Hsa_circRNA_0001971 contributes to oral squamous cell carcinoma progression via miR‐186‐5p/Fibronectin type III domain containing 3B axis

Abstract Background Circular RNAs (circRNAs) are closely associated with the progression of oral squamous cell carcinoma (OSCC). circRNA_0001971 has been proved to accelerate the OSCC development. Here, we aim to identify the new molecular mechanism of hsa_circRNA_0001971 (circRNA_0001971) in OSCC. Methods The levels of circRNA_0001971, miR‐186‐5p, and fibronectin type III domain containing 3B (FNDC3B) in tissues and cells were verified by qRT‐PCR or Western blotting. The interaction between circRNA_0001971, miR‐186‐5p, and FNDC3B was identified by bioinformatics analysis, luciferase assay, and RIP assay. The effect of circRNA_0001971/miR‐186‐5p/FNDC3B axis on OSCC cell proliferation, migration, and invasion by cell functional experiments including CCK8, wound healing, and transwell assays. Results Our study displayed that circRNA_0001971 and FNDC3B were elevated in OSCC, whereas miR‐186‐5p was declined in OSCC. Silencing circRNA_0001971 attenuated the malignancy of OSCC cells by suppressing proliferation, migration, and invasion. In OSCC cells, circRNA_0001971 sponged miR‐186‐5p to enhance FNDC3B. Due to the interaction between circRNA_0001971, miR‐186‐5p, and FNDC3B, FNDC3B overexpression relieved the negative function of silencing circRNA_0001971 in OSCC cells. Conclusion Overall, our study discovered that circRNA_0001971 was a tumor promoter in OSCC progression by targeting miR‐186‐5p/FNDC3B axis.

poor prognosis of OSCC. 5,6 Therefore, the study on the molecular mechanisms underlying OSCC progression is worthy and important to identify novel therapeutic targets.
Circular RNAs (circRNAs) featured by covalently closed-loop structures are considered as competitive endogenous RNAs (ceR-NAs) of microRNA (miRNAs) to affect gene expression. 7,8 circRNAs previously regarded as accessory substance of splicing errors are proved to serve key roles in multiple human disease with the deep study on circRNA function. [9][10][11][12] During OSCC development, cir-cRNAs are pointed out to act as key roles by ceRNA mechanism. For example, circIGHG contributed to OSCC progression and induced epithelial-to-mesenchymal transition via sponging miR-142-5p to regulate IGF2BP3 expression. 13 Another circRNA circ_100533 was proved to be a tumor suppressor in OSCC by sponging miR-933/GNAS axis. 14 Hsa_circ_0001971 (circ_0001971), a novel cir-cRNA, was only found in OSCC to enhance the OSCC progression by sponging miR-194/miR-204. 15 However, the regulatory mechanism of circRNAs is complex, which may involve multiple miRNAs to regulating different expressions of genes. Therefore, the molecular mechanism of circ_0001971 is still necessary to be deeply explored.
Our study was to probe the role of circ_0001971 in OSCC progression and provide a novel molecular axis of circ_0001971 regulating OSCC progression by the ceRNA mechanism. Our findings might provide novel evidence for circ_0001971 potential as a target of OSCC treatment.

| Clinical samples collection
Thirty-four paired OSCC samples and adjacent normal samples were collected from the patients diagnosed with OSCC in our hospital between January 2020 and May 2021. Each participant in our study signed informed consent, and the ethics committee of our hospital approved our study. The inclusion criteria in our study were as follows (1): All patients were diagnosed with OSCC for the first time (2); all the patients had not received chemotherapy and radiation before surgery; and (3) all patients signed informed consent. The exclusion criteria in our study were as follows (1)

Proliferation of SCC-4 and HSC-3 cells was assessed via Cell
Counting

| Luciferase assay
The binding sites of miR-186-5p for circRNA_0001971 and FNDC3B were predicted by circInteractome and TargetScan, respectively.
According to the prediction, the wild-type circRNA_0001971 (WT

| RIP assay
Magna RIP Kit (Millipore, USA) was purchased for performing RIP assay. The OSCC cells transfected with miR-186-5p mimic or mimic-NC for 48 h were collected and lysed with RIP buffer. Then, the magnetic beads coated with anti-Ago2 or anti-IgG were added to cell lysates at 4°C overnight. Finally, the immunoprecipitated RNA was isolated to measure the enrichment of circRNA_0001971 by qRT-PCR. The sequences of circRNA_0001971 primers are listed in Table 2.

| Western blotting
For detecting the protein expression, RIPA lysis buffer (Beyotime, China) was first used to isolate the total proteins from cells. Then, the isolated proteins were separated by 10% SDS-PAGE followed by transferring onto PVDF membranes (Beyotime, China). Next, the membranes after blocking with fat-free milk were incubated with anti-

| Statistical analysis
All experimental values were analyzed as mean ± SD from three independent experiments. The differences for two groups were identified by paired student's t test, whereas ANOVA was applied to identify the differences for >2 groups. p value < 0.05 was statistically significant.

| The role of circRNA_0001971 in OSCC cells
After performing qRT-PCR, the level of circRNA_0001971 in OSCC tissues and cells was found to be upregulated compared with adjacent normal tissues or HOEC ( Figure 1A, B). Since the expression of circRNA_0001971 increased by almost fourfold in SCC-4 and HSC-3 OSCC cells, the transfection efficiency of two siRNAs targeting cir-cRNA_0001971 was determined in these cell lines ( Figure 1C). Then, the CCK8 assay displayed that silencing circRNA_0001971 suppressed cell proliferation in both two OSCC cells ( Figure 1D). The wound closure rate detected by wound-healing assay was decreased by approximately 50% in silencing circRNA_0001971 group, suggesting that circRNA_0001971 knockdown inhibited cell migration in OSCC cells ( Figure 1E). Moreover, the transwell assay discovered that silencing circRNA_0001971 impaired invasion ability of SCC-4 and HSC-3 cells ( Figure 1F). Our data proved the inhibitory role of silencing circRNA_0001971 in OSCC cells.

