miR-1292-5p. The primer sequences were as follows: circ_0000039: F: 5 ${}^{\prime}$ -GCTACAAGCACACCACTTCCA-3 ${}^{\prime}$ and R: 5 ${}^{\prime}$ -GAGAAGCCAATGGAGGGACT-3 ${}^{\prime}$ ; DEK: F: 5 ${}^{\prime}$ -ACGGAACAGTTCTGGAATGG-3 ${}^{\prime}$ and R: 5 ${}^{\prime}$ -TTTGGTGGCTCCTCTTCACT-3 ${}^{\prime}$ ; miR-1292-5p: F: 5 ${}^{\prime}$ -GGGTTCCGGCAGACGCTG-3 ${}^{\prime}$ and R: 5 ${}^{\prime}$ -CAGTGCGTGTCGTGGAGT-3 ${}^{\prime}$ ; GAPDH: F: 5 ${}^{\prime}$ -GACAGCCGCATCTTCT-3 ${}^{\prime}$ and R: 5 ${}^{\prime}$ -GCGCCCAATACGACCAAATC-3 ${}^{\prime}$ ; U6: F: 5 ${}^{\prime}$ -CTCGCTTCGGCAGCACA-3 ${}^{\prime}$ and R: 5 ${}^{\prime}$ -AACGCTTCACGAATTTGCGT-3 ${}^{\prime}$ .

2.4 Cell transfection

Circ_0000039 overexpressing plasmid pcDNA3.1-circ_0000039 (pcDNA3.1-circ), DEK overexpressing plasmid pcDNA3.1-DEK (DEK) and control plasmid pcDNA3.1, siRNA against circ_0000039 (si-circ) and control siRNA (si-NC), miR-1292-5p mimics and inhibitors, and controls (NC mimics or NC inhibitors) were available from GenePharma (Shanghai, China). AGS and SGC-7901 cells were inoculated in 24-well plates at 3 $\times$ 10 ${}^{5}$ cells/well. Then pcDNA3.1-circ, DEK or pcDNA3.1 and miR-1292-5p mimics or NC mimics were transfected (or co-transfected) into AGS cells, and si-circ, si-NC, miR-1292-5p inhibitors, NC inhibitors or pcDNA3.1-DEK were transfected (or co-transfected) into SGC-7901 cells using Lipofectamine ${}^{\text{\textregistered}}$ 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instruction.

2.5 Cell proliferation detection

Cell proliferation was assessed using the Cell Counting Kit 8 (CCK-8) experiment. Transfected cells were inoculated in 96-well plates at 1 $\times$ 10 ${}^{3}$ cells/well and cultured for 24 h, and 10 $\mu$ L of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well. After the cells were incubated for 1 h, the value of OD ${}_{\text{450\;nm}}$ was measured with a spectrophotometer (Bio-Rad, CA, USA). Similarly, the values of OD ${}_{\text{450\;nm}}$ at 48 h, 72 h and 96 h after inoculation were detected, respectively.

2.6 Scratch healing experiment

Scratch healing experiment was employed to detect the motility of cancer cells. In short, after the cells cultured in a 6-well plate covered the bottom of the wells, a sterilized pipette was used to make a wound on the bottom of the wells. After that, the cells were gently washed with PBS twice, and serum-free DMEM was added. Then the scratch was observed and photographed under a microscope. After cell culture was continued for 24 h, the healing of scratch was observed and photographed, and the average scratch healing rate was calculated. Scratch healing rate (%) $=$ (0 h scratch width – 24 h scratch width)/0 h scratch width $\times$ 100%.

2.7 Cell migration and invasion experiment

Figure 1.

Circ_0000039 and DEK expression were up-regulated in the GC tissues A-B. qRT-PCR was used to detect the expression levels of circ_0000039 and DEK in GC tissues and matched adjacent non-tumor tissues of GC patients, the results of which showed that both circ_0000039 and DEK were up-regulated in GC samples. C. Expression correlation between circ_0000039 and DEK in GC samples was analyzed by Pearson’s correlation analysis, which indicated that circ_0000039 and DEK were positively correlated in GC samples. D. qRT-PCR was used to detect the expression level of circ_0000039 in normal gastric mucosal epithelial cell lines (GES-1) and GC cell lines (MGC-803, SGC-7901, BGC-823, and AGS), which implied that the expression of circ_0000039 was up-regulated in GC cell lines. * $P<$ 0.05, ** $P<$ 0.01, and *** $P<$ 0.001 vs. control.

