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Downregulation of circ-RANBP9 in laryngeal cancer and its clinical significance

Background Laryngeal cancer (LC) is a common malignant tumor of the head and neck. As circular RNAs (circRNAs) and other non-coding RNAs are involved in various malignant processes, we analyzed circRNAs to better understand LC and explored specific tumor markers. Methods High-throughput sequence was performed to analyze the differential circular RNAs in four coupled laryngeal cancers and para-cancerous tissues. The differential expression of selected circ-RANBP9 in laryngeal cancer tissues and cells was verified by RT-qPCR assay. CCK8, EDU, Transwell and wound healing assays were used to confirm the biological function of circ-RANBP9 in laryngeal cancer. Western blot assay was performed to identify the effects of circ-RANBP9 having on the epithelial to mesenchymal transition process. One-way AN0VA was used to analyze the correlation between the expression of circ-RANBP9 and clinicopathological parameters of the included patients. Kaplan-Meier analysis was used to investigate whether the expression level of circ-RANBP9 correlated with survival in LC patients. Bioinformatic analyses were also conducted to predict the functions and possible signaling pathways of the targeted mRNAs of circ-RANBP9 via co-expression and competing endogenous RNA network. Results We found a transcript from RNA sequence data, termed hsa_circ_0001578, which is a circRNA spliced from RANBP9. Circ-RANBP9 was downregulated in the LC cell lines tissues, relating to a better prognosis. Circ-RANBP9 was found to inhibit the proliferation, migration, and invasion ability of LC, exerting a suppressive role in the epithelial to mesenchymal transition process as well. For the diagnostic value of circ-RANBP9, the sensitivity and the specificity were 0.979 and 0.553, respectively. Circ-RANBP9 downregulation was significantly correlated with differentiation (P=0.031), T-stage (P=0.018), lymphatic metastasis (P=0.046), and clinical stage (P=0.003). Circ-RANBP9 was involved in insulin-like growth factor receptor binding, cell polarity, focal adhesion, and MAPK signaling pathways. CeRNA analysis identified the possible involvement of circ-RANBP9 in the ECM-receptor interaction, cAMP, calcium, and Wnt signaling pathways by harboring miRNA genes. Conclusions Circ-RANBP9 was confirmed to play important roles in inhibiting laryngeal cancers. Circ-RANBP9 was also validated to be associated with the clinicopathological parameters and diagnostic value, suggesting that circ-RANBP9 is a promising biomarker for LC prognosis and early diagnosis.


Introduction
Laryngeal cancer (LC) is the second most common malignancy of the head and neck, accounting for 30-50% of these tumors (1). Laryngeal squamous cell carcinoma accounts for 85-90% of all LC cases (2). The initiation and progression of LC is a complicated process driven by various pathogenetic factors and dysregulation of multiple genes (3). Smoking and heavy alcohol consumption have been deemed to be the most significant risk factors for LC, and HPV infection, air pollution and sex hormone levels have also been considered as the risk factors for LC (4). With respect to dysregulated genes, accumulating studies have shown that the dysregulation of multiple genes contribute to the malignant progression of various tumors, especially LC (5). For instance, hsa_circ_0057481 was upregulated in LC, and promoted LC proliferation and migration by modulating the miR-200c/ZEB1 axis (6). Currently, surgery and radiotherapy are the main treatment methods for LC, but the results of these treatments are frequently unsatisfactory. Due to the difficulty in early diagnosis, a large number of patients are diagnosed with an advanced stage or lymph node metastasis. Furthermore, the 5-year survival rate for patients in advanced stages is less than 50% (7). Therefore, it is particularly important to explore new biomarkers for the early diagnosis of LC.
Due to the rapid development of high-throughput sequencing, a multitude of non-coding RNAs have been proven to participate in the tumorigenesis and progression of malignant tumors (8). Circular RNAs (circRNAs), which are a new type of non-coding RNA with strong stability, high tissue specificity, and high expression abundance, have attracted more and more attention recently. The majority of previous studies have shown that circRNAs act as microRNA sponges, which regulate tumorigenesis though ceRNA network (9). Additionally, circRNAs were also found to play important roles in gene splicing, transcription and translation (10,11). For example, circITGA7 was found to promote the transcription of its parent gene integrin alpha 7 (ITGA7) through suppressing a transcription factor RAS responsive element-binding protein 1 (RREB1) (10). Moreover, circ-UBR5 was observed to be involved in RNA splicing regulatory process by interacting with splicing regulatory factor QKI, NOVA alternative splicing regulator 1 (NOVA1) and U1 small nuclear RNA (snRNA) in the NSCLC cells (11). Interestingly, accumulating studies have focus on the RBP mechanism of RNA interacting with protein and on how circular RNA regulate the RNA binding protein (12,13). For instance, circACC1 binds to β and γ subunits of AMP activated protein kinase (AMPK) to assemble a large complex, thus stabilizing and enhancing the enzymatic activity of AMPK holoenzyme (13). So far, there have been many studies on molecular mechanisms investigations of LC (14). And a few studies have been proven that circular RNAs play a very important role in the pathogenesis of LC (15,16). However, different from those studies on LC, our study proposed a novel circular RNA, analyzing its prognostic value and diagnostic value, and made a comprehensive analysis for its downstream signaling pathway. Therefore, in this study, we aimed to analyze circRNAs to better understand the pathogenesis and progression of LC, and explored specific tumor biomarkers that can help facilitate the early diagnosis of LC. We present the following article in accordance with the MDAR reporting checklist (available at http://dx.doi.org/10.21037/ atm-21-567). The patients who were included in the study underwent total or partial laryngectomy and did not receive any treatment before diagnosis. The diagnosis of LC was based on postoperative pathology. Inclusion criteria: (I) clear pathological diagnosis of laryngeal cancer; (II) new cases of LC; (III) the patient did not receive any radiotherapy, chemotherapy or other anticancer treatments before surgery.  , and informed consent was obtained from all patients. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013).

