CircFNTA promotes tumorigenesis and progression of gastric cancer via miR‐604/miR‐647/SCN8A axis

Gastric cancer (GC) is a major contributor to cancer‐related deaths and is characterized by high heterogeneity in epidemiology and histopathology worldwide. Increasing evidence indicates that circular RNAs (circRNAs) play multifaceted roles in cellular processes in human cancers. Here, we demonstrated that circFNTA high expression increases the proliferation, metastasis, and epithelial–mesenchymal transition process and tumorigenicity of GC cells. First, we found that circFNTA was upregulated in GC cells and tissues, and the high circFNTA levels were positively associated with the poor prognosis in GC patients. Using luciferase reporter and RNA‐pull down assays, we elucidated that circFNTA sponged two microRNAs, miR‐604 and miR‐647. In addition, the proliferation and metastatic ability of GC cell reduction caused by silencing circFNTA was hindered by inhibitors of miR‐604 and miR‐647. Moreover, SCN8A was predicted by miRDB as a common target gene of miR‐604 and miR‐647, which was then verified by the luciferase reporter assay. Knockdown of circFNTA causes messenger RNA and protein levels in SCN8A to be downregulated in GC cells. However, this effect was overturned by cotransfection miR‐604 and miR‐647. Also, we identified that SCN8A was downregulated in GC tissues, which was positively correlated with circFNTA expression. In rescue experiments, the attenuated cell proliferation and metastatic ability caused by circFNTA knockdown was reversed by miR‐604 and miR‐647 inhibitors and SCN8A overexpression. Collectively, our findings suggest an oncogenic role of circFNTA in GC progression and elucidate that circFNTA exerts its function by modulating the miR‐604/miR‐647/SCN8A axis.

HP infection. [6]The first-line treatments, such as endoscopic resection, surgery, systemic chemotherapy, and immune-targeted therapies including trastuzumab and ramucirumab, have improved the survival of GC patients. [7]However, because of the high rate of recurrence and distant metastasis, the median survival of patients with advanced GC is less than 1 year. [8]Integrative genomic analysis reveals great heterogeneity in GC and various GC subtypes in different continents. [9]Given the low survival rate of patients with GC, it is urgent to identify novel therapeutic targets for developing new treatments.
Circular RNAs (circRNAs) are conserved single-stranded noncoding RNAs, named under the circular structure generated by back-splicing from exons of their parental genes. [10]An increasing number of circRNAs has been found to be dysregulated in human cancers, acting either as oncogenes or tumor suppressors in tumorigenesis and cancer progression. [11][15] Growing evidence suggests that circRNAs regulate the development and progression of cancers through distinct mechanisms.Typically, circRNAs sponge microRNAs (miRNAs) to regulate the expression or stability of target genes, thereby regulating the tumorigenesis and progression of human cancers.For example, circDLG1 facilitates the immune evasion of GC cells by sponging miR-141-3p and upregulating CXCL12 expression. [16]Recently, circFNTA has been found to be upregulated in invasive bladder cancer, promoting cell proliferation and invasion by sponging miRNA-451a and suppressing S1PR3 expression. [17]Additionally, circFNTA has recently been implicated in cisplatin resistance in bladder cancer. [18]However, the function of circFNTA and the pertinent molecular mechanism in GC progression remain unexplored.
Here, we aim to investigate the function of circFNTA in GC progression.Considering its high expression in GC, we hypothesized that circFNTA may act as a competing endogenous RNA (ceRNA) to facilitate tumor development and progression in GC.To test this, we used a combination of molecular, biochemistry, and bioinformatics approaches to explore the intrinsic molecular mechanism of circFNTA.Our work provides new insights into the etiology of GC development, which have new implications for formulating new therapy for GC.