In migration experiment, after AGS and SGC-7901 cells were dispersed with 0.25% trypsin, the cells were centrifuged, resuspended, and dispersed with serum-free medium. 8 $\mu$ M pore size Transwell chambers (Corning, Beijing, China) placed on a 24-well plate were used. 5 $\times$ 10 ${}^{4}$ GC cells/well were inoculated into the chambers; meanwhile, 600 $\mu$ L medium supplemented with 10% FBS was added in each well of the 24-well plate. After the incubation at 37 ${}^{\circ}$ C for 24 h, the cells that failed to migrate were removed from the chambers. Subsequently, the Transwell membrane was fixed with 4% paraformaldehyde for 10 min and then stained with 0.5% crystal violet solution. After the chambers were gently washed and dried, the migrated cells were observed, counted and photographed. In invasion assay, Matrigel ${}^{\text{\textregistered}}$ (BD, San Jose, CA, USA) was used to cover the Transwell membrane before the cells were inoculated.

2.8 Dual-luciferase reporter experiment

Luciferase reporter gene assay was performed using the dual-luciferase reporter assay system (Promega, Madison, WI, USA). The target fragments of wild type circ_0000039 and mutant circ_0000039 were constructed and integrated into pGL3 vector (Promega, Madison, WI, USA) to construct pGL3-circ_0000039-wild type (circ_0000039-WT) and pGL3-circ_0000039-mutant (circ_0000039-MUT) reporter vectors. Circ_ 0000039-WT or circ_0000039-MUT, and miR-1292-5p mimics or NC mimics were co-transfected into AGS and SGC-7901 cells. After 48 h of transfection, the luciferase activity of the cells in each group was determined according to manufacturer’s instruction.

2.9 Western blot

AGS cells transfected with pcDNA-3.1-circ and miR-1292-5p mimics, and SGC-7901 cells transfected with si-circ and miR-1292-5p inhibitors were collected. The medium was discarded and the cells were rinsed once with PBS. The total protein was extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). After the protein samples were denatured, SDS-PAGE was performed, and the protein was transferred to a PVDF membrane (Millipore, Bedford, MA, USA) by electrophoresis. After blocking the PVDF membrane with 5% skim milk solution for 2 h, primary antibodies DEK antibody (ab166624, 1: 1000, Abcam, Shanghai, China) and $\beta$ -actin antibody (ab8226, 1: 2000, Abcam, Shanghai, China) were added and incubated overnight at 4 ${}^{\circ}$ C. Subsequently, the membrane was rinsed 3 times with TBST for 15 min each time, and then incubated with the HRP-labeled secondary antibody (ab205718, 1:2000, Abcam, Shanghai, China) for 2 h at room temperature, followed by being washed three times with TBST. The protein was imaged using the ECL kit (Amersham Pharmacia Biotech, Little Chalfont, UK).

Figure 2.

Effect of circ_0000039 overexpression on the growth, migration and invasion of AGS and SGC-7901 cells. A. Circ_0000039 overexpression and knockdown cell line were constructed, and the expression level of circ_0000039 in cells was detected by qRT-PCR. B. CCK-8 experiments were used to analyze the effect of circ_0000039 on the proliferation of AGS and SGC-7901 cells, which indicated that circ_0000039 overexpression promoted the proliferation of AGS cells, and circ_0000039 knockdown repressed the proliferation of SGC-7901 cells. C. The effect of circ_0000039 expression on AGS and SGC-7901 cell movement was analyzed by scratch healing experiment, which indicated that circ_0000039 overexpression promoted the motility of AGS cells, and circ_0000039 knockdown inhibited the motility of SGC-7901 cells. D. Transwell assay was used to analyze the effect of circ_0000039 on the migration and invasion of AGS and SGC-7901 cells, and the results suggested that circ_0000039 overexpression promoted the migration and invasion of AGS cells, and circ_0000039 knockdown inhibited the migration and invasion of SGC-7901 cells. * $P<$ 0.05, ** $P<$ 0.01, and *** $P<$ 0.001 vs. control.

