Recent researches indicate the key role of circRNA in gastric cancer (GC) progression. However, the role of circ_0026359 in GC progression remains unclear. This study aims to analyze the role of circ_0026359 in GC development and the underlying mechanism. The results showed that compared with controls, GC tissues and cells displayed high circ_0026359 and HOXA9 expression, and low miR-140-3p expression. Depletion of circ_0026359 repressed cell proliferation, migration, invasion and glycolysis, and induced cell apoptosis. Circ_0026359 knockdown inhibited neoplasm growth in vivo. Under-expression of miR-140-3p, a target miRNA of circ_0026359, relieved the effects of circ_0026359 knockdown on GC progression. Additionally, HOXA9 was regulated by the circ_0026359/miR-140-3p axis. Thus, circ_0026359 absence inhibited GC progression by miR-140-3p/HOXA9 pathway, which provided an effective therapeutic target for GC.
1. Circ_0026359 was upregulated in GC tissue specimens.
3. Circ_0026359 was associated with miR-140-3p/HOXAP axis.
According to the statistics reported in 2018, gastric cancer (GC) is the 3rd leading cause of cancer-related death for the combination of both sexes globally . GC malignant progression is commonly induced by some factors like smoking, excessive salt accumulation and transgenation [2, 3]. Owing to the improvement of nutrition intake and newly developed therapeutic strategies, the survival of GC cases has steadily increased . However, the survival time is less than 1 year for advanced GC patients . As a result, investigating the mechanism underlying GC development is necessary to develop efficient therapeutic strategies.
CircRNA stably exists in tissues and cells and has no 5’ or 3’ polarities structure . CircRNA is formed by back-splicing events and is featured by a covalently closed cyclic structure . CircRNAs show a great possibility in acting as cancer biomarkers owing to their high stability, abundance as well as conservation in different types of cancer cells. Additionally, some strategies based on circRNA levels have been suggested for the therapy of GC [8,9,10]. CircRNA was also involved in the cellular processes of cancers , which suggested the key roles of circRNA in GC development. Most of them are able to mediate GC-linked signaling cascades by acting as sponges for microRNAs (miRNAs) [12,13,14]. Circ_0026359, located in chr12:52,628,938–52,642,709 + and generated from keratin 7 (KRT7), is significantly upregulated in GC tissues than most other circRNAs, as compared with normal gastric tissues . Given that circ_0026359 is associated with cisplatin sensitivity in GC , the circRNA is chosen for further study.
MiRNA virtually participates in post-transcriptional regulation and inhibits gene expression by targeting non-coding sequence of interest gene . Abnormal miRNA level is associated as the main mechanism behind the pathophysiological processes of cancers, including GC . Owing to its stability as well as easy and reproducible production, miRNA has the potential as a biomarker in GC [19, 20]. The above evidence manifest the importance of miRNA in GC development. Based on bioinformatics predictions, we found miR-140-3p might be a target miRNA of circ_0026359, and homeobox A9 (HOXA9) might be targeted by miR-140-3p. Importantly, previous data have shown that miR-140-3p acts as a repressor and HOXA9 knockdown inhibits cell proliferation and metastasis in GC [21, 22]. These data imply the possibility of circ_0026359/miR-140-3p/HOXA9 pathway regulating GC malignant progression; however, there is no study focusing on the role of the potential mechanism in gastric cancer progression.
Thus, whether circ_0026359 role in GC cell processes was attributed to miR-140-3p/HOXA9 axis was explored in the study. The molecular mechanism of the circRNA mediating GC development was tried to determine using the circ_0026359/miR-140-3p/HOXA9 signaling cascade.
Materials and methods
With the approval of the Ethics Committee of the First People’s Hospital of JingMen, we collected GC tissues (N = 30) and matched normal gastric tissues (N = 30) from GC patients during surgery in the First People’s Hospital of JingMen. The signed written informed consent was provided by the GC sufferers. All tissues were kept at -80˚C.
Procell (Wuhan, China) provided human AGS cells, HGC-27 cells, and GES1 cells. AGS cells were maintained in Ham’s F12K (Procell), and other types of cells were cultured in RPMI-1640 (Biosun, Shanghai, China) at 37˚C.
