Circular RNA ABCB10 contributes to laryngeal squamous cell carcinoma (LSCC) progression by modulating the miR-588/CXCR4 axis

Laryngeal squamous cell carcinoma (LSCC) is a common head and neck cancer with a high metastasis and poor prognosis. Circular RNAs (circRNAs) are a type of non-coding RNAs (ncRNAs) with regulatory function and broadly participate in cancer development. However, the correlation of circular RNA ABCB10 (circABCB10) with LSCC remains unclear. Here, we were interested in the role of circABCB10 in the modulation of LSCC progression. Our data demonstrated that the depletion of circABCB10 significantly inhibited the proliferation and induced the apoptosis of LSCC cells. Meanwhile, circABCB10 knockdown was able to remarkably reduce the invasion and migration of LSCC cells. Mechanically, circABCB10 served as a sponge for microRNAs-588 (miR-588) and miR-588 could target and down-regulated chemokine receptor 4 (CXCR4) expression in LSCC cells. The overexpression of CXCR4 or miR-588 inhibitor could reverse circABCB10 depletion-attenuated malignant phenotypes of LSCC cells. Functionally, the depletion of circABCB10 alleviated the tumor growth of LSCC cells in the tumorigenicity analysis of nude mice. The CXCR4 expression was decreased while the miR-588 expression was enhanced by circABCB10 depletion in vivo. Thus, we concluded that circABCB10 was involved in the malignant progression of LSCC by regulating miR-588/CXCR4 axis. Our finding provides new insights into the mechanism of circRHOT1 contributing to the development of LSCC. CircABCB10 and miR-588 may be used as potential targets for the treatment of LSCC.


INTRODUCTION
Laryngeal squamous cell carcinoma (LSCC) is a prevalent and severe malignancy [1,2]. Despite the improvement of current therapies, such as radiotherapy, chemotherapy, and surgery, the survival rate for advanced LSCC patients remains unsatisfactory [3,4]. Surgery and radiotherapy are broadly used, and chemotherapy is applied in several curative approaches [3,4]. The high rate of LSCC recurrence is usually caused by chemotherapy resistance and high invasiveness [5,6]. Consequently, it is required for the profoundly comprehend the mechanisms of LSCC progression and carcinogenesis for the development of more efficient therapeutic strategies.
Another well-recognized small ncRNAs called microRNAs with about 22 nucleotides target 3′ untranslated region (UTR) of various genes to inhibit their expression in many critical biological processes [15][16][17]. Multiple miRNAs have been identified in the modulation of LSCC. For instance, miR-613 inhibits the progression of LSCC by targeting PDK1 [18]. MiR-154 reduces the LSCC growth by inhibiting GALNT7 [19].
Meanwhile, it has been demonstrated that miR-588 serves as a tumor inhibitor in various cancers, such as lung and breast cancer [20,21]. Moreover, chemokine receptor 4 (CXCR4) is abnormally expressed in LSCC and contributes to LSCC malignant progression [22,23]. However, the influence of circABCB10 and miR-588 on CXCR4 in LSCC development remains obscure.
We tried to explore the roles of circABCB10 in LSCC and we reported the new functions of circABCB10 in promoting malignant progression of LSCC via the miR-588/CXCR4 axis.

CircABCB10 promotes proliferation and inhibits apoptosis of LSCC cells
We evaluated the effect of circABCB10 on LSCC cell proliferation. MTT assays demonstrated that the circABCB10 depletion repressed cell viabilities ( Figure  1A and 1B). Consistently, the colony numbers were suppressed by circABCB10 knockdown (Figure 1C and 1D). Furthermore, cell apoptosis was promoted by the circABCB10 depletion in the cells ( Figure 1E and 1F), suggesting that circABCB10 increases proliferation and represses apoptosis of LSCC cells.

CircABCB10 enhances invasion/migration ability of LSCC cells
We then investigated functions of circABCB10 in modulation of migration/invasion abilities. Transwell assays showed that the capability of migration/invasion was suppressed by depletion of circABCB10 ( Figure  2A and 2B). The knockdown of circABCB10 AGING significantly increased wound proportion ( Figure 2C and 2D), indicating that circABCB10 is able to induce the migration/invasion of LSCC cells.

CircABCB10 is able to sponge miR-588 in LSCC cells
Next, we further investigated the mechanism underlying circABCB10-regulated LSCC progression. We found the potential interaction between circABCB10 and miR-588 using a bioinformatic analysis ( Figure 3A). The AMC-HN-8 and Hep-2 cells were treated with miR-588 mimic and presented enhanced miR-588 expression ( Figure 3B). The miR-588 significantly inhibited luciferase activity of wild type circABCB10, but not circABCB10 mutant ( Figure 3C and 3D). Consistently, the depletion of circABCB10 remarkably promoted miR-588 expression in the AMC-HN-8 and Hep-2 cells ( Figure  3E and 3F), suggesting that circABCB10 is able to sponge miR-588 in LSCC cells.