Cell culture
The human LC cell lines LCC, and LLN were purchased from the Institute of Basic Medical Sciences (Beijing,

Sequencing analysis
Five out of the 47 pairs of specimens were selected for whole-transcriptome sequencing using the Illumina HiSeq X Ten instrument. CIRI (17) and find_circ (18) were used to predict circRNAs. Differentially expressed transcripts and genes were identified by the criteria of |log2(fold-change)| ≥1 and a P value ≤0.05 between the paired samples.

Plasmids, siRNAs and transfection
A cDNA fragment that contained the sequence of circ-RANBP9 was cloned into the PLCDH vector (GenePharma) to overexpress circ-RANBP9. The siRNA of circ-RANBP9 was synthesized by GenePharma (Suzhou, China). The plasmids and circ-RANBP9 siRNA was transiently transfected into cells using the X-tremeGENE siRNA or DNA Transfection Reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions. The sequences of plasmids, siRNAs are shown in Table S1.
The primers in our study were synthesized by Shanghai Shenggong Company (Shanghai, China). The PCR program was set at 95 ℃ for 5 min, then 95 ℃ (5 sec) and 60 ℃ (34 sec) in a total of 40 cycles. The 2 −ΔΔCt method was used to analyze RNA expression levels (19).

RNA fluorescence in situ hybridization (RNA-FISH)
Cells were fixed on slides with 4% paraformaldehyde for 20 minutes.
Then, the circ-RANBP9 probe was hybridized overnight at a concentration of 6 ng/µL. The hsa_circ_0001578 probe sequence for FISH was 5'-GCTTCCTTTGCCTGCTGA AAAATGTG-3'. Images were captured using a fluorescence microscope (Eclipse CI; Nikon Corporation).

EdU assay
Twenty-four hours after transfection, the transfected cells were seeded into 12-well plates at a density of 1×10 4 cells per well, cultrred with EDU for 2 h, and then stained with the Cell Light EdU Apollo488 In Vitro Imaging Kit (RiboBio). The percentage of EdU positive cells were defined as proliferation rate in EdU assay. The images were obtained using the Olympus microscope (Olympus, Tokyo, Japan). Independent proliferation assays were conducted three times.

Cell migration and invasion assay
Transwell chambers containing a polycarbonate membrane with 8.0 µm pores (Corning, NY, USA) were placed in a 24well plate. For the migration assays, transfected cells were digested and diluted to a density of 5×10 4 cells per 200 µL and then added to upper chamber, medium with 20% FBS was added to the lower chamber. After incubation for 24-48 h, the cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet. For invasion assays, the polycarbonate membrane was coated with Matrigel matrix (BD Bioscience). For quantification, cells were counted in five randomly selected fields using a microscope (IX51, Olympus, Japan). Independent transwell assays were performed three times. The data was shown as mean ± standard deviation (SD).