Vector establishment and cell transfection
GC cells at ~ 70% confluence were selected for cell transfection using TurboFect reagent (Thermo Fisher, Waltham, MA, USA) in accordance with the standard instructions. GenePharma (Shanghai, China) provided the small hairpin RNA targeting circ_0026359 (sh-circ_0026359, 5’-GATCCGTGATCCACGTGGTGCGGTTTCAAGAG AAACCGCACCACGTGGATCACTTTTTG-3’ and 5’-AATTCAAAAAGTGATCCACGTGGTGCGGTTTCACTTC AAACCGCACCACGTGGATCACG-3’), the mimics and inhibitors of miR-140-3p (miR-140-3p, 5’-UACCACAGGGUAGAACCACGG-3’ and anti-miR-140-3p, 5’-CCGUGGUUCUACCCUGUGGUA-3’), and the respective controls. The coding sequence of HOXA9 generated by polymerase chain reaction (PCR) as well as pcDNA 3.0 vector (pcDNA; Genomeditech; Shanghai, China) were used to build HOXA9 overexpression plasmid (HOXA9).
The extracted RNA using TsingZol (Tsingke, Shanghai, China) was employed for reverse transcription with EasyScript® cDNA Synthesis SuperMix (TransGen, Beijing, China) or miRNA reverse transcription reagents (TaKaRa, Dalian, China). An aliquot of 50–80 ng cDNA was reacted on a Bio-Rad 96-well Machine with qRT-PCR Mix (Tsingke) to perform a quantification analysis of circRNA/miRNA/mRNA. Finally, gene expression was analyzed using the 2−∆∆Ct method. The primer sequences are displayed in Table 1.
Identification of circ_0026359 location and stability
Circ_0026359 location was confirmed by the nucleocytoplasmic separation assay. In brief, the cultured GC cells were lysed on ice. Then, the PARIS™ Kit (Thermo Fisher) was used to extract nucleocytoplasmic RNA according to user’s manual. Expression of nucleocytoplasmic circ_0026359 was confirmed by qRT-PCR.
For identifying the stability of circ_0026359, 50 ng/mL Actinomycin D (Seebio Biotech, Shanghai, China) was incubated with AGS and HGC-27 cells for different time points. Then, RNA was isolated, and circ_0026359 and KRT7 levels were detected by qRT-PCR.
GC cells were plated in tissue culture microplates, and treated with sh-circ_0026359, miR-140-3p, anti-miR-140-3p or HOXA9. Forty-eight hours later, cell counting kit-8 reagent (Abcam, Cambridge, MA, USA) was added into each well and incubated for 2 h. The absorbance at 460 nm (A460) was measured using a microplate reader (Thermo Fisher).
Cell colony formation assay
GC cells stably expressing plasmids or oligonucleotides were diluted in media and placed in 6-well plates. About 2-week culture later, cell supernatant was removed. Paraformaldehyde (Seebio Biotech) was used to incubate the cells. After the cells were washed twice with deionized water, the number of colonies containing > 50 cells was determined.
Flow cytometry analysis
The cells were fixed with ethanol (Millipore, Bradford, MA, USA) overnight at 4˚C, and then harvested. Afterward, staining buffer, propidium iodide (Beyotime, Shanghai, China) and RNase A (Beyotime) were incubated with the cells. Flow cytometer was employed to assess DNA content and cell apoptotic rate.
Western blot analysis
Protein Extraction Kit (Phygene, Fuzhou, China) was used to prepare protein lysates. After measuring protein concentration, protein samples were added into each lane of polyacrylamide gels. After that, skimmed milk (Solarbio, Beijing, China) was utilized to block aspecific signals, and the membranes were incubated with anti-CyclinD1 (1:100; Abcam), anti-Bax (1:800; Otwo Biotech, Shenzhen, China), anti-HOXA9 (1:800; Otwo Biotech), β-actin (1:1000; Abcam), and secondary antibodies (1:10,000; Abcam). The protein blots were developed using eyoECL Plus.
Cells were suspended in FBS-free medium and dropwise added into the upper chambers coated with Matrigel (Corning, Madison, New York, USA). After 24 h later, the cells on the top surface of the lower chambers were treated with paraformaldehyde (Seebio Biotech) and crystal violet (Seebio Biotech), and photographed under a microscope (100 × ; Nikon, Tokyo, Japan). Cell migration assay was implemented in accordance with the above steps except that the upper chambers were not pre-coated with Matrigel.
Glucose consumption and lactate production
Cells were resuspended in Assay buffer (Abcam) and homogenized by pipetting up and down the tubes. Then, the supernatant was transferred to another tube, and endogenous LDH or enzymes were removed using Deproteinizing Sample Preparation Kit. The Reaction Mix or Background Reaction Mix was added into the test wells following the guidebook. Samples were assessed on microplate reader.
