hsa_circ_0023305 Enhances Laryngeal Squamous Cell Carcinoma Progression and Modulates TRPM7 via miR-218-5p Sponging

Recently, circular RNAs have been shown to function as critical regulators of many human cancers. However, the circRNA mechanism in laryngeal squamous cell carcinoma (LSCC) remains elusive. Recent investigations using bioinformatics analysis revealed high expression of hsa_circ_0023305 in LSCC tissues compared to normal tissues. Furthermore, we discovered that hsa_circ_0023305 expression level was positively correlated to tumor/node/metastasis (TNM) stage as well as lymph node metastasis in LSCC. Moreover, higher hsa_circ_0023305 levels were correlated to poorer LSCC patient outcomes. Knockdown of hsa_circ_0023305 significantly inhibited LSCC cell proliferation, invasion, and migration abilities. Our team validated that hsa_circ_0023305 functioned as a miR-218-5p sponge from a mechanistic perspective, targeting the melastatin-related transient receptor potential 7 (TRPM7) in LSCC cells. TRPM7 regulates a nonselective cation channel and promotes cancer proliferation and metastasis. Our data demonstrated that miR-218-5p was downregulated in LSCC and that miR-218-5p upregulation repressed LSCC proliferation and invasion both in vivo and in vitro. Additionally, we found that hsa_circ_0023305-mediated upregulation of TRPM7 inhibited miR-218-5p and contributed to LSCC migration, proliferation, and invasion. In summary, these data propose a new mechanism by which the hsa_circ_0023305/miR-218-5p/TRPM7 network enhances LSCC progression.


Introduction
Laryngeal cancer is a malignancy of the larynx that accounts for 1-5% of total malignant tumor incidence. Laryngeal squamous cell carcinoma (LSCC) is the most general class of laryngeal cancer [1]. Currently, LSCC treatment primarily includes radiation therapy, surgery, and chemotherapy along with biological therapy [2]. The fiveyear survival rate of LSCC is greater than 90% with early treatment; metastasis and recurrence are the main factors that influence LSCC prognosis. Poor differentiation and lymph node metastases are associated with reduced fiveyear survival rate [3]. As such, early diagnosis and treatment are extremely indispensable for lowering laryngeal cancer mortality.
Recently, circular RNAs (circRNAs) have received attention regarding their correlation with human diseases, particularly cancer, suggesting a potential use for circRNAs as novel biomarkers and therapeutic targets [4,5]. circRNAs are an abundant type of endogenous RNA and have recently been rediscovered and reevaluated for their essential functions in the regulation of gene expression [5]. circRNAs primarily function as noncoding RNA molecules that are comprised of a covalently closed continuous loop without 5′-3′ polarity or a poly-A tail. circRNAs have a relatively jarless framework, and their expression is tissue-specific in the eukaryotic transcriptome. Emerging evidence has identified the presence of endogenous circRNAs in mammalian cells [6]. These circRNAs regulate gene expression at the posttranscriptional or transcriptional level by binding to and inhibiting the function of microRNAs (miRNAs) and other molecules [6]. Previous studies have found that cir-cRNA_100290 overexpression enhances LSCC progression via the miR-136-5p/RAP2C axis [7]. The circular RNA cir-cCORO1C promotes LSCC progression via modulation of the let-7c-5p/PBX3 axis [8].
Our study with high-throughput sequencing identified many circRNAs with abnormal expression. Among these circRNAs, hsa_circ_0023305 expression was increased in LSCC tissues. However, the precise relationship between hsa_circ_0023305 expression and LSCC progression remains unclear. Therefore, the current study is aimed at revealing the regulatory mechanism concerning hsa_circ_ 0023305 expression in LSCC.

Materials and Methods
2.1. Tissue Samples. In total, we obtained 30 paired LSCC tissues along with adjacent nontumor tissues from patients in the hospital. No patients received chemotherapy and/or radiotherapy prior to surgery. We obtained written informed consent from all patients. The Ethics Committee in Shanghai Gongli Hospital approved the investigation.