Wound healing assay
Cells were seeded in a 6-well plate and transfected when cell confluence reached 60-70%. When the cells reached confluency, the well was scratched with a 200 µL pipette tip, three lines at least acrossing each well. The cells were incubated with 1% FBS. The images were obtained at 0 and 48 h after healing using a microscope (IX51, Olympus, Japan).

Statistical analysis
Statistical significance was determined using Student's t-test, with SPSS 22.0 (IBM, Armonk, NY,USA). MeV4 (http://mev.tm4.org) and GraphPad Prism 6 (GraphPad, San Diego, USA) were used to generate figures. P<0.05 was considered to indicate statistical significance, and P<0.01 was considered to indicate high significance.

Differentially expressed circRNAs
We conducted high-throughput transcriptome sequencing on 5 pairs of LC tissues. According to the criteria of fold-change ≥2.0 and P<0.05, 77 differentially expressed circRNAs were found in the LC samples. Compared with adjacent normal tissues, 22 circRNAs were upregulated and 55 circRNAs were downregulated in LC tissues. The significantly differentially expressed circRNAs are shown in the heatmap in Figure 1.

Downregulation of circ-RANBP9
Our sequencing data demonstrated that expression of circ-RANBP9 was low in LC ( Table 1). We used RT-qPCR to assess the differential expression of circ-RANBP9 in the cell lines TU212, LCC, LLN, and HaCat. Our results revealed that circ-RANBP9 expression was downregulated in all 3 LC cell lines by 3.2-fold compared with that of HaCat cells ( Figure 2A). We also validated the differential expression of circ-RANBP9 in 47 pairs of cancer and non-cancer tissue samples, and observed low circ-RANBP9 expression in most LC tissues ( Figure 2B,C). Furthermore, we constructed a receiver operating characteristic curve to examine the diagnostic value of circ-RANBP9. The cutoff value was 0.0007 ( Figure 2D), the area under the curve was 0.716, and the sensitivity and the specificity were 0.979 and 0.553, respectively. Importantly, we found that higher expression of circ-RANBP9 was related to the higher survival rates (hazard ratio =0.3336, P=0.0290) ( Figure 2E).  N1  N3  N4  N5  T1  T3  T4  T5   0  5 The main characteristics of circ-RANBP9 Next, we confirmed the characteristics of circ-RANBP9. According to circBase, circ-RANBP9 is spliced from the RANBP9 gene, and is rooted in chr6:13639794|13644961 with a length of 598 nt ( Figure 3A). Localization of circ-RANBP9 was validated by FISH analysis, which indicated that it was localized both in the nucleus and cytoplasm of TU212 and LCC cells ( Figure 3B). Circ-RANBP9 was resistant to RNase R digestion, demonstrating the cyclic structure by RT-qPCR ( Figure 3C,D).

The anticancer role of circ-RANBP9
To validate the functional role of circ-RANBP9 in LC, we used silenced or overexpressed circ-RANBP9 by transfection with si-RNA or RANBP9, respectively. Transfection efficiency was determined using RT-qPCR ( Figure 4A). Subsequently, the effects of circ-RANBP9 on LC cell proliferation, migration, and invasion were assessed. Overexpression of RANBP9 markedly inhibited cell proliferation, whereas knockdown of circ-RANBP9 significantly enhanced cell viability ( Figure 4B). The EDU assay was performed to observe the effects of circ-RANBP9 on LC cells, and revealed that higher circ-RANBP9 inhibited cell proliferation, whereas cell viability was enhanced by knockdown of circ-RANBP9 ( Figure 4C). Additionally, the transwell assay was conducted in LCC cells transfected with siRNA or RANBP9, and showed that the overexpression of circ-RANBP9 significantly inhibited the invasiveness and mobility of LC cells, whereas knockdown of circ-RANBP9 had the opposite effect ( Figure 4D). Also, overexpression significantly increased the wound healing ability of cells, whereas downregulation of circ-RANBP9 significantly inhibited cell motility ( Figure 4E). Our results confirm the role of circ-RANBP9 in inhibiting LC cell growth. Furthermore, the western bolt assay was used to observe the effects of circ-RANBP9 on the epithelial to mesenchymal transition process, and demonstrated that higher circ-RANBP9 inhibited the epithelial to mesenchymal transition process, whereas lower circ-RANBP9 promoted the epithelial to mesenchymal transition process ( Figure 4F). The results indicated that circ-RANBP9 inhibited the metastasis of laryngeal cancer.