2.10 Statistical analysis

All experiments were conducted in triplicate and repeated three times. All statistical analyses were performed using SPSS 20.0 software (IBM, SPSS, Chicago, IL, USA). Data were expressed as mean $\pm$ SD. Statistical significance between two groups was analyzed using the Student’s $t$ -test. The correlation between circ_0000039 and miR-1291-5p or DEK was analyzed using the Pearson’s correlation analysis. To analyze the correlations between circ 0000039 and the pathological characteristics of the patients, Fisher’s exact test was used. $P<$ 0.05 signified statistical significance.

Table 1
Association between circ_0000039 expressions and clinicopathologic features

Parameters	Cases	Hsa_circ_0000039 relative expression		Chi-square value	$P$ value
		High expression	Low expression
	All cases	27	16	11
Gender
Male	19	11	8	0.043	0.836
Female	8	5	3
Age (years)
$\geqslant$ 55	18	10	8	0.019	0.890
$<$ 55	9	6	3
T stage
T1 $+$ T2	13	6	7	0.890	0.345
T3 $+$ T4	14	10	4
Lymphatic invasion
Negative	4	1	3	0.921	0.337
Positive	23	15	8
Tumor size
$\leqslant$ 3 cm	12	5	8	2.984	0.084
$>$ 3 cm	15	11	3
Differentiation status
Poor	16	13	3	5.790	0.016
Moderate/well	11	3	8

3. Results

3.1 Circ_0000039 and DEK expressions were up-regulated in the GC tissues

First of all, the expression level of circ_0000039 in GC samples and adjacent non-tumor tissue samples was detected by qRT-PCR. It was revealed that circ_0000039 was highly expressed in GC tissues (Fig. 1A). Meanwhile, the expression of DEK was also analyzed by qRT-PCR, and the data demonstrated that DEK expression in GC tissue was also remarkably higher than that of non-tumor tissues (Fig. 1B). Importantly, correlation analysis implied that circ_0000039 expression was positively correlated with DEK expression in GC tissues (Fig. 1C). In addition, the expression of circ_0000039 in GC cell lines was significantly increased compared to in human normal gastric mucosal epithelial cell line GES-1, further confirming that circ_0000039 expression was up-regulated in GC (Fig. 1D). Subsequently, the relationship between circ_0000039 expression and different clinicopathological characteristics was analyzed by Chi-square test, and the results showed that the expression of circ_0000039 was closely related to poor differentiation of tumor tissues (Table 1). Among the GC cell lines, the expression of circ_0000039 is relative higher in SGC-7901 cell line, and relatively lower in AGS cell line. Thus, AGS and SGC-7901 cells were chosen to be applied to the following gain-of-function and loss-of-function experiments respectively.

Figure 3.

Circ_0000039 targeted miR-1292-5p. A. Sequence of targeted binding sites of circ_0000039 and miR-1292-5p. B. qRT-PCR was used to detect the expression level of miR-1292-5p in GC tissues and matched adjacent non-tumor tissues of GC patients, and the results suggested that miR-1292-5p was significantly reduced in GC tissues. C. The correlation between circ_0000039 and miR-1292-5p expressions was analyzed, and the results showed that circ_0000039 expression and miR-1292 expression were negatively correlated. D. qRT-PCR was used to detect the expression level of miR-1292-5p in AGS and SGC-7901 cell lines after circ_0000039 was overexpressed or knocked down, and as shown, circ_0000039 overexpression repressed the expression of miR-1292-5p and circ_0000039 promoted the expression of miR-1292-5p. E. Dual-luciferase reporter assay was used to verify the relationship between circ_0000039 and miR-1292-5p in AGS and SGC-7901 cells. * $P<$ 0.05, ** $P<$ 0.01, and *** $P<$ 0.001 vs. control.