Cell Culture and Transfection.
We received AMC-HN-8 and Hep-2 cells from Cell Bank of Chinese Academy of Sciences (Shanghai, China). We maintained cells in Dulbecco's modified Eagle medium (DMEM, Gibco, CA, USA) with 10% fetal bovine serum (FBS, Gibco) at 37°C in a humidified atmosphere with 5% CO 2 .
2.6. Cell Counting Kit-8 (CCK-8) Assay. Two days after transfection, we collected cells and plated them in 96-well plates at 2,000 cells/well. After culturing for 0, 1, 2, or 3 d, we added 10 μL CCK-8 solution (Gibco, Logan, Utah, USA) per well and incubated for 1 h. Then, we measured the optical density (OD) value of each well using a microplate reader with 490 nm wavelength.

Colony Formation
Assay. Cells were collected 48 h after transfection, seeded at a density of 200 cells/well in a 6well plate, and cultured with complete medium for 10 d. We fixed cell colonies with 4% paraformaldehyde for 15 minutes and stained them with 0.1% crystal violet for 5 minutes. We photographed and counted colonies in a light selective environment.
2.8. Transwell Assay. Transfected cells were incubated with serum-free DMEM for 0.5 d prior to the Transwell assay. We stored Matrigel (Corning Life Sciences, Corning, NY, U.S.A.) at 4°C overnight for experiments. We placed Transwell chambers into each well of a 24-well plate and added DMEM (500 μL; containing 20% FBS) to the lower chambers. We also added 1 × 10 5 transfected cells in 100 μL serum-free DMEM to the upper chambers to assess migration ability. Alternatively, we added cells into upper chambers coated with 10 μL Matrigel to assess invasion. After 2 d of conventional incubation, we removed the Transwell chambers. We lightly wiped the inner cells away and placed chambers in a new 24-well plate with 4% paraformaldehyde (600 μL). Cells were fixed for 5 minutes and then stained with 0.1% crystal violet for 10 minutes. Lastly, a technician counted cells in five random fields using an inverted microscope.
2.9. Western Blots. Protein was extracted from cells or tissues with RIPA lysis buffer, and Western blots were completed. Primary antibodies were against TRPM7 and GAPDH (Cell

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Wisconsin, USA). The binding sites were cloned in downstream of the firefly luciferase 3 ′ -UTR or hsa_circ_ 0023305 as a primary luciferase signal. Renilla luciferase was used as the normalization signal. Constructs were labeled TRPM7-Wt/hsa_circ_0023305-Wt and TRPM7-Mut/hsa_circ_0023305-Mut. The psi-CHECK vector supplied the Renilla luciferase normalization signal to compensate for alterations resulting from transfection and harvesting efficiencies. Transfection into HEK293 cells was achieved using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, USA). We detected Renilla and firefly luciferase activities 1 d after transfection using the Dual-Luciferase Reporter Assay System (Promega, Mannheim, Germany) and a luminometer (Molecular Devices, U.S.A.). Relative Renilla luciferase activities were detected according to the manufacturer's instructions (Promega, Mannheim, Germany).
2.12. Statistical Analysis. Data are reported as the mean ± standard deviation (SD). A statistician utilized GraphPad Prism (GraphPad, La Jolla, USA) to detect differences between groups. P values ≤ 0.05 were considered to be statistically significant.

High Expression of hsa_circ_0023305
Correlates to Poor LSCC Prognosis. RT-qPCR detection found that hsa_circ_ 0023305 expression was increased in LSCC tissues compared to adjacent tissue (Figure 1(a)). Higher hsa_circ_ 0023305 expression was also correlated to more advanced tumor stage (Figure 1(b), Table 1) as well as lymph node metastasis ( Figure 1(c), Table 1). To detect the interplay between hsa_circ_0023305 and prognosis in laryngeal cancer patients, we plotted Kaplan-Meier survival curves. High hsa_circ_0023305 expression may be associated with the poor prognosis of laryngeal carcinoma. Specifically, higher hsa_circ_0023305 levels correlated with worse prognoses (Figure 1(d)). Fluorescence in situ hybridization (FISH) analysis demonstrated that hsa_circ_0023305 expression was increased in LSCC tissues compared to adjacent tissue and hsa_circ_0023305 was primarily located in cytoplasm (Figure 1(e)). Bioinformatics analysis found that hsa_circ_ 0023305 was comprised of four exons from the CCND1 gene. The mature hsa_circ_0023305 was located in chr11:69457798-69469242, and the spliced length was 3882 bp (Figure 1(f)).