Correlation between circ-RANBP9 level and clinical parameters
We investigated the relationship between circ-RANBP9 expression and the clinicopathological parameters of the included patients (

Bioinformatics analysis
Pearson's correlation coefficient was calculated to assess the correlation between circ-RANBP9 and differentially expressed mRNAs, and transcripts were identified according to the criteria of P>0.05 or P<−0.05. GO ( Figure 5A) and KEGG ( Figure 5B) analyses from the co-expressed mRNAs were performed to predict possible functions and pathways. Insulin-like growth factor receptor binding and regulation of establishment or maintenance of cell polarity were identified as significant GO items. Focal adhesion and MAPK pathways were considered to be the primary enrichment signaling pathways. The ceRNA network was also constructed to predict miRNAs and the corresponding targeted mRNAs related to circ-RANBP9. As shown in Figure 6, we found that circ-RANBP9 could target JAK3, FOXN1, MYC, and APC2 by harboring miR-4746-3p, miR-4757-5p, miR-3131, and miR-611. Subsequently, we conducted GO and KEGG analyses based on the mRNAs in the ceRNA network ( Figure 7A,B) and found that circ-RANBP9 exerted anticancer activity through the extracellular matrix (ECM)-receptor interaction, cAMP, calcium, and Wnt signaling pathways. Four proteins were found to be the possible target RNA-binding proteins of circ-RANBP9:AGO2, EIF4A3, HuR, and U2AF65 ( Table 3).