| Human tissues
The 60 GC samples and corresponding nontumorous tissues were obtained from the patients by surgery at the Second Affiliated Hospital of Soochow University between April 2016 and March 2020.These patients had no experience with chemotherapy, radiotherapy, or any other medical interventions before the surgery.The GC diagnosis was confirmed by histopathological examination.Paired adjacent nontumor tissues were derived from an area 5 cm away from the GC foci and were confirmed to be free of tumor cells.Fresh tissue samples were carefully collected and quickly stored in liquid nitrogen.Clinicopathological characteristics including age, sex, smoking status, HP infection, tumor stage, histological grade, lymph node metastasis and distant metastasis were curated (Table 1).All patients were informed of the purpose and significance of this study and signed the consent form before surgery.
All experiments were approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University (approval number: [EC-SH-2021-0117]).All procedures were performed in accordance with the ethical guidelines of the Declaration of Helsinki. [20]3 | Short hairpin RNA (shRNA) transfection and DNA constructs shRNAs targeting circFNTA, miR-604 inhibitors, miR-647 inhibitors, miR-604 mimics, and miR-647 mimics were designed by Genechem.

| Cell viability and colony formation assays
To perform the cell viability assay, 100 µL of cells (1 × 10 4 cells/mL) were seeded in each well of 96-well plates.The next day, the cell culture in each well was supplemented with 10 µL of Cell Counting Kit-8 (CCK-8) (#40203ES88; Yeasen) and was further incubated for 2 h, followed by the measurement of the 450 nm absorbance at 0, 24, 48, and 72 h. [22]For colony formation assay, cells (1 × 10 3 cells/well) were seeded in 12-well plates with triplicate for each group.After 10-14 days, colonies were fixed with methanol (10 min) and stained with 1% crystal violet (30 min).The colonies were then counted using Image J.Each experiment was repeated three times. [23]

| Cell migration, invasion, and wound-healing assays
To assess cell migrative ability, cells were digested by trypsin.Then, 4 × 10 4 cells suspended in 200 µL of fetal bovine serum free-medium were applied to the upper chamber without Matrigel coat.To explore cell invasive ability, the upper chamber coated with Matrigel was seeded with 200 µL of suspended 8 × 10 4 cells.Then, the lower chamber of each well was supplemented with 800 μL of the medium containing 10% fetal bovine serum.After 24 h, the cells on the upper chamber surface were removed with a cotton swab, and cells on the lower chamber surface were fixed with methanol and stained with crystal violet.The cells within five random fields were photographed and counted under a microscope.To perform the wound-healing assay, cells were seeded in six-well plates.The next day, a 200 μL pipette tip was used to scratch, and the cells were cultured in fetal bovine serum and photographed under a microscope at the corresponding time points. [24,25]

| RNA extraction and real-time polymerase chain reaction (RT-qPCR)
The cell and tissue total RNAs were extracted with RNAiso Plus (#9109; Takara).The RNA integrity was examined by 1% formaldehyde-denatured gel electrophoresis and the RNA concentration was determined based on the OD260/280 values measured by a NanoDrop spectrophotometer. [27]

| Luciferase reporter assay
The sequence of circFNTA was subcloned into the psiCHECK2 plasmid.Subsequently, the vectors containing the miR-604 and miR-647 mimics were cotransfected into BGC-823 and MKN-45 cell lines.

| Tumor xenografts in nude mice
To establish a tumor xenograft model, 200 µL of BGC-823 cells (5 × 10 6 cells/mL) were inoculated subcutaneously into a 6-week-old BALB/c nude mouse (the Second Affiliated Hospital of Soochow University).The tumor volume was calculated once a week using the formula: (length × width × width)/2.After 5 weeks, the mice were killed by carbon dioxide asphyxiation, and the tumors were surgically dissected and weighed. [23]Animal administration was viewed and approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University (approval number: [EC-SA-2021-0526]).

| Statistical analyses
The data of at least three replicates were displayed with mean ± standard deviation.Statistical significances between the two groups were detected using the unpaired two-tailed Student's t-test in IBM SPSS 22.0, and the results were plotted in GraphPad Prism 8. [31] The significant level was set at 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).