Mouse model assay
Under the agreement of the Animal Care and Use Committee of The First People’s Hospital of JingMen, the assay was implemented on 12 five-week-old male BALB/c nude mice (Charles River, Beijing, China). These mice were randomized into 2 groups: (1) sh-con group (injected with AGS cells stably expressing sh-con); (2) sh-circ_0026359 group (injected with AGS cells with circ_0026359 knockdown). The above cell suspensions containing 5 × 106 cells were injected into the upper back of nude mice. The volume (V) of peritoneal nodules was monitored once a week. On the 28th day, mice were anesthetized with xylazine, the forming tumors were harvested. Stable AGS cell line was established by GenePharma Co., Ltd.
Immunohistochemistry (IHC) assay
Ki-67 expression was detected by IHC assay according to the published methods . In short, the 4-µm-thick neoplasms from the mouse model assay were fixed, and dehydrated. The sections were heated at 80 °C and then incubated with anti-Ki-67 (1:80; Affinity, Nanjing, China) and secondary antibody (1:100; Affinity). Then, the samples were counterstained using hematoxylin (Phygene). The immunoreactivity was observed under a microscope.
Dual-luciferase reporter assay
The sequences of circ_0026359 and HOXA9 3’-untranslated region (3’UTR) containing the binding sites of miR-140-3p were amplified to establish the wild-type (WT) vectors (circ_0026359 WT and HOXA9 3’UTR WT). The mutant (MUT) plasmids including circ_0026359 MUT and HOXA9 3’UTR MUT were built by Yeasen (Shanghai, China). Then, the above vectors, miR-140-3p and miR-con were transfected into GC cells using TurboFect reagent (Thermo Fisher). Fluorescence signals were detected using a Dual-Lucy Assay Kit (Solarbio).
The results from 3 independent duplicate assays were expressed as means ± standard deviations. The significant differences between the 2 groups with Student’s t-tests or Wilcoxon rank-sum test, while among 3 or more groups with one-way analysis of variance. Log-rank test was used for comparing the disparities in overall survival curve, and a chi-square test was employed for comparing groups between low and high circ_0026359 expression. P value < 0.05 indicated statistical significance.
Circ_0026359 expression in GC tissues and cells
As shown in Fig. 1A and B, circ_0026359 was overexpressed in GC tissues and AGS and HGC-27 cells when compared with the normal gastric tissues and human healthy gastric mucosa cells (GES1), respectively. Moreover, the patients with high circ_0026359 expression had a poorer overall survival (Fig. 1C). Meanwhile, circ_0026359 expression was significantly associated with tumor size, lymph node metastasis and tumor-node-metastasis stage in GC patients (Table 2). Subsequently, we found the high expression of circ_0026359 in the cytoplasm in comparison with its expression in cell nuclei (Fig. 1D and E), which suggested the circRNA mainly functioned in a post-transcriptional level. Further data exhibited that the transcript half-life of linear KRT7 was about 12 h, while that of circ_0026359 exceeded 36 h (Fig. 1F and G). Thus, circ_0026359 might be associated with GC progression.
Circ_0026359 knockdown inhibited AGS and HGC-27 cell tumor properties
Circ_0026359 expression was significantly downregulated by sh-circ_0026359 (Fig. 2A). Then, the study found circ_0026359 depletion inhibited cell viability and cell proliferation (Fig. 2B-D). On the contrary, cell apoptosis was promoted by reducing circ_0026359 expression (Fig. 2E). In addition, circ_0026359 knockdown led to the downregulation of CyclinD1 and the upregulation of Bax (Fig. 2F), which certified the repressing role of circ_0026359 silencing in cell growth. Additionally, cell migration and invasion were inhibited by reducing circ_0026359 expression (Fig. 2G and H). Further, we found circ_0026359 absence inhibited glucose uptake and lactate production (Fig. 2I). Thus, all these findings demonstrated the repressing role of circ_0026359 knockdown in GC cell tumor properties.
Circ_0026359 silencing inhibited tumor formation in vivo
A mouse model assay was conducted to validate the repressing role of circ_0026359 silencing in GC cell processes. The volume and weight of the tumors from the sh-circ_0026359 group were smaller or lighter than in those tumors from the sh-con group (Fig. 3A and B). Comparative qRT-PCR analysis of the neoplasm samples displayed the reduced expression of circ_0026359 in the sh-circ_0026359 group (Fig. 3C). Additionally, it was found using IHC assay that the tumor samples from the sh-circ_0026359 group showed fewer Ki-67-positive cells (Fig. 3D). All in all, these observations elucidated circ_0026359 silencing inhibited tumor formation.
