Hsa_circ_0000285 contributes to gastric cancer progression by upregulating FN1 through the inhibition of miR‐1278

Abstract Background Gastric cancer (GC) is one of the most severe cancers worldwide, particularly in China. Circular RNA (circRNA) plays an essential role in GC. Hsa_circ_0000285 regulates the progression of several cancers. However, its role in GC has not been reported. This study elucidated the molecular mechanism and role of hsa_circ_0000285 in GC progression. Methods GC cells were transfected with silencers of hsa_circ_0000285 and fibronectin 1 (FN1), an inhibitor of miR‐1278, and their negative controls (NC). Mice were injected with short hairpin (sh) RNAs targeting hsa_circ_0000285 or NC. The expression levels of hsa_circ_0000285, miR‐1278, and FN1 were assessed using western blotting and reverse transcription quantitative real‐time polymerase chain reaction (qRT‐PCR). Several assays were used to evaluate cell proliferation, invasion, and apoptosis. Tumor burden was also analyzed. The interactions between miR‐1278, hsa_circ_0000285, and FN1 were ascertained using dual‐luciferase reporter assays. An RNA immunoprecipitation (RIP) assay was used to assess the enrichment of hsa_circ_0000285 and miR‐1278 in GC. Results Hsa_circ_0000285 was significantly overexpressed in the GC tissues. Silencing hsa_circ_0000285 inhibited cell proliferation and invasion, promoted apoptosis, and inhibited tumor development. Hsa_circ_0000285 sponged miR‐1278. Inhibition of miR‐1278 in vitro reversed the effects of hsa_circ_0000285 silencing on GC progression. MiR‐1278 targeted FN1, and silencing FN1 neutralized the effects of miR‐1278 inhibitors on GC progression. Conclusions The hsa_circ_0000285/miR‐1278/FN1 axis regulated GC progression. In addition, it may serve as a potential therapeutic biomarker for GC.


| INTRODUC TI ON
Gastric cancer (GC) is one of the most common malignant tumors. In China, it is the second most common malignant tumor of the digestive tract. 1 Its incidence significantly increases among people aged ≥50 years. 2 In addition, its incidence among men is 2.4 times than that among women. 3 Several factors, including environment, diet, infection, genetics, and immunity, are possible causes of GC. 2 China has a high incidence of GC, and its annual prevalence and mortality rates are much higher than those of other countries. 4 The 5 years relative survival rate is as low as 10% in patients diagnosed with advanced-stage GC. 5,6 Metastasis is a significant cause of death in patients with GC. However, the process of invasion and metastasis in GC is complicated. Therefore, an in-depth analysis of the molecular mechanisms underlying GC progression is necessary.
Circular RNA (circRNA) is a type of single-stranded RNA that features a covalently closed ring structure without 5' caps and 3' poly (A) tails, and it can be divided into noncoding and coding cir-cRNAs. 7,8 Several circRNAs have been identified recently using next-generation sequencing. 9 CircRNA is expressed in mammals and can influence the process of life through a complex regulatory network. 10 CircRNAs can regulate the expression of microRNAs (miRNAs), thereby affecting the expression of miRNAs downstream of the target mRNA. 11,12 Therefore, circRNAs can influence the evolution, growth, and invasion of cells through circRNA/miRNA/ mRNA regulatory mechanisms. This regulatory network helps classify histology, regulate disease progression, and explore strategies in GC. [13][14][15] For example, the hsa_circ_0004771/miR-149-5p/AKT1/ mTOR 16 and CircPDSS1 (hsa_circ_0093398)/miR-186-5p/NEK2 17 pathways have been shown to promote GC progression.
Hsa_hsa_circ_0000285, a circRNA generated from homeodomain interacting protein kinase 3 (HIPK3), suppresses the progression of various cancers, including cervical cancer, 18 osteosarcoma, 19,20 thyroid cancer, 21 and hepatocellular carcinoma. 22 However, whether and how it affects GC has rarely been studied. In this study, we evaluated the regulation of hsa_circ_0000285 in GC and investigated whether and how it influences GC via the circRNA/miRNA/mRNA axis. Our current study provides a new view of the function of hsa_ circ_0000285 in GC. In addition, a potential therapeutic target for GC has been discovered.

| Clinical samples and ethics statements
GC tissues (n = 40) and their corresponding normal adjacent tissues (n = 40) were collected from patients admitted to the Chengdu Fifth People's Hospital. The tissue specimens were quickly subjected to liquid nitrogen freezing and then kept at −80°C. None of the patients had received radiotherapy or chemotherapy prior to surgery.
Histopathological evaluation was conducted to verify whether the patients had GC. Written informed consent was obtained from all the patients. This study was approved by the Ethics Committee of Chengdu Fifth People's Hospital, China. The human tissues involved in this study were handled appropriately and strictly in compliance with the standards of the Declaration of Helsinki.