| RESULTS
We first detected the high expression of circFNTA using the GEO database and then validated the expression in GC samples and cell lines.
Considering the high expression, we hypothesized that circFNTA may act as a ceRNA to regulate GC progression.We then explored the relevant molecular mechanism.We verified the binding between circFNTA, miR-604/miR-647, and SCN8A by luciferase reporter assay and RNA pulldown assay.Moreover, we investigated the function of circFNTA/miR-604/miR-647/SCN8A in BGC-823 and MKN-45 cells by CCK-8 assay, colony formation assay, transwell assay, and western blot.

| CircFNTA expression in GC tissues and prognosis of patients with GC
We downloaded the GEO database, GSE78092, to screen differentially expressed circRNAs (Figure 1A) and found that circFNTA Abbreviation: qPCR, quantitative polymerase chain reaction.
expression was significantly upregulated in GC samples relative to the normal ones (Figure 1B).To further confirm the expression pattern of circFNTA in GC tissues, 60 pairs of GC and control samples were collected.RT-qPCR results showed that circFNTA expression was significantly higher in GC tissues relative to the normal tissues (Figure 1C), and the high circFNTA levels were positively associated with the poor prognosis in GC patients (Figure 1D).Similarly, based on the median value of circFNTA expression, we divided the GC samples into low and high circFNTA groups.The expression level of circFNTA was significantly correlated with histological grade, lymph node metastasis, distant metastasis, and HP infection in GC patients (Table 1).Furthermore, we performed RT-qPCR to examine circFNTA levels in a gastric mucosal cell line (GES-1) and four GC lines (AGS, SGC-7901, BGC-823, and MKN-45).CircFNTA was upregulated in all GC cell lines tested compared with the normal cell line (Figure 1E).
CircFNTA is formed by the back splicing of exons of the FNTA gene located in chromosome 8p11.21. Figure 1F shows the predicted structure of circFNTA.To confirm the circular nature of circFNTA, divergent primers and convergent primers were synthesized to amplify circFNTA from cDNA and genomic DNA (gDNA).The PCR using convergent primers amplified circFNTA from both cDNA and gDNA, while the PCR using divergent primers only amplified circFNTA from cDNA (Figure 1G), suggesting that circFNTA was spliced from the exon two to the six of the FNTA gene.
Moreover, RNase R effectively digested FNTA messenger RNA (mRNA), but had little effect on circFNTA (Figure 1H), thereof confirming that the principal structure of circFNTA is circular.
Collectively, these results suggest that circFNTA is upregulated in GC cell lines and tissues, which is associated with the poor survival of GC patients.2G).In addition, we detected the expression of circFNTA in the mouse groups when knocking out circFNTA (Supporting Information: Figure S1A).The results showed that the level of circFNTA was significantly reduced.Collectively, results indicate that circFNTA knockdown suppresses cell proliferative and metastatic capacity.

| CircFNTA acts as a sponge of miR-604 and miR-647 in GC cells
As circRNAs typically sponge miRNAs to exert their regulation, we next sought to identify the target miRNA(s) for circFNTA in GC cells.First, we used the Circular RNA Interactome to predict the miRNA(s) that could be sponged by circFNTA and found that both miR-604 and miR-647 contain complementary sequences to circFNTA, suggesting that these two miRNAs might be sponged by circFNTA (Figure 3A).To confirm this, we transfected BGC-823 and MKN-45 cells with the plasmids of wt or mut circFNTA together with the mimics of miR-604 or miR-647 and performed luciferase reporter and RNA pulldown assays.As shown in Figure 3B, overexpression of miR-604 and miR-647 obviously decreased the luciferase activity in the cells cotransfected with circFNTA wt.The RNA pulldown assay showed that both miR-604 and miR-647 were enriched in the circFNTA probe fraction (Figure 3C).Moreover, circFNTA knockdown significantly increased the expression levels of miR-604 and miR-647 in both cell lines (Figure 3D).To further confirm whether circFNTA promotes GC progression by regulating miR-604 and miR-647, we suppressed the expression of miR-604 and miR-647 using their inhibitors in BGC-823 and MKN-45 cells carrying circFNTA knockdown.The inhibition of the proliferation and clone-forming capability conferred by circFNTA knockdown could be partially rescued by miR-604 or miR-647 inhibition (Figure 3E,F).Meanwhile, the inhibited migrative and invasive ability caused by circFNTA knockdown could be partially rescued by suppressing the expression of miR-604 or miR-647 (Figure 3G).We performed RT-qPCR to confirm the expression of miR-604 and miR-647 in 60 pairs of GC and corresponding normal samples.
miR-604 and miR-647 expression were significantly downregulated in GC tissues relative to the normal tissues (Figure 3H).In addition, we detected the expression of miR-604 and miR-647 in the mouse groups when knocking out circFNTA (Supporting Information: Figure S1B).The results showed that the levels of miR-604 and miR-647 were significantly increased.Collectively, the above results suggest that circFNTA functions as an oncogene in GC cells by regulating miR-604 and miR-647.