Discussion
A multitude of non-coding RNAs have been found, which can be attributed to the rapid development of highthroughput sequencing (20). Although non-coding RNAs    were once considered as meaningless transcriptional noise, relevant studies have confirmed their involvement in disease progression (8,21). CircRNAs, a new type of non-coding RNA, have closed-loop strings formed by 3'-5' terminal reverse splicing (18). CircRNAs can act as sponges for miRNAs and participate in the mechanisms of ceRNAs (22). They also encode proteins at internal ribosome entry sites and regulate transcription factors by binding to proteins. CircRNA dysregulation has been observed in tumors and is involved in tumor pathogenesis. For example, circAGFG1 expression is upregulated in breast cancer tissues and is related to both clinical stage and poor prognosis (23). However, the mechanism of action of most circRNAs is not well known. In our sequencing results, we found that circ-RANBP9 expression was low in cancer tissue samples compared with that in normal tissue samples. Furthermore, qRT-PCR was used to assess the differential expression of circ-RANBP9 in LC cell lines and tissue samples.  Despite the fact that non-coding RNAs have no proteincoding functions, their functions can, nevertheless, be predicted through ceRNA mechanisms or RNA-binding proteins. According to the parent gene theory (24), the coexpression network was used to predict circRNA function by means of co-expressed mRNAs. KEGG analysis of co-expressed mRNAs indicated that the predominantly enriched signaling pathways were the MAPK and focal adhesion pathways. The MAPK signaling pathway is linked to cell growth, differentiation, apoptosis, and cancer progression (25). Four cascades exist in the MAPK signaling pathway: ERK1/2, p38 MAPK, c-Jun N-terminal kinase (JNK), and ERK5 (26). It has been shown that p38is involved in the invasion and metastasis of breast cancer (27), while ERK5 is involved in both cell proliferation in prostate cancer (28) and cell proliferation, migration, and invasion in bladder cancer (29). The focal adhesion signaling pathway has also been found to be related to the tumorigenesis and progression of various cancers (30). Our study showed that circ-RANBP9 inhibited the proliferation, invasion, and migration of LC cells. Combined with the analysis above, we speculated that circ-RANBP9 might have anticancer effects on LC through the MAPK signaling pathway or the focal adhesion signaling pathway.
CeRNA analysis demonstrated that circ-RANBP9 could harbor miR-4746-3p, miR-4757-5p, miR-3131, and miR-611.The KEGG analysis of the ceRNA network then revealed enrichment in the ECM-receptor interaction, cAMP, calcium, and Wnt signaling pathways. The pathways mentioned above have been shown to be dysregulated in multiple malignant tumors. For example, the cAMP/ PKA pathway is abnormally activated and involved in both angiogenesis and vasculogenic mimicry in colorectal cancer (31). The Wnt/β-catenin signaling pathway is associated with a variety of human tumors. β-catenin is a primary component of the Wnt/β-catenin pathway and regulates downstream target genes through their translocation from the cytoplasm to the nucleus (32). Approximately 40-70% of hepatocellular carcinomas show β-catenin nuclear accumulation and abnormal Wnt/ β-catenin activation (33,34). Our results showed that circ-RANBP9 expression was lower in LC and that it exerted Italic P values indicate statistical significance. One-way ANOVA was used to analyze the correlation between the expression level of circ-RANBP9 and clinicopathological parameters of the patients. *, P<0.05. The ECM-receptor interaction pathway is a common signaling pathway that was found in both the KEGG analysis and the ceRNA network. The shared mRNA was LAMA2, which also showed lower expression in the sequencing data. Therefore, we suspect that circ-RANBP9 may regulate the pathogenesis of LC by harboring hsa-miR-5787 to suppress the expression of LAMA2.
In addition, the circular RNA interactome was used to predict the possible RNA-binding proteins of circ-RANBP9. Among these proteins, HuR has been shown to bind to the long non-coding RNA (lncRNA) OIP5-AS1 and is associated with the proliferation of cervical carcinoma cells (35). U2AF65 was also found to be an RNA-binding protein of lncRNA NEAT1and is related to the proliferation and invasion of hepatocellular carcinoma cells (36). Thus, we speculate that circ-RANBP9 regulates LC through the RNA-binding proteins that were predicted using the circular RNA interactome.
Recently, a few studies have investigated the molecular mechanisms affecting laryngeal cancer. For example, circ_0004507 was found to contribute to laryngeal cancer progression and cisplatin resistance by sponging miR-873 to upregulate multidrug resistance 1 and multidrug resistance protein 1 (37). Similarly, knockdown of hsa_ circ_0023028 inhibits cell proliferation, migration, and invasion in laryngeal cancer by sponging miR-194-5p (38). However, the precise pathogenesis underlying LC remains largely unknown. Its frequent recurrence and high mortality may be attributed to resistance to both chemotherapy and radiotherapy. Early diagnosis can lead to an improved quality of life and better outcomes for patients with LC (39). The diagnosis of LC depends largely on its signs and symptoms, along with auxiliary examinations. The symptoms are frequently nonspecific, which may result in misdiagnosis. Despite its traumatic nature, conventional biopsy of the tumor is the best method for the accurate diagnosis of LC. Clinical examinations such as computed tomography, magnetic resonance imaging, or positron emission tomography are not sufficient for an accurate diagnosis (40,41). Thus far, no ideal biomarker has been found to replace these clinical examinations for LC and improve diagnostic accuracy. The results of our study suggest that circ-RANBP9 could have 97.9% accuracy and 55.3% specificity for the diagnosis of LC, with an area under the curve of 0.716. These results indicate that circ-RANBP9 could be useful for the diagnosis of LC. Our results showed that higher expression of circ-RANBP9 was correlated with a better outcome. Specifically, considering that circ-RANBP9 inhibited the invasion and migration capacity through functional assays, combing with the fact that circ-RANBP9 also inhibited epithelial to mesenchymal transition process, we believe that circ-RANBP9 can inhibit the metastasis of laryngeal cancer. Furthermore, through analysis of clinicopathological characteristics, we uncovered that the lower expression of circ-RANBP9 was correlated with differentiation level, T stage, lymphatic metastasis and clinical stage, indicating that circ-RANBP9 may be considered a valuable biomarker for the prediction of clinical outcomes. Furthermore, we can investigate novel medicine targeting the molecular axis of circ-RANBP9, hsa-miR-5787, and LAMA2 for precision treatment. Then the expression level of circ-RANBP9 should be considered as prognostic index for predicting outcome. In addition, as accumulating studies showing that dysregulated genes were related to risk stratification and local regional metastasis in tumors (42), combining with our results that circ-RANBP9 was related to the clinicopathological characteristics and involved in the metastasis of LC, we should speculate that circ-RANBP9 may be used as a potential biomarker for risk stratification and local regional metastasis in patients with laryngeal cancer. Certainly, it needs further identification. Our findings may therefore improve the early diagnosis, early treatment, and overall prognosis for LC.

Conclusions
Our results are the first to indicate that circ-RANBP9 plays a crucial role in the pathogenesis and progression of LC and has the potential to be used as a valuable biomarker for early diagnosis and prognosis. Furthermore, circ-RANBP9 may be a useful target for the development of future therapies. Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the noncommercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.