MiR-588 is able to target CXCR4 in LSCC cells
Next, we also revealed the potential miR-588-binding site within CXCR4 3′UTR in a bioinformatic analysis ( Figure 4A). The miR-588 mimic inhibited the luciferase activities of wild type CXCR4, but not CXCR4 mutant ( Figure 4B). In addition, the levels of CXCR4 were remarkably down-regulated by miR-588 mimic in the cells ( Figure 4C and 4D). Moreover, depletion of circABCB10 significantly reduced expression of CXCR4, and miR-588 inhibitor reversed the result ( Figure 4E). The migration and invasion were examined by wound healing assays in the cells. The wound healing proportion was shown. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01. AGING CircABCB10 contributes to LSCC progression by miR-588/CXCR4 axis Next, we analyzed the function of circABCB10/miR-588/CXCR4 axis in the regulation of LSCC malignant progression. As expected, the CXCR4 overexpression or miR-588 inhibitor could increase the circABCB10 depletion-inhibited cell viability in the AMC-HN-8 and Hep-2 cells ( Figure 5A and 5B). Furthermore, apoptosis was increased by depletion of circABCB10, in which overexpression of CXCR4 or miR-588 inhibitor could reverse this phenotype in the AMC-HN-8 and Hep-2 cells ( Figure 5C and 5D). Together these data indicate that circABCB10 enhances LSCC malignant progression by regulating miR-588/CXCR4 axis.

CircABCB10 promotes the cell proliferation of LSCC in vivo
Next, the function of circABCB10 in regulating LSCC cell growth was explored in vivo. Tumorigenicity analysis showed that the circABCB10 depletion attenuated the tumor growth, as presented by the decreased tumor size ( Figure 6A), repressed tumor volume ( Figure 6B), and reduced tumor weight ( Figure  6C). As expected, the miR-588 expression was upregulated and CXCR4 expression was down-regulated by circABCB10 knockdown in the tumor tissues of the mice ( Figure 6D and 6E). Taken together, these data suggest that circABCB10 promotes the tumor growth of LSCC in vivo.

DISCUSSION
LSCC is the prevailing head and neck cancer with high mortality and widely affects modern people [2]. CircRNAs exert important roles in development of LSCC (Liang, 2017 #5). Nevertheless, the effect of circABCB10 on the tumorigenesis of LSCC is still poorly investigated. We firstly reported that circABCB10 promoted malignant progression of LSCC by the miR-588/CXCR4 axis.

AGING
Many previous investigations have shown several circRNAs in the LSCC pathogenesis. It has been reported that circMYLK promotes LSCC progression by miRNA-195/cyclin D1 signaling [24]. The upregulation of circRNA100290 enhances the LSCC development [25]. Circ103862 contributes to the invasion of LSCC [26]. Besides, circABCB10 enhances migration and proliferation of lung cancer by miR-1252/FOXR2 signaling [27]. CircABCB10 increases the progression of non-small cell lung cancer [28]. CircABCB10 contributes to invasion and proliferation of esophageal squamous cell carcinoma cells by targeting miRNA-670-3p [29]. We demonstrated that circABCB10 enhanced LSCC progression. CircABCB10 promoted the cell growth of LSCC in vivo. It demonstrates the critical roles of circABCB10 in the LSCC, indicating informative evidence of the function of circRNAs in modulating LSCC.
As the interacted non-coding regulators of circRNAs, miRNAs are also well-described in LSCC development. MiRNA-26a represses tumorigenesis and proliferation of LSCC by inhibiting CKS2 [30]. CircFLNA elevation promotes LSCC migration [31]. MiRNA-195 suppresses cell invasion, migration, and proliferation in LSCC by down-regulating ROCK1 [32]. MiR-588 as a tumor suppressor is involved in long non-coding RNA GSEC-mediated osteosarcoma progression [33]. Furthermore, it has been reported that CXCR4 contributes to LSCC metastasis by regulating MMP-13/ERK1/2/AP-1 signaling [22]. CXCR4 induces angiogenesis of LSCC [34]. Our mechanical investigation revealed that circABCB10 could serve as a sponge for miR-588 and miR-588 was able to target CXCR4 in LSCC cells. The overexpression of CXCR4 or miR-588 inhibitor could reverse circABCB10 knockdown-inhibited LSCC malignant phenotypes in vitro. These data reveal an unreported association of circABCB10 with miR-588 and CXCR4, elucidating a new mechanism involving circABCB10, miR-588, and CXCR4 in LSCC pathogenesis.
In this study, we identified the new function of circular RNA circABCB10 in the development of LSCC, and we found that miR-588/CXCR4 axis was involved in circABCB10-meidiated LSCC progression. Despite the innovation of this study, there are still some limitations of this study. The significance and the relationship of circABCB10, miR-588, and CXCR4 in the clinical LSCC samples were elusive, which deserve to be investigated. Meanwhile, the miR-588/CXCR4 axis might be just one of the downstream factors of circABCB10 in regulating LSCC progression. Other potential mechanisms need to be explored in the future study.  Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01. AGING Moreover, we identified that circABCB10 contributed to the LSCC cell invasion, migration, proliferation, and repressed LSCC cell apoptosis in vitro. CircABCB10 was also involved in growth of LSCC cells in vivo. It implies that targeting circABCB10 may be a potential therapeutic strategy in the clinic, and more evidence needs to be found to validate its therapeutic value. Meanwhile, the expression of circABCB10 in the clinical LSCC samples and the correlation of circABCB10 with the survival rate of LSCC patients needed to analyze to evaluate the diagnostic and prognostic significance of circABCB10.
In conclusion, we discovered that circABCB10 was involved in the malignant progression of LSCC by regulating the miR-588/CXCR4 axis. CircABCB10 and miR-588 may serve as targets for LSCC therapy.