Circ_0026359 targeted miR-140-3p
The target miRNA of circ_0026359 was identified in this part. miR-140-3p could potentially bind to circ_0026359 (Fig. 4A). Subsequently, the luciferase activity of circ_0026359 WT was significantly inhibited by miR-140-3p mimics, but the luciferase activity of circ_0026359 MUT had no response to miR-140-3p overexpression (Fig. 4B and C). The above evidence demonstrated that circ_0026359 bound to miR-140-3p. Besides, the low expression of miR-140-3p was found in GC tissues and AGS and HGC-27 cells (Fig. 4D and E). Further, the study disclosed the negative correlation of circ_0026359 with miR-140-3p expression in clinical GC tissue specimens (Fig. 4F).
Circ_0026359 knockdown inhibited GC cell processes through miR-140-3p
We silenced circ_0026359 and miR-140-3p to demonstrate the consequential effect on GC cell processes. Comparative qRT-PCR analysis showed that miR-140-3p expression was significantly increased after circ_0026359 knockdown, where the effect was rescued after cotransfection of sh-circ_0026359 and miR-140-3p inhibitors (Fig. 5A). Then, we found the repressive effects of circ_0026359 silencing on cell viability and proliferation were attenuated after miR-140-3p absence (Fig. 5B-D). Consistently, miR-140-3p inhibitors restored circ_0026359 knockdown-induced cell apoptosis (Fig. 5 E). In support, the reduced expression of CyclinD1 and increased expression of Bax caused by circ_0026359 depletion were relieved by miR-140-3p inhibitors (Fig. 5F). Additionally, circ_0026359 absence-induced cell migration and invasion inhibition were remitted via decreasing miR-140-3p (Fig. 5G). Data from Fig. 5H exhibited that circ_0026359 knockdown decreased glucose consumption and lactate production, whereas miR-140-3p depletion counteracted these effects. Therefore, all data suggested circ_0026359 regulated GC cell processes by binding to miR-140-3p.
MiR-140-3p targeted HOXA9
HOXA9 carried the binding sites of miR-140-3p (Fig. 6A), and miR-140-3p mimics dramatically inhibited the luciferase activity of HOXA9 3’UTR WT rather than that of HOXA9 3’UTR MUT (Fig. 6B and C). Comparatively, HOXA9 was highly expressed in GC tissues and cells (AGS and HGC-27) as compared with normal gastric tissues or GES1 cells (Fig. 6D, F and G). In support, we identified the negative correlation of miR-140-3p with HOXA9 expression in clinical GC tissues (Fig. 6E).
MiR-140-3p suppressed GC cell malignancy by negatively regulating HOXA9 expression
The high efficiency of miR-140-3p overexpression was shown in Fig. 7A. Then, miR-140-3p mimics reduced HOXA9 production, whereas the effect was relieved after upregulation of HOXA9 expression (Fig. 7B). Additionally, the decreased cell viability and proliferation by miR-140-3p were attenuated by increasing HOXA9 expression (Fig. 7C-E). The promoting effect of miR-140-3p mimics on cell apoptosis was also restored after HOXA9 introduction (Fig. 7F). In support, HOXA9 reintroduction relieved miR-140-3p-mediated CyclinD1 and Bax expression (Fig. 7G). Figure 7H showed that miR-140-3p mimics repressed cell migration and invasion, which was remitted by increasing HOXA9 expression. Moreover, HOXA9 overexpression rescued the inhibitory impacts of miR-140-3p mimics on glucose uptake and lactate production (Fig. 7I). By the large, all data manifested miR-140-3p regulated GC cell growth by binding to HOXA9.
Circ_0026359 regulated HOXA9 expression by interacting with miR-140-3p
To demonstrate whether circ_0026359 could regulate HOXA9 expression through miR-140-3p, we silenced circ_0026359 and miR-140-3p. Circ_0026359 absence reduced HOXA9 expression, but the effect was rescued by miR-140-3p inhibitors (Fig. 8A and B). The finding indicated that circ_0026359 regulated HOXA9 by associating with miR-140-3p.