3.2 The effects of circ_0000039 on AGS and SGC-7901 cell proliferation and invasion in vitro

Next, pcDNA3.1-circ_0000039 overexpressing vector was transfected into AGS cells and circ_0000039 overexpression model was constructed. Circ_0000039 siRNA was transfected into GC cell line SGC-7901, and the low expression model of circ_0000039 was constructed. The transfection efficiency was determined 24 h after the transfection (Fig. 2A). CCK-8 experiments implied that overexpression of circ_0000039 facilitated the growth of AGS cells, and knocking down circ_0000039 inhibited the proliferation of SGC-7901 cells (Fig. 2B). The results of scratch healing and Transwell experiments revealed that circ_0000039 overexpression enhanced the motility, migration and invasion of AGS cells, while circ_0000039 knockdown inhibited these malignant biological behaviors of SGC-7901 cells (Fig. 2C–D). These results suggested that circ_0000039 was a cancer-promoting factor in GC.

3.3 Circ_0000039 acted as a miRNA sponge for miR-1292-5p in AGS and SGC-7901 cells

The potential targets of circ_0000039 were searched in the Circinteractome. Circ_0000039 was predicted to have a binding site for miR-1292-5p (Fig. 3A). The expression of miR-1292-5p in GC tissues and adjacent non-tumor tissues was detected by qRT-PCR. Compared with in adjacent non-tumor tissues, miR-1292-5p expression in GC tissues was significantly reduced (Fig. 3B), and the expression of circ_0000039 in GC tissues was negatively correlated with the expression of miR-1292-5p (Fig 3C). Moreover, circ_0000039 overexpression in AGS cells markedly inhibited miR-1292-5p expression, and inhibition of circ_0000039 significantly increased the expression of miR-1292-5p in SGC-7901 cells (Fig 3D). Furthermore, dual-luciferase reporter gene experiments confirmed that miR-1292-5p mimics significantly inhibited the luciferase activity of wild-type circ_0000039 reporter in AGS and SGC-7901 cell lines, but could not significantly reduce the luciferase activity of the mutant circ_0000039 reporter, validating the binding relationship between circ_0000039 and miR-1292-5p (Fig. 3E).

3.4 MiR-1292-5p overexpression partly reversed the oncogenic effect of circ_0000039

To pinpoint the role of circ_0000039/miR-1292-5p axis in GC cells, miR-1292-5p mimics were transfected into AGS cells overexpressing circ_0000039, and SGC-7901 with circ_0000039 knocked down cells was transfected with miR-1292-5p inhibitors (Fig. 4A). After that, the biological behaviors of the AGS and SGC-7901 cells were detected by CCK-8 experiment, scratch healing experiment, and Transwell experiment, respectively. The results implied that overexpression of miR-1292-5p significantly inhibited the promotion of AGS cell proliferation, movement, migration and invasion induced by circ_0000039 overexpression (Fig. 4B–D). Conversely, the knockdown of circ_0000039 caused the inhibition of SGC-7901 cell proliferation, movement, migration, and invasion, which was partially attenuated by miR-1292-5p inhibitors (Fig. 4B–D). These results further indicated that circ_0000039 participated in regulating GC cell proliferation and metastasis via adsorbing miR-1292-5p.

Figure 4.

MiR-1292-5p overexpression inhibited the oncogenic effect of circ_0000039. A. circ_0000039 overexpressed AGS cells were transfected with miR-1292-5p mimics; SGC-7901 cells with circ_0000039 knockdown were transfected with miR-1292-5p inhibitors. qRT-PCR was used to detect the expression level of miR-1292-5p. B. CCK-8 experiments were used to analyze the proliferation of AGS and SGC-7901 cells, and the results showed that miR-1292-5p mimics partly abolished the function of circ_0000039 overexpression, and miR-1292-5p inhibitors partly reversed the effects of circ_0000039 knockdown. C. Scratch healing experiments were used to detect the movement of AGS and SGC-7901 cells, and the results showed that miR-1292-5p mimics partly abolished the function of circ_0000039 overexpression, and miR-1292-5p inhibitors partly reversed the effects of circ_0000039 knockdown. D. Transwell assay was used to analyze the migration and invasion of AGS and SGC-7901 cell lines, and the results showed that miR-1292-5p mimics partly abolished the function of circ_0000039 overexpression, and miR-1292-5p inhibitors partly reversed the effects of circ_0000039 knockdown. * $P<$ 0.05, ** $P<$ 0.01, and *** $P<$ 0.001 vs. control.