| Cell culture and transfection
The GC cell line and human gastric epithelial cell line GES-1 were

| Subcellular localization of hsa_circ_0000285
In accordance with the manufacturer's instructions, cytoplasmic and nuclear RNAs were extracted using the PARIS Kit (Thermo Fisher Scientific, Inc.; Cat#:AM1921). The distribution of hsa_circ_0000285 within the nucleus and cytoplasm was assessed by qRT-PCR and was normalized to U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively.

| Evaluation of proliferation, invasion, and apoptosis
Cell proliferation, invasion, and apoptosis were evaluated to determine the functions of hsa_circ_0000285, miR-1278, and FN1 in GC progression.
Cell proliferation was evaluated using the cell counting kit-8 The cells were washed, fixed, and stained with glutaraldehyde and 0.1% crystal violet. Finally, cells from five randomly selected views were observed and counted using a 400× microscope.

| Dual-luciferase reporter experiment
The miR-1278 complementary sites on FN1 and hsa_circ_0000285 were identified using StarBase (http://starb ase.sysu.edu.cn) and Circular RNA Interactome (https://circi ntera ctome.nia.nih.gov), respectively. In accordance with the binding sequences of miR-1278, psiCheck-2 vectors (Promega, Madison, WI, USA) were used to construct wild-type (WT) and mutant (Mut) hsa_circ_0000285 and Transfection was conducted in accordance with the product protocol. At 48 h post-transfection, luciferase activity was determined using a dual-luciferase reporter assay system (Promega). Relative luciferase activity was normalized to that of Renilla.

| RNA immunoprecipitation (RIP) assay
This experiment employed the Magna RIP RNA-binding protein immunoprecipitation kit (Sigma-Aldrich). HGC-27 and GTL-16 cells were disintegrated in lysis buffer and exposed to magnetic beads with anti-IgG (NC) and Anti-Ago2 coating. The RNAs attached to the beads were eluted and purified before they were evaluated using qRT-PCR.

| Western blotting
Radioimmunoprecipitation assay (RIPA) lysis buffer (Cat#: R0278, Sigma) was used for protein extraction. The proteins were quantified using a BCA kit (Solarbio). Western blotting was conducted as previously described. 23

| Tumor xenograft mice model
Sequences of small hairpin RNAs for hsa_circ_0000285 (sh-circ) and NC (sh-NC) were designed and manufactured by GenePharma.

| Hsa_circ_0000285 is significantly overexpressed in GC
Hsa_circ_0000285 expression level in GC was evaluated and its expression level in tumors was found to be approximately six times that in normal tissues (p < 0.0001; Figure 1A).  Figure 1C). We found that hsa_circ_0000285 was predominantly localized within the cytoplasm of both HGC-27 and GTL-16 cells, indicating that hsa_circ_0000285 may function as a competing endogenous RNA (ceRNA) that plays a regulatory role in the development of GC. Additionally, the circRNA structure of hsa_circ_0000285 was identified using RNase R. Hsa_circ_0000285 exhibited better resistance to RNase R digestion than GAPDH, which has a linear structure ( Figure 1D). This confirms the closed structure of hsa_circ_0000285.
Overall, significant upregulation of hsa_circ_0000285 was observed in vitro and in GC clinical samples.

| Silencing hsa_circ_0000285 retards the proliferative and invasive capabilities of cells, stimulates apoptosis, and inhibits tumor development
The role of hsa_circ_0000285 in the regulation of proliferation, invasion, apoptosis, and tumor development was assessed. Transfection efficacy was ascertained by measuring hsa_circ_0000285 expression levels in the si-NC and si-circ groups. qRT-PCR analysis revealed downregulated hsa_circ_0000285 expression after silencing, indicating successful transfection (p < 0.001; Figure 2A). In the HGC-27 and GTL-16 cell lines, cell proliferation was greatly repressed in the si-circ group after 72 h of incubation compared to that in the si-NC group (p < 0.001; Figure 2B). In addition, silencing hsa_circ_0000285 significantly decreased the number of invasive HGC-27 and GTL-16 cells compared to that in the control group (p < 0.001; Figure 2C). Additionally, the apoptotic rate was higher in the si-circ group than in the si-NC group ( Figure 2D). in the in vivo analysis, the mice in the sh-circ group manifested smaller and lighter tumors than the mice in the sh-NC group ( Figure 2E). Altogether, in vivo and in vitro experiments showed that silencing hsa_circ_0000285 suppressed the proliferation and invasion of cells, boosted apoptotic rates, and inhibited tumor development.