| miR-186-5p was sponged by circRNA_0001971 in OSCC cells
circInteractome was an online tool to predict the downstream miR-NAs of circRNA_0001971, as well as GSE82064 downloaded from GEO database aimed to screen the downregulated miRNAs in OSCC samples with adj.p < 0.05 and log 2 FC < −1. It was found that only one miRNA (miR-186-5p) was overlapped from circInteractome and GSE82064 (Figure 2A). According to the prediction by circInteractome, we designed the WT and MUT circRNA_0001971 vectors, and the sequences of WT and MUT circRNA_0001971 are shown in Figure 2B. The luciferase assay showed that co-transfection of WT circRNA_0001971 vectors and miR-186-5p mimic reduced the luciferase activity ( Figure 2C). The RIP assay further identified circRNA_0001971 as a sponge of miR-186-5p ( Figure 2D). In our clinical samples, miR-186-5p expression decreased by 50% in OSCC samples ( Figure 2E), as well as the level of miR-186-5p was negatively correlated to that of circRNA_0001971 in OSCC samples ( Figure 2F). These findings suggested that circRNA_0001971 could sponge miR-186-5p in OSCC cells.

| FNDC3B was a target of miR-186-5p in OSCC cells
To further explore the targets of miR-186-5p, TargetScan was used to predict the target genes for miR-186-5p, as well as GEPIA, an online tool, was applied to verify the upregulated mRNAs in OSCC with adj.p < 0.01 and log 2 FC > 1. As shown in Figure 3A, ten mRNAs were overlapped from TargetScan and GEPIA. According to the data from TCGA, FNDC3B was upregulated in head and neck squamous cell carcinoma ( Figure 3B) and was found to be an oncogene in tongue squamous cell carcinoma. 19 Therefore, FNDC3B was identified as the interested gene. The sequences of WT and MUT FNDC3B vectors are displayed in Figure 3C. After performing luciferase assay, the luciferase activity reduced the most in co-transfection of miR-186-5p mimic and WT FNDC3B group, and MUT1 or MUT2 FNDC3B together with miR-

186-5p mimic group induced a slight decrease in luciferase activity
( Figure 3D). By qRT-PCR, FNDC3B was found to be upregulated by 3.5-fold in OSCC samples ( Figure 3E), and its level was negatively correlated to miR-186-5p level in OSCC samples ( Figure 3F). All the data proved that FNDC3B was a target gene of miR-186-5p.

| FNDC3B overexpression partly abrogated the effect of circRNA_0001971 knockdown on OSCC cells
The result from Western blotting proved that circRNA_0001971 knock- Increasing studies reveal that the abnormal expression of cir-cRNAs is closely associated with OSCC progression; hence, circRNAs are considered as the key biomarkers of OSCC. 14,[20][21][22] For instance, circ_0008309 with low expression was proved to be correlated with pathological differentiation, and circ_0008309 upregulated ATXN1 expression by sponging miR-136-5p and miR-382-5p in OSCC cells. 22 circ-PVT1 functioning as a miR-106a-5p sponge promoted OSCC cell growth and metastasis by upregulating HK2. 23 Our study revealed that circRNA_0001971 was elevated in OSCC, and silencing cir-cRNA_0001971 suppressed the malignancy of OSCC cells, which was consistent with the conclusion from X Tan et al. 15 7,24 Here, we found miR-186-5p was a downstream of circRNA_0001971 in OSCC cells by bioinformatics analysis, luciferase assay, and RIP assay. miR-186-5p, a member of miRNAs, has been studied in human cancer, and its anticancer role is proved in various cancer types. For example, the anticancer roles of miR-186-5p were observed in gastric cancer, 25 colorectal cancer, 26 and ovarian cancer. 27 In OSCC, multiple studies found miR-186 was a tumor suppressor in OSCC by targeting FUT8 28 and SHP2. 29 Here, we for the first time proved that miR-186-5p was proved to be downregulated in OSCC samples, and FNDC3B was a target of miR-186-5p in OSCC cells.
Fibronectin type III domain containing 3B (FNDC3B) has been identified as a modulator of osteoblast and adipocyte differentiation. 19 In recent years, many evidence proved the positive role of FNDC3B in colorectal cancer, 30 cervical cancer, 31 glioblastoma, 32 and tongue squamous cell carcinoma cells. 19 However, FNDC3B has not been explored in OSCC. In this study, FNDC3B with high expression was observed in OSCC samples, and its overexpression partly overturned the effect of circRNA_0001971 on OSCC progression in vitro owing to FNDC3B as the downstream of circRNA_0001971/ miR-186-5p.
Our study proved the role of circRNA_0001971/miR-186-5p/ In summary, our study discovered that circRNA_0001971 was upregulated in OSCC and accelerated the progression of OSCC in vitro by ceRNA mechanism to sponge miR-186-5p/FNDC3B axis.
Our conclusion might provide novel evidence for circ_0001971 potential as a target of OSCC therapy.

ACK N OWLED G EM ENT
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interests.

AUTH O R CO NTR I B UTI O N S
JHZ designed this study, performed the experiments, and drafted the article. YJP analyzed the data and provided the funding. SJJ collected materials and resources. JL reviewed and edited the article.

I N FO R M E D CO N S E NT
Informed consent was obtained from all patients.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data and materials in the current study are available from the corresponding author if request is reasonable.