3.5 DEK overexpression restored the oncogenic effect of circ_0000039

Targeted binding between the 3’UTR of DEK and miR-1292-5p is validated by a previous study [25] (Fig. 5A). In this work, qRT-PCR and Western blot were employed to detect DEK expression in AGS and SGC-7901 cell lines after circ_0000039 and miR-1292-5p expressions were artificially regulated. The results showed that overexpression of circ_0000039 enhanced the expressions of DEK mRNA and protein, while the transfection of miR-1292-5p mimics significantly inhibited the expression of DEK (Fig. 5B–C). Subsequently, pcDNA3.1-DEK overexpressing plasmid was transfected into the SGC-7901 cells with circ_0000039 knockdown (Fig. 5D). The results of the CCK-8 experiment, the scratch healing experiment, and the Transwell experiment showed that the knockdown of circ_0000039 inhibited the proliferation, motility, migration, and invasion of SGC-7901 cells (vs. NC SGC-7901 cell line), while the overexpression of DEK partially restored these biological processes of SGC-7901 cells (vs. NC $+$ si-circ_0000039 group) (Fig. 5E–G). These results suggested that the oncogenic effect of circ_0000039 was partly dependent on DEK.

Figure 5.

DEK overexpression restored the oncogenic effect of circ_0000039. A. The sequence of targeted binding sites of DEK and miR-1292-5p was predicted by bioinformatics analysis. B-C. qRT-PCR and Western blot were used to analyze the expressions of DEK mRNA and protein in AGS and SGC-7901 cells after the transfection, and the results showed that miR-1292-5p mimics and inhibitors repressed and promoted the expression of DEK respectively, and circ_0000039 overexpression and knockdown respectively counteracted the function of miR-1292-5p mimics and inhibitors. D. pcDNA3.1-DEK was transfected into SGC-7901 cells with circ_0000039 knocked down, and the expression of DEK was detected by qRT-PCR, and the results showed that circ_0000039 knockdown repressed the expression of DEK, and after pcDNA3.1-DEK was transfected, the expression of DEK was restored. E. CCK-8 was used to detect the proliferation of SGC-7901 cells, and the results suggested circ_0000039 knockdown repressed the proliferation, while DEK overexpression reversed it. F. Scratch healing experiment was used to analyze the movement of SGC-7901 cells, and the results suggested circ_0000039 knockdown repressed the motility, while DEK overexpression reversed it. G. Transwell assay was used to analyze the migration and invasion of SGC-7901 cells, and the results suggested circ_0000039 knockdown repressed the migration and invasion, while DEK overexpression reversed it. * $P<$ 0.05, ** $P<$ 0.01, and *** $P<$ 0.001 vs. control.

4. Discussion

CircRNAs were originally thought to be byproducts of RNA transcription and splicing and have low expression abundance. However, recent studies authenticate that circRNA possesses a highly stable and specific circular structure and are promising biomarker for the diagnosis and prognosis evaluation of cancers [26, 27]. Functionally, circRNAs can regulate key biological behaviors in human malignancies, such as cell proliferation, migration, and invasion [28, 29, 30, 31]. For instance, it is revealed that circ_0067934 is up-regulated in esophageal squamous cell carcinoma and is involved in promoting cancer cell proliferation and migration [32]. The inhibition of circPVRL3 enhances the proliferation and migration of GC cells [33]. Nevertheless, the role of circ_0000039 in GC has not been studied previously. In this study, for the first time, it was found that circ_0000039 expression in human GC tissues was remarkably higher than that in non-tumor tissues; up-regulation of circ_0000039 expression was significantly related to poor differentiation of tumor tissues. Further experiments showed that over-expression of circ_0000039 enhanced the proliferation, motility, migration and invasion of GC cells. These demonstrations implied that circ_0000039 played a role as a novel oncogenic circRNA in promoting the development of GC.