| Hsa_circ_0000285 sponges miR-1278
The abundance of hsa_circ_0000285 in the cytoplasm enables it to serve as an miRNA sponge. Hence, its potential hsa_circ_0000285 targets were identified using the Circular RNA interactome.
Common binding sites were observed between miR-1278 and  Figure 3F). These findings confirm that hsa_circ_0000285 sponges miR-1278.

| Inhibiting miR-1278 reverses the suppressive effects of hsa_circ_0000285 silencing on GC progression in vitro
The regulatory roles of miR-1278 and hsa_circ_0000285 in GC progression were also investigated. Figure 4A shows that miR-1278 expression was remarkably upregulated after silencing

| Silencing FN1 counteracts the effects caused by miR-1278 inhibitor in the progression of GC in vitro
The effects of silencing FN1 and inhibiting miR-1278 inhibition were investigated. First, the expression levels of FN1 protein in the si-NC,   In thyroid cancer, hsa_circ_0000285 sponges miR-599 and enhances cell metastasis. 21 These trends in circRNA expression in other cancers were consistent with those observed in our present study.
When hsa_circ_0000285 was silenced, cell proliferation and invasion were attenuated, apoptosis was enhanced, and tumor growth in vivo was suppressed. These findings regarding the functions of hsa_ circ_0000285 in GC are consistent with those in the aforementioned cancers. However, further research is required to explore the underlying mechanisms.
Considering the sponging relationship between circRNAs and miRNAs, the corresponding miRNAs sponged by hsa_circ_0000285 were predicted. MiR-1278 has been predicted to be sponged by Similarly, in another study, miR-1278 was sponged by circNEURL4 (hsa_circ_0041821), inhibiting the proliferation and invasion of papillary thyroid carcinoma. 27 These findings suggest that circ-NEURL4 is a potential prognostic biomarker for papillary thyroid carcinoma. 27 In contrast, Du et al. 26  The molecular mechanism underlying the role of miR-1278 in GC progression was further investigated by predicting its target genes. We verified that FN1 is a miR-1278 target gene that counteracts the effects of miR-1278 in GC progression. In this study, the targeting relationship between miR-1278 and FN1 was revealed for the first time. FN1 is a member of the integrin receptor family that plays essential roles in the adhesion, growth, differentiation, and migration of cells by mediating the interaction between the extracellular matrix and cells. 29,30 FN1 is involved in the progression of various cancers, such as esophageal squamous cell carcinoma, 31 breast cancer, 32 colorectal carcinogenesis, 33 and nasopharyngeal carcinoma. 34 A previous study confirmed that FN1 knockdown could abate GC cell proliferation, migration, and invasion. 35 It was also reported that FN1 could act as a prognostic biomarker 36,37 and affect the clinicopathological parameters and prognosis of patients with GC. 38 In this study, upregulation of FN1 was observed in GC cell lines and tumors, which is consistent with the results of other studies. [35][36][37]39 We also demonstrated that silencing FN1 repressed cell proliferation and invasion but boosted apoptosis.
Considering all our findings, we conclude that hsa_circ_0000285 contributes to the progression of GC by upregulating FN1 through the inhibition of miR-1278.
Our present study lacks validation from in vivo experiments.
Therefore, further animal studies are necessary. Moreover, the number of clinical samples used in this study was insufficient.
Considering the complexity of the molecular mechanisms, our present study cannot fully elucidate the role of the hsa_circ_0000285/ miR-1278/FN1 axis in the progression of GC.

| CON CLUS ION
Silencing hsa_circ_0000285 suppressed the proliferative and invasive capacities of GC cells, stimulated apoptosis, and impeded tumor development. In addition, the sponging and targeting relationships between miR-1278, hsa_circ_0000285, and FN1 were confirmed. Our work revealed the regulatory role of the hsa_ circ_0000285/miR-1278/FN1 axis in GC progression. This axis may contribute to the identification of potential therapeutic targets in GC.

AUTH O R CO NTR I B UTI O N S
MT and HH conducted the experiments and data analysis. XW devised and designed the study. YLZ obtained the data. XW and MQW performed the data analysis and interpretation. All authors read and approved the manuscript.

ACK N OWLED G EM ENTS
None.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets that have been used and/or analyzed in this study are available from the corresponding author upon reasonable request.

E TH I C A L A PPROVA L
The present study was approved by the Ethics Committee of Chengdu Fifth People's Hospital (Chengdu, China). The processing of clinical tissue samples is in strict compliance with the ethical standards of the Declaration of Helsinki. All patients signed a written informed consent. All animal experiments were in strict compliance with the ARRIVE guidelines and were carried out in accordance with National Research Council's Guide for the Care and Use of Laboratory Animals.

CO N S E NT TO PA RTI CI PATE
All patients provided a written informed consent.

CO N S E NT FO R PU B LI C ATI O N
All participants provided consent for publication.