MTT assays
For MTT assay, AMC-HN-8 and Hep-2 cells were placed in each well of a 96-well plate, followed by the treatment. After 12 hours incubation, 10 μL MTT solution was added in the medium to incubate for another 4 hours, followed by replacement with 150 μL DMSO. The absorbance at 570 nm was detected by a microplate reader.

Colony formation assays
For colony formation assay, 1 × 10 3 AMC-HN-8 and Hep-2 cells were suspended as single cell and seeded into a 6-well plate with 1000 cells per well, and incubated for 2 weeks to form visible colonies. The colonies were stained by 1% crystal violate resolved in methanol for 30 minutes, and photographed.

Transwell assays
The migration and invasion ability of AMC-HN-8 and Hep-2 cells were determined via using a transwell chamber (Corning, USA). To detect migration, AMC-HN-8 and Hep-2 cells (1 × 10 5 cells/well) were seeded into the upper chambers with FBS-free medium, while the lower chambers were filled with complete DMEM medium. After 24 hours incubation, the membranes of upper chambers were fixed by 4% paraformaldehyde for 15 min, and stained by 0.5% crystal violet for 30 minutes. For cell invasion, the process was similar with that of migration experiment, only that the upper chambers were coated with Matrigel (BD Bioscience, USA).

Wound healing assay
Wound healing experiment was also performed to detect cell migration. In short, cells were planted in a 6well plate at a density of 3 × 10 5 cells per well, and incubated overnight to form a monolayer confluence. Then the monolayer was scratched by a 200 μL pipette tip, and washed with PBS to remove debris. FBS-free medium was added to the wells and cells were cultured for 12 hours. The width of wound was captured and measured at 0, 6, and 12 hours under a microscope.

Analysis of cell apoptosis
To determine apoptotic cells, we adopted an Annexin V FITC Apoptosis Kit (CST, USA). In short, 2 × 10 5 AMC-HN-8 and Hep-2 cells were placed in each well of a 6well plate, digested and suspended in binding buffer. Subsequently, the cells were stained by Annexin-V for 20 minutes at room temperature, followed by staining with PI. The samples were analyzed on a C6 flow cytometer (BD Biosciences, USA).

Luciferase reporter gene assay
The sequence of circABCB10 and 3′UTR region of CXCR4 were cloned into the pmiR-Glo vector (Promega, USA). Cells were transfected with the WT or Mut along with d miR-588mimics or NC for 24 hours. The cells were lysed and the luciferase activity was measured by a dual luciferase detection kit (Promega, USA).

Western blot analysis
Total protein was extracted from AMC-HN-8 and Hep-2 cells using a RIPA lysis reagent (Sigma, USA), quantified by using a BCA kit (Sigma, USA), separated in the SDS-PAGE gel, and transferred to PVDF membranes. Subsequently, the blots were soaked in blocking buffer for 15 minutes, and incubated in diluted primary antibodies against CXCR4 (Abcam, USA) and β-actin (Abcam, USA), overnight at 4°C. Next day, the blots were washed with PBS and incubated with HRPconjugated secondary ani-mouse or anti-rabbit antibody, accordingly. All antibodies used in this work was purchased from Abcam (USA) and diluted as instructions of the manufacturer. The blots were visualized by using the ECL solution (Beyyotime, China) and captured in a gel imaging system (BioRad, USA).

Analysis of tumorigenicity in nude mice
AMC-HN-8 cells (1 × 10 7 , 100 μL) were subcutaneously injected into the left fat pad of Balb/c nude mice (n = 5) aged 4-week. Five days after inoculation, the mice were sacrificed and tumors were scaled. The tumor size (volume = 0.5 × width 2 × length) and body weight were measured at the indicated time points. Animal care and method procedure were authorized by the Animal Ethics Committee of Cangzhou Central Hospital, was carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

Statistical analysis
Each experiment was repeated three times and the data was presented as means ± SD. Statistical analysis was carried out by using a Graphpad prism. Mann-Whitney U and one-way ANOVA were adopted to evaluate differences between two or more groups. P < 0.05 was set as threshold for statical significance.

AUTHOR CONTRIBUTIONS
Jin Zhao and Xing-De Li designed and performed experiments, analysed data and wrote the paper; Ming Wang and Li-Na Song designed and performed experiments; Mei-Jiao Zhao designed experiments, analysed data and wrote the paper.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest related to this study.