Competitive endogenous RNAs (ceRNAs) include mRNA, pseudogenic RNAs, long noncoding RNAs (lncRNAs) and circRNAs, which are transcripts that competitively sponge miRNAs to reduce their expression and function, and regulate the expression of downstream genes. Recent studies reveal that circRNAs figure prominently in regulating gene expression by targeting specific miRNAs [34]. Accumulating data demonstrate that ceRNA mechanism in which circRNAs participate is involved in promoting or inhibiting the tumorigenesis and development of malignancies [35]. For instance, circ_103809 can sponge miR-620 and negatively regulate its expression, thereby further inhibiting the proliferation and invasion of liver cancer cells [36]. In addition, circPSMC3 is identified as a molecular sponge for miR-296-5p to regulate the expression of PTEN and further inhibit the tumorigenesis of GC [37]. In this study, bioinformatics analysis revealed that circ_0000039 could probably target miR-1292-5p, and dual-luciferase and in vitro experiments validated this prediction. It was also demonstrated that miR-1292-5p overexpression repressed the oncogenic effect of circ_0000039. This phenomenon indicated that miR-1292-5p was a crucial downstream miRNA by which circ_0000039 exerted its function in GC.

Proto-oncogene DEK is a highly conserved chromatin-bound protein, and its high expression helps keep genome stability and facilitates the DNA double strand breaks repair of cells [22, 38]. It is reported that DEK is involved in the tumorigenesis and development of diverse tumors. DEK is verified to facilitate the invasion of breast cancer cells via modulating $\beta$ -catenin nuclear translocation [39]. Additionally, DEK facilitates cancer cell proliferation in hepatocellular carcinoma by up-regulating cell cycle-related CDK signaling, and promotes cancer cell migration and EMT via regulating the $\beta$ -catenin/E-cadherin axis [24]. In GC, DEK expression is reported to be up-regulated in both cancer tissues and the plasma of the patients, and DEK promotes cancer cell migration via increasing the expression level of matrix metalloproteinases MMP-2/MMP-9, and high expression of DEK protein predicts adverse prognosis [40, 41]. In this study, it was found that the DEK was overexpressed in GC tissues, which is consistent with previous reports [40, 41]. It was also demonstrated that the expression of DEK in GC tissues was positively related to circ_0000039 expression, and further rescue assays suggested that DEK overexpression rescued the malignant biological behaviors of GC cells after circ_0000039 was knocked down. Considering that DEK is proved to be a target of miR-1292-5p by our data and a previous study [25], we concluded that circ_0000039 facilitated GC progression via regulating miR-1292-5p/DEK axis.

In conclusion, this study finds that circ_0000039 expression is significantly elevated in GC tissues and closely associated with the differentiation status of tumor tissues. Our data also reveal that circ_0000039 is an oncogenic factor in GC, which can modulate miR-1292-5/DEK axis to affect the malignant biological behaviors of GC cells, providing a better understanding of the mechanism of GC carcinogenesis and development.

Footnotes

Funding

This study is supported by the Research Funding for Clinical Researchers of Caoxian People’s Hospital (Approval No. CXRMYY-17075).

References

Jemal

Bray

Center

M.M.

Ferlay

Ward

and Forman

, Global cancer statistics, CA: A Cancer Journal for Clinicians 61(2) (2011), 69–90.

Nagini

, Carcinoma of the stomach: A review of epidemiology, pathogenesis, molecular genetics and chemoprevention, World Journal of Gastrointestinal Oncology 4(7) (2012), 156–169.

Torre

L.A.

Bray

Siegel

R.L.

Ferlay

Lortet-Tieulent

and Jemal

, Global cancer statistics, 2012, CA: A Cancer Journal for Clinicians 65(2) (2015), 87–108.

Ferlay

Soerjomataram

Dikshit

et al., Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012, International Journal of Cancer 136(5) (2015), E359–386.

Strong

V.E.

A.W.

Selby

L.V.

et al., Differences in gastric cancer survival between the U.S. and China, Journal of Surgical Oncology 112(1) (2015), 31–37.

Allemani

Weir

H.K.

Carreira

et al., Global surveillance of cancer survival 1995–2009: Analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD-2), Lancet (London, England) 385(9972), (2015), 977–1010.

Chen

L.L.

and Yang

, Regulation of circRNA biogenesis, RNA Biology 12(4) (2015), 381–388.