hsa_circ_0023305 Downregulation Suppresses LSCC Cell
Proliferation and Growth. To better analyze the regulatory effect of hsa_circ_0023305 on LSCC cell proliferation and tumor growth, we constructed a lentiviral vector with hsa_ circ_0023305 interference stability. RT-qPCR analyses showed that hsa_circ_0023305 expression was significantly decreased in Hep-2 and AMC-HN-8 cells (Figure 2(a)). CCK8 analysis showed that silencing of hsa_circ_0023305 inhibited LSCC cell proliferation in both AMC-HN-8 and Hep-2 cells (Figures 2(b) and 2(c)). Plate cloning experiments were carried out to explore proliferative capacity in AMC-HN-8 and Hep-2 cell lines. Knockdown of hsa_ circ_0023305 attenuated the proliferative capacity of AMC-HN-8 and Hep-2 cells compared to the sh-NC group (Figures 2(d) and 2(e)).
We also explored the impact of hsa_circ_0023305 expression on tumor growth in vivo using AMC-HN-8 cells and discovered that tumor volume (Figures 2(f) and 2(g)) and weight (Figure 2(h)) were clearly decreased in the sh-hsa_circ_0023305 group compared to the sh-NC group. Immunohistochemical detection showed that Ki67 expression was decreased in the sh-hsa_circ_0023305 group compared to the sh-NC group (Figures 2(i) and 2(j)). Together, our data suggest that hsa_circ_0023305 downregulation suppresses LSCC cell proliferation and tumor growth.

hsa_circ_0023305 Downregulation Decreases LSCC Cell
Invasion In Vivo and In Vitro. Transwell invasion and migration studies found that silencing of hsa_circ_0023305 decreased LSCC cell invasion and migration in Hep-2 and AMC-HN-8 cells (Figures 3(a)-3(d)). Whole body imaging in mice illustrated that hsa_circ_0023305 knockdown inhibited tumor metastasis and development (Figures 3(e) and 3(f  )). Living imaging detection shows the pulmonary metastasis and found that hsa_circ_0023305 silence decreased the pulmonary metastasis ability by decreasing the numbers of metastatic foci in lung tissues after HE staining analysis     (Figure 4(a)). This suggested that miR-218-5p was a downstream target of hsa_circ_0023305. We next constructed WT and MUT hsa_circ_0023305 luciferase reporter plasmids (Figure 4(b)). The data suggest that luciferase activity decreased after cotransfection with WT hsa_circ_0023305 and miR-218-5p mimics (Figure 4(c)), suggesting that hsa_circ_0023305 interacted directly with miR-218-5p. Bioinformatics analysis characterized TRPM7 as a miR-218-5p downstream target (Figure 4(d)). Luciferase reporter assay results validated that miR-218-5p was able to bind to the TRPM7 3 ′ -UTR (Figure 4(e)).   gene regulatory potential, including regulation of tumor formation and aggressiveness [8]. Previous studies have shown circRNAs participate in the development of resistance to treatment by modulating regulatory pathways and cellular processes, including the mitogen-activated protein kinase pathway, epithelial-mesenchymal transition, apoptosis, and autophagy [9]. The study also found that circRNA regulates gene expression through various mechanisms, such as acting as an "miRNA sponge," regulating transcription and splicing, interacting with RNA-binding proteins, and even translating polypeptides [10]. Our study showed that hsa_circ_ 0023305 expression was increased in LSCC patients and higher hsa_circ_0023305 expression correlated to poorer prognosis, increased tumor stage, and more lymph node metastasis among LSCC patients. Additionally, higher hsa_ circ_0023305 expression was associated with poorer overall survival (OS) of LSCC patients, suggesting that hsa_circ_ 0023305 plays an important role in LSCC. Bioinformatics analysis (https://starbase.sysu.edu.cn/) also found that hsa_ circ_0023305 can interact with RNA-binding proteins including ELAVL1, U2AF2, TAF15, RBFOX2, and IGF2BP2. But the mechanism of circRNAs to interact with proteins, thereby binding, sequestering, or translocating proteins to particular subcellular fractions, allows circRNAs to act as dynamic scaffolds that modulate protein-protein interaction still largely unclear. Downregulation of hsa_circ_0023305 suppressed LSCC cell proliferation and invasion both in vitro and in vivo. Bioinformatics results identified miR-218-5p as a hsa_circ_ 0023305 downstream target. Luciferase reporter assays validated the relationship between hsa_circ_0023305 and miR-218-5p. Previous studies have reported abnormal miR-218-5p expression in some diseases, including bladder cancer [11], non-small-cell lung cancer [12], gastric cancer [13], and pancreatic cancer [14]. Upregulation of miR-218-5p suppressed cancer proliferation and invasion [15]. The study also suggested that miR-218-5p was downregulated in invasive front cells and that this miRNA negatively regulated oral squamous cell carcinoma invasiveness through targeting of the CD44-Rho kinase (ROCK) pathway [16]. The present study discovered that silencing of hsa_circ_ 0023305 promoted miR-218-5p expression but inhibition of miR-218-5p restored the proliferative and invasive capacity of these cells.