| SCN8A is a common target gene of miR-604 and miR-647
We next used the web-based algorithm, miRDB, to predict the downstream target gene(s) of miR-604 and miR-647.The prediction identified complementary sequences between SCN8A and the two miRNAs (Figure 4A).Then, we employed the luciferase reporter assay to verify the predicted interaction.Figure 4B shows that both BGC-823 and MKN-45 cells cotransfected of SCN8A wt with either miR-604 or miR-647 mimics showed reduced luciferase activity, whereas the cells transfected with the SCN8A mut in which the putative miRNA binding sites were mutated with miRNA mimics exhibited normal luciferase activity.Meanwhile, the reduced expression levels of SCN8A mRNA and protein caused by circFNTA knockdown could be rescued by miR-604 or miR-647 knockdown (Figure 4C,D).To confirm the expression pattern of SCN8A in GC patients, we performed RT-qPCR to quantify SCN8A's transcripts in 60 pairs of GC and corresponding normal samples.SCN8A expression was significantly upregulated in GC tissues relative to the normal tissues (Figure 4E).Interestingly, the Spearman analysis revealed that the  GC exhibits high genetic heterogeneity. [32]Generally, cir-cRNAs can resist RNase R and RNA exonuclease degradation due to their circular structures. [33,34]As such, circRNAs are much more stable than linear RNAs with a long half-life.Over the past two decades, bioinformatics analysis of high-throughput sequencing data and experimental analyses have implied that dysregulated circRNAs play multifaceted roles in GC progression. [35]For example, hsa_circ_0000190 is downregulated in GC patients and its expression levels are correlated with the diameter, metastasis, and TNM stage of tumors. [36]Moreover, circPVRL3, a circRNA derived from gene PVRL3, is involved in GC progression, and its expression is negatively associated with prognosis. [37]Therefore, circRNA may be considered a noninvasive biomarker or promising therapeutic target for GC patients.Recently, two studies suggested that circFNTA could be a tumor promoter in bladder cancer. [17,18]In this study, we found that circFNTA was overexpressed in GC tissues and cells, which was positively associated with an unfavorable prognosis.
Further, we demonstrated that circFNTA increased cell proliferation and enhanced metastatic potential.Clearly, our results together with the aforementioned studies indicate that circFNTA plays a tumor-promoting role in human cancers.
It has been widely reported that the leading mechanism of circRNA is to serve as miRNA sponges through base-pairing, thus regulating the expression of downstream target genes.In this research, we demonstrated that circFNTA accelerates GC progression through sponging miR-604 and miR-647.These two miRNAs, in particular, miR-647, have been suggested as tumor suppressors in various types of cancers.For example, miR-647 is sponged by circNOLC1 and circ-FAM53B to regulate PAQR4 and MDM2 expression in prostate cancer and ovarian cancer, respectively. [38,39] addition, miR-647 is found to be downregulated in many other tumor types, such as glioma, [40] cervical cancer, [41] and hepatocellular carcinoma. [42]Moreover, miR-647 is downregulated in patients with metastatic GC, and miR-647 overexpression leads to decreased metastasis capability and increased vincristine sensitivity. [43]In contrast to the reports on the function of miR-647 in cancers, the receptor (EGFR) compared with the patients with wt EGFR. [44]nsistent with these studies, our results suggest that miR-604 and miR-647 may function as tumor suppressors in GC. [45][46][47] Typically, mRNAs serve as downstream targets of miRNAs. [48]Here, we illustrated that SCN8A expression was upregulated by circFNTA through sponging both miR-604 and miR-647.SCN8A is a member of the sodium channel family that has been implicated in several cellular processes in human cancers. [49]For example, SCN8A expression is upregulated in colorectal cancer, resulting in enhanced tumorigenesis and deteriorated oxaliplatin-induced chronic neuropathic pain.Moreover, SCN8A expression level is negatively associated with the overall survival of patients with ovarian cancer. [50]However, SCN8A appears to be downregulated in colorectal cancer, playing a negative role in cancer progression. [51]Therefore, the roles of SCN8A in human cancers are a live debate.In this study, we found that as the common target of miR-604 and miR-647, SCN8A was highly expressed in GC tissues, which was positively correlated with the expression of circFNTA, favoring a tumorpromoting role of SCN8A in GC.