Yang

et al., Circular RNA: A new star of noncoding RNAs, Cancer Letters 365(2) (2015), 141–148.

Yao

J.T.

Zhao

S.H.

Liu

Q.P.

et al., Over-expression of CircRNA_100876 in non-small cell lung cancer and its prognostic value, Pathology, Research and Practice 213(5) (2017), 453–456.

10.

Q.G.

Wang

Z.G.

et al., Circular RNA cSMARCA5 inhibits growth and metastasis in hepatocellular carcinoma, Journal of Hepatology 68(6) (2018), 1214–1227.

11.

Zhang

Liu

et al., CircRNA_100269 is downregulated in gastric cancer and suppresses tumor cell growth by targeting miR-630, Aging 9(6) (2017), 1585–1594.

12.

Hsiao

K.Y.

Lin

Y.C.

Gupta

S.K.

et al., Noncoding Effects of Circular RNA CCDC66 Promote Colon Cancer Growth and Metastasis, Cancer Res 77(9) (2017), 2339–2350.

13.

Chen

Zhao

Xiao

and Guo

, Using circular RNA hsa_circ_0000190 as a new biomarker in the diagnosis of gastric cancer, Clinica Chimica Acta; International Journal of Clinical Chemistry 466 (2017), 167–171.

14.

Huang

Y.R.

Liang

L.C.

Huang

and Zhu

Z.Q.

, Circular RNA hsa_circ_0000745 may serve as a diagnostic marker for gastric cancer, World J Gastroenterol 23(34) (2017), 6330–6338.

15.

Yang

Zhou

et al., Circular RNAs in cancer: Novel insights into origins, properties, functions and implications, American Journal of Cancer Research 5(2) (2015), 472–480.

16.

Cao

Wang

and Zhang

, Hsa_circ_ 101280 promotes hepatocellular carcinoma by regulating miR-375/JAK2, Immunology and Cell Biology 97(2) (2019), 218–228.

17.

Liu

Cai

et al., Circ-BPTF promotes bladder cancer progression and recurrence through the miR-31-5p/RAB27A axis, Aging 10(8) (2018), 1964–1976.

18.

Guan

Y.J.

J.Y.

and Song

, Identification of circRNA-miRNA-mRNA regulatory network in gastric cancer by analysis of microarray data, Cancer Cell International 19 (2019), 183.

19.

Kappes

Burger

Baack

Fackelmayer

F.O.

and Gruss

, Subcellular localization of the human proto-oncogene protein DEK, The Journal of Biological Chemistry 276(28) (2001), 26317–26323.

20.

Kappes

Waldmann

Mathew

et al., The DEK oncoprotein is a Su(var) that is essential to heterochromatin integrity, Genes & Development 25(7) (2011), 673–678.

21.

Sawatsubashi

Murata

Lim

et al., A histone chaperone, DEK, transcriptionally coactivates a nuclear receptor, Genes & Development 24(2) (2010), 159–170.

22.

Kavanaugh

G.M.

Wise-Draper

T.M.

Morreale

R.J.

et al., The human DEK oncogene regulates DNA damage response signaling and repair, Nucleic Acids Research 39(17) (2011), 7465–7476.

23.

Zou

Yao

Zhang

Gan

and Tang

, Silencing DEK downregulates cervical cancer tumorigenesis and metastasis via the DEK/p-Ser9-GSK-3beta/p-Tyr216-GSK-3beta/beta-catenin axis, Oncology Reports 38(2) (2017), 1035–1042.

24.

Huang

Zhang

et al., Critical role of DEK and its regulation in tumorigenesis and metastasis of hepatocellular carcinoma, Oncotarget 7(18) (2016), 26844–26855.

25.

Hui

Zan

Song

Zhang

and Dong

, MicroRNA-1292-5p inhibits cell growth, migration and invasion of gastric carcinoma by targeting DEK, American Journal of Cancer Research 8(7) (2018), 1228–1238.

26.

Memczak

Jens

Elefsinioti

et al., Circular RNAs are a large class of animal RNAs with regulatory potency, Nature 495(7441) (2013), 333–338.

27.