Discussion
Previous studies have identified TRPM7 as a miR-218-5p downstream target, and this was validated by luciferase reporter assay. TRPM7 regulates a nonselective cation channel and enhances cancer metastasis. TRPM7 expression was negatively correlated to E-cadherin expression, but positively correlated to Vimentin, N-cadherin, and Twist expression in ovarian cancer. Depletion of TRPM7 inhibited invasion and migration in OVCAR3 and SKOV3 cells via regulation of phosphoinositide 3-kinsae (PI3K)/protein kinase B (AKT) oncogenic signaling [17]. A study has found that aberrantly expressed circRNAs can mediate cancer progression through regulation of the activity of major signaling cascades, such as the VEGF, WNT/β-catenin, MAPK, PI3K/AKT, and Notch signaling pathways, as well as by interfering with signaling crosstalk [18]. Additional studies have demonstrated that TRPM7 is aberrantly expressed in human diseases such as cancer. In cancer cells, TRPM7 functions to promote survival, cell cycle progression, migration, growth, proliferation, invasion, and epithelial-mesenchymal transition (EMT) [19]. In this study, we discovered that silencing of hsa_ circ_0023305 decreased TRPM7 expression, but TRPM7 expression was restored in these cells upon inhibition of miR-218-5p. Thus, overexpression of TRPM7 restored the proliferative and invasive ability of cells with silenced expression of hsa_circ_0023305.

Conclusions
In summary, hsa_circ_0023305 expression was increased in the LSCC patients and the high expression of hsa_circ_ 0023305 could enhance LSCC cell development and metastasis by upregulating TRPM7. We discovered for the first time that hsa_circ_0023305 binds directly to miR-218-5p and regulated the expression of TRPM7. We supposed that targeting of hsa_circ_0023305 expression may prove to be a novel insight for early diagnosis and genetic therapeutic strategy for LSCC.

Data Availability
Data produced and analyzed in the study are contained in the paper.

Conflicts of Interest
None is declared.

Authors' Contributions
Yi Zhang, Xiaoping Chen and Kaisai Tian conducted the study. Enhui Zhou, Xiaocheng Xue and Shiling Yan carried out assays and collected all samples. Yixin Chen, Peipei Qiao and Liyun Yang conducted the statistical analyses. Xiaoping Chen participated in research coordination and acted as a technical consultant. Yi Zhang drafted the paper. All authors read and approved the final version.