| CONCLUSION
In sum, we find that circFNTA is significantly upregulated in GC tissues and plays oncogenic roles by modulating the miR-604/miR-647/SCN8A signaling pathway, which implicates circFNTA for the diagnosis and treatment of GC.
cDNA was obtained from 1 µg of RNA through reverse transcription by PrimeScript RT Master Mix (#RR036A; Takara) and HiScript III RT SuperMix (#R323-01; Vazyme).The cDNA was diluted 10-fold by double-distilled water and used for RT-qPCR with Chamq Universal SYBR qPCR Master Mix (#Q711-03; Vazyme).The 2 C − t ∆∆ method was employed to calculate the relative gene expression.The primers were synthesized by GENEWIZ and the sequences were detailed in

1
CircFNTA is upregulated in GC tissues and the high expression level of circFNTA is associated with poor prognosis.(A, B).Volcano plot (A) and heatmap (B) showing differentially expressed circRNAs in three paired GC samples.(C) RT-qPCR quantifying circFNTA expression in 60 pairs of GC samples and matched normal samples.(D) KM-plot analysis evaluating the relationship between the expression level of circFNTA and the overall survival of GC patients.(E) CircFNTA expression in a gastric mucosal cell line and four GC cell lines.(F) Schematic representation of circFNTA synthesis.(G) Amplification of circFNTA from genomic DNA and cDNA.(H) RT-qPCR assessing the expression of circFNTA and FNTA in BGC-823 and MKN-45 cell lines treated with RNase R for 30 min.cDNA, complementary DNA; circRNA, circular RNA; GC, gastric cancer; RT-qPCR, real-time polymerase chain reaction.

F I G U R E 2 | 7 of 12 3. 2 |
Figure1F, knockdown circFNTA increased the expression of E-cad, while decreasing the expression of N-cad and Vimentin.To verify the in vivo results, we subcutaneously inoculated 6-week-old BALB/c nude mice with BGC-823 cells carrying circFNTA knockdown.The cancer cells with circFNTA knockdown exhibited significantly slow growth rates as reflected by the reduced weight and volume compared to the control (Figure2G).In addition, we detected the expression of circFNTA in the CircFNTA acts as a sponge of miR-604 and miR-647 in GC cells.(A) Complementary sequences between circFNTA, miR-604, and miR-647.(B) Luciferase reporter assays showing the relative luciferase activity in BGC-823 and MKN-45 cells transfected with ±circFNTA wt or ±circFNTA mut and ±miR-604 or miR-647 mimics.(C) RNA pulldown assays showing the enrichment of miR-604 and miR-647 by circFNTA probe.(D) RT-qPCR showing the expression levels of miR-604 and miR-647 in BGC-823 and MKN-45 cells with circFNTA knockdown.(E, F) Cell proliferation and colon formation abilities determined by CCK-8 assays (E) and colony formation assays (F) in BGC-823 and MKN-45 cells transfected with ±sh-circFNTA and ±miR-604 or miR-647 inhibitors.(G) Cell migrative and invasive abilities evaluated by the transwell assay in BGC-823 and MKN-45 cells transfected with ±sh-circFNTA and ±miR-604 or miR-647 inhibitors.(H) miR-604 and miR-647 expression quantified by RT-qPCR in 60 pairs of GC samples and matched normal samples.CCK-8, Cell Counting Kit-8; GC, gastric cancer; RT-qPCR, real-time polymerase chain reaction; wt, wild-type.expressions of circFNTA and SCN8A were positively correlated (Figure4F).Together, these results indicate that SCN8A serves as the common target gene of miR-604 and miR-647.