Wang

Sun

Tao

Fei

and Chang

, Androgen receptor (AR) promotes clear cell renal cell carcinoma (ccRCC) migration and invasion via altering the circHIAT1/miR-195-5p/29a-3p/29c-3p/CDC42 signals, Cancer Letters 394 (2017), 1–12.

28.

Taborda

M.I.

Ramirez

and Bernal

, Circular RNAs in colorectal cancer: Possible roles in regulation of cancer cells, World Journal of Gastrointestinal Oncology 9(2) (2017), 62–69.

29.

Chen

Tan

and Liu

, Circular RNAs: A new frontier in the study of human diseases, Journal of Medical Genetics 53(6) (2016), 359–365.

30.

Jiang

Chen

et al., CircIRAK3 sponges miR-3607 to facilitate breast cancer metastasis, Cancer Letters 430 (2018), 179–192.

31.

Wang

Zhang

et al., Circular RNA circITGA7 inhibits colorectal cancer growth and metastasis by modulating the Ras pathway and upregulating transcription of its host gene ITGA7, The Journal of Pathology 246(2) (2018), 166–179.

32.

Xia

Qiu

Chen

et al., Circular RNA has_circ_ 0067934 is upregulated in esophageal squamous cell carcinoma and promoted proliferation, Sci Rep 6 (2016), 35576.

33.

Sun

H.D.

Z.P.

Sun

Z.Q.

et al., Down-regulation of circPVRL3 promotes the proliferation and migration of gastric cancer cells, Sci Rep 8(1) (2018), 10111.

34.

Rusinek

Swierniak

Chmielik

et al., BRAFV600E-associated gene expression profile: Early changes in the transcriptome, based on a transgenic mouse model of papillary thyroid carcinoma, PLoS One 10(12) (2015), e0143688.

35.

Rupaimoole

and Slack

F.J.

, MicroRNA therapeutics: Towards a new era for the management of cancer and other diseases, Nature reviews, Drug Discovery 16(3) (2017), 203–222.

36.

and Shen

, Circular RNA hsa_circ_103809 suppresses hepatocellular carcinoma proliferation and invasion by sponging miR-620, European Review for Medical and Pharmacological Sciences 23(2) (2019), 555–566.

37.

Rong

Zhang

et al., CircPSMC3 suppresses the proliferation and metastasis of gastric cancer by acting as a competitive endogenous RNA through sponging miR-296-5p, Mol Cancer 18(1) (2019), 25.

38.

Smith

E.A.

Gole

Willis

N.A.

et al., DEK is required for homologous recombination repair of DNA breaks, Sci Rep 20(7) (2017), 44662.

39.

L.M. Privette Vinnedge McClaine

Wagh

P.K.

Wikenheiser-Brokamp

K.A.

Waltz

S.E.

and Wells

S.I.

, the human DEK oncogene stimulates beta-catenin signaling, invasion and mammosphere formation in breast cancer, Oncogene 30(24) (2011), 2741–2752.

40.

Lee

K.F.

Tsai

M.M.

Tsai

C.Y.

Huang

C.G.

et al., DEK is a potential biomarker associated with malignant phenotype in gastric cancer tissues and plasma, Int J Mol Sci 20(22) (2019).

41.

Piao

Shang

Liu

et al., High expression of DEK predicts poor prognosis of gastric adenocarcinoma, Diagn Pathol 9 (2014), 67.

Circular RNA circ_0000039 enhances gastric cancer progression through miR-1292-5p/DEK axis

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Clinical samples

2.2 Cell lines and culture

2.3 RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

2.4 Cell transfection

2.5 Cell proliferation detection

2.6 Scratch healing experiment

2.7 Cell migration and invasion experiment

2.9 Western blot

Table 1 Association between circ_0000039 expressions and clinicopathologic features

3.1 Circ_0000039 and DEK expressions were up-regulated in the GC tissues

3.3 Circ_0000039 acted as a miRNA sponge for miR-1292-5p in AGS and SGC-7901 cells

3.4 MiR-1292-5p overexpression partly reversed the oncogenic effect of circ_0000039

Footnotes

Funding

References

Table 1
Association between circ_0000039 expressions and clinicopathologic features