As compared to some other cancers, clinical outcome of GC sufferers remains unsatisfactory. Much work suggested the potential of circRNAs as therapeutic targets for GC. Cross-sectional researches indicate that a potential mechanistic route for circRNA to mediate malignant progression of GC cells is to modulate mRNA and some signaling pathways via trapping miRNA [13, 24]. In this study, we found high circ_0026359 expression in GC patients and cells, and circ_0026359 silencing inhibited GC malignant progression. The underlying mechanism involved miR-140-3p and HOXA9.
The detailed mechanism related to circRNA in GC development has been partly unveiled. Circ_001569 contributed to GC cell proliferation by binding to miR-145 . Circ_100782 accelerated GC cell motility by adsorbing miR-574-3p . From the data of Fang and his colleagues, we found miR-149/Wnt1 feedback loop participated in circ_0044516-mediated GC cell growth . Role of circ_0026359 in GC progression is rarely reported. Recent research displayed that poor survival of GC cases involved the increased expression of circ_0026359 ; however, the inner mechanism was unknown. In this research, we found that circ_0026359 was overexpressed in GC cases and cells, which was in line with the reported data . Additionally, circ_0026359 expression was negatively associated with the overall survival of GC patients. Circ_0026359 silencing inhibited cell proliferation, metastasis and tumor formation, and promoted cell apoptosis. During cancer development, cancer cells are inclined to produce energy from glucose metabolism to resist metabolic stresses. The phenomenon is known as the “Warburg effect”, which usually causes the increase of glucose uptake as well as accumulation of ATP and lactate production . Herein, it was found that circ_0026359 absence repressed glycolysis in GC cells.
miR-140-3p carried the complementary sequence of circ_0026359, as determined by a bioinformatics tool. MiR-140-3p can repress the cell processes of colorectal cancer , cutaneous melanoma  and breast cancer . In GC, Zhang and his colleagues indicated the low expression of miR-140-3p in GC tissues and proved that miR-140-3p inhibited GC cell growth through interaction with circRNA ATPase family AAA domain containing 1 (circATAD1) . In addition, recent research unveiled that miR-140-3p impeded GC cell growth and metastasis by reducing B-cell lymphoma-2 and increasing beclin 1 production . Agreeing with the above data, the research also reported the repressing role of miR-140-3p in GC progression. Different from the published data, we found that miR-140-3p inhibited glycolysis. Importantly, the study provided evidence that circ_0026359 regulated GC development by absorbing miR-140-3p.
HOXA9 is a member of HOX genes and can encode proteins that play essential parts in regulating the development of cancers at an organismal and evolutionary level. Based on our reviews of literature, HOXA9 is one of the most commonly altered genes in cancer progression . Previous reports demonstrated that HOXA9 acted as a suppressor in the progression of cutaneous squamous cell carcinoma , and breast cancer , and had an oncogenic function in hematologic cancer  and osteosarcoma . According to the data reported in 2017, HOXA9 was upregulated in GC tissues and was related to lymphaden metastasis, cell differentiation, and poor clinical outcome of GC patients . The data from Yu et al. implied that HOXA9 increased GC cell proliferation and metastasis . Agreeing with the above opinions, our data also reported the augment of HOXA9 expression in gastric cancer tissue specimens and the promoting effects of HOXA9 on cellular proliferation and motility. Beyond that, the study also showed that HOXA9 inhibited cell apoptosis and promoted glucose metabolism. Very importantly, the study elucidated that HOXA9 promoted GC cell growth via binding to miR-140-3p. Also, circ_0026359 mediated HOXA9 expression through the regulation of miR-140-3p.
However, the data about the function of miR-140-3p/HOAX9 in tumor tumorigenesis of GC in vivo are lacking in the present research. Additionally, the relevant clinical trials verifying the novel mechanism are absent in this research.
Availability of data and materials
The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.
Polymerase chain reaction
CircRNA ATPase family AAA domain containing 1
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Conceptualization and Methodology: JL and LL; Formal analysis and Data curation: YS, YZ and XT; Validation and Investigation: SL and JL; Writing—original draft preparation and Writing—review and editing: SL, JL, LL and YiS; All authors read and approved the final manuscript.
The underlying mechanism of circ_0026359 regulating GC progression. The pathogenesis of GC involved high circ_0026359 upregulated. The increased circ_0026359 expression induced HOXA9 production in a miR-140-3p-dependent manner, further increasing GC cell proliferation, migration, invasion and glycolysis and decreasing cell apoptosis.
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