3. 5 |
CircFNTA promotes GC cells proliferation and metastasis via miR-604/miR-647/SCN8A axisTo explore whether circFNTA exerted biological functions through regulating the miR-604/miR-647/SCN8A axis in GC cells, we overexpressed SCN8A in circFNTA knockdown cell lines.Immunoblotting confirmed that the inhibition of SCN8A expression conferred by circFNTA knockdown was effectively reversed by SCN8A overexpression (Figure5A).More importantly, the inhibited proliferative capability of BGC-823 and MKN-45 cells caused by circFNTA knockdown was partially rescued by SCN8A overexpression (Figure5B,C).Similarly, the suppressed cell migrative and invasive capacities could also be rescued by SCN8A overexpression (Figure5D).Collectively, these data suggest that circFNTA promotes the proliferation and metastasis of GC cells through regulating the miR-604/miR-647/SCN8A signaling pathway.4| DISCUSSIONIn the current study, we found that the expression of circFNTA was elevated in GC patients.Functional experiments indicated that circFNTA facilitated the proliferation and metastasis of cancer cells both in vitro and in vivo.Mechanistic investigations revealed that circFNTA exerted oncogenic function by increasing the expression of SCN8A through sponging miR-604 and miR-647.F I G U R E 4 SCN8A is the common target gene of miR-604 and miR-647.(A) The complementary sequences between SCN8A and miR-604 and miR-647.(B) Luciferase reporter assays show the relative luciferase activity in BGC-823 and MKN-45 cells transfected with ±SCN8A wt or ±SCN8A mut and ±miR-604 or miR-647 mimics.(C, D) RT-qPCR and immunoblotting showing the expression levels of SCN8A mRNA (C) and protein (D), respectively, in cells transfected with ±sh-circFNTA and ±miR-604 or miR-647 inhibitors.(E) SCN8A expression quantified by RT-qPCR in 60 pairs of GC samples and matched normal samples.(F) The Spearman analysis shows the correlation between the expression of circFNTA and SCN8A.GC, gastric cancer; mRNA, messenger RNA; mut, mutant; RT-qPCR, real-time polymerase chain reaction; wt, wild-type.
expression and function of miR-604 have been relatively understudied.The downregulation of miR-604 has been reported in nonsmall-cell lung cancer patients defective in epidermal growth factor F I G U R E 5 CircFNTA promotes GC cells proliferation and metastasis via miR-604/miR-647/SCN8A axis.(A) SCN8A expression in BGC-823 and MKN-45 cells with circFNTA knockdown and ±SCN8A overexpression.(B, C) Cell proliferation and colony formation abilities determined by the CCK-8 assay (B) and the colony formation assay (C) in cells with circFNTA knockdown ±SCN8A overexpression.(D) Cell migrative and invasive abilities evaluated by the transwell assay in cells with circFNTA knockdown ±SCN8A overexpression.CCK-8, Cell Counting Kit-8; GC, gastric cancer.

Table 2 .
T A B L E 1 Clinicopathological data of 60 GC patients.