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. 2021 Aug 26;20(18):1799–1811. doi: 10.1080/15384101.2021.1959973

Hsa_circ_0006232 promotes laryngeal squamous cell cancer progression through FUS-mediated EZH2 stabilization

Tianyi Wu a, Guangke Wang a, Xianting Zeng b, Zhanwei Sun a, Shichao Li a, Weiwei Wang a, Boyu Yu a,✉
PMCID: PMC8525971  PMID: 34437823

ABSTRACT

Laryngeal squamous cell cancer (LSCC) is one of the most common malignant tumors in head and neck tumors. Our previous study has revealed that hsa_circ_0006232 is abnormally expressed in LSCC. This study attempts to verify the biological role of hsa_circ_0006232 in LSCC. We found that compared with human bronchial epithelial cells, hsa_circ_0006232 was highly expressed in human LSCC cells (AMC-HN-8 and TU686). Moreover, hsa_circ_0006232 promoted proliferation, migration and invasion of AMC-HN-8 and TU686 cells. Hsa_circ_0006232 promoted the expression of enhancer of zeste homolog 2 (EZH2) and repressed the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Fused in sarcoma (FUS) interacted with hsa_circ_0006232 and EZH2, and FUS promoted the stabilization of EZH2. Hsa_circ_0006232 inhibited PTEN by promoting FUS expression. Moreover, we constructed a tumor xenograft model by injection of AMC-HN-8 cells with hsa_circ_0006232 knockdown, and we found that hsa_circ_0006232 deficiency decreased tumor growth in mice. Hsa_circ_0006232 silencing repressed EZH2 expression and enhanced PTEN expression in tumor tissues. In conclusion, our data have demonstrated that Hsa_circ_0006232 promotes proliferation, migration and invasion of LSCC cells, and accelerates tumor growth of LSCC through FUS-mediated EZH2 stabilization. Thus, hsa_circ_0006232 may be a novel therapeutic target in LSCC treatment.

KEYWORDS: Hsa_circ_0006232, proliferation, migration, invasion, laryngeal squamous cell cancer

Introduction

Laryngeal squamous cell cancer (LSCC) is one of the most common malignant tumors in head and neck tumors [1]. With the rapid advancement of industrialization and urbanization, the environmental pollution caused by them has become more and more serious, which has caused the incidence of LSCC in the world to increase year by year. Approximately more than 150,000 patients are diagnosed with LSCC every year [1]. Although surgical treatment, adjuvant radiotherapy and chemotherapy drugs and targeted drugs have been widely used in clinical treatment of LSCC, the mortality of LSCC is still high [2]. Therefore, strengthening the research on the pathogenesis of LSCC and seeking new therapeutic targets are of great significance for LSCC treatment.

Circular RNA (circRNA) is a kind of endogenous non-coding RNA with extensiveness and diversity [3]. CircRNA is stable due to its special ring structure, and it also has many characteristics such as richness, conservation and tissue specificity. CircRNA participates in multiple regulatory mechanisms, such as sponge of microRNA (miRNA), protein interactions, and gene transcriptional and translational regulation [4,5]. Thus, circRNA participates in the transcription and translation of key genes, thereby regulating the occurrence, development and prognosis of many tumors [6,7]. There are many circRNAs are associated with the progression of LSCC. CircRNA_103862 acts as a sponge of miR-493-5p to promote GOLM1 expression, and then circRNA_103862/miR-493-5p/GOLM1 axis takes part in promoting proliferation of LSCC cells [8]. Circ_0000218 is highly expressed LSCC cells, and circ_0000218 functions as a carcinogenic factor in LSCC by regulating miR-139-3p/Smad3 axis [9]. CircCORO1C is up-regulated in LSCC cells and tissues, and up-regulation of CircCORO1C participates in the malignant progression and poor prognosis of LSCC via let-7c-5p/PBX3 axis [10]. In addition to serving as a competing endogenous RNA mechanism, some circRNAs containing protein-binding sequences can interact with RNA binding proteins to play a role. For instance, circ-Amotl1 can interact with both PDK1 and AKT1, and thus promotes the cardio-protective nuclear translocation of pAKT [11]. CircFOXO3 can combine with CDK2 and P21 proteins to form circFOXO3-CDK2-P21 complex, thereby affecting the cell cycle [12].

Previously, we have used GeneChip technology to screen multiple abnormally expressed circRNAs from tumor tissues and adjacent tissues of LSCC patients, among which hsa_circ_0006232 is abnormally expressed in LSCC tumor tissues [13]. However, whether hsa_circ_0006232 can participate in the progression of LSCC has not been reported, and we have conducted research on this topic. We have used Circinteractome database to predict the potential target protein of hsa_circ_000623, revealing that hsa_circ_000623 may interact with the RNA-binding protein fused in sarcoma (FUS). FUS belongs to the FET protein family, which participates in the regulation of tumorigenesis and development through regulating intracellular RNA transport, mRNA synthesis, alternative splicing, etc. [14–16]. Thus, we suspected that hsa_circ_0006232 may interact with FUS and then regulate the expression of its downstream genes. A previous study has reported that FUS promotes the expression of enhancer of zeste homolog 2 (EZH2) by stabilizing EZH2 mRNA [17]. EZH2 is a catalytic subunit of polycomb repressive complex 2, and functions as histone H3K27 methyltransferase. EZH2 is highly expressed in LSCC, and EZH2 promotes proliferation, migration and invasion of LSCC cells by regulating the histone methylation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) [18,19]. Therefore, we speculated that hsa_circ_0006232 was highly expressed in LSCC, and promoted the expression of EZH2 by interacting with FUS. EZH2 inhibited the expression of PTEN by regulating methylation of PTEN, thereby promoting the proliferation, migration and invasion of LSCC cells and tumor growth of LSCC in mice. We verified this hypothesis through in vivo and in vitro assays.

Materials and methods

Cell culture

Human bronchial epithelial cells (HBECs) (Cat: PCS-300-010; ATCC, Manassas, VA, USA) and human laryngeal carcinoma cell lines, AMC-HN-8 (Cat: BFN60808789) and TU686 (Cat: BFN608007264) (BLUEFBIO, Shanghai, China) were cultured in Dulbecco’s modified eagle medium (DMEM) (Solarbio, Beijing, China). Cells were incubated in DMEM containing 10% fetal bovine serum (Solarbio) and 1% penicillin/streptomycin (Solarbio) at 37°C and 5% CO2.

Cell transfection

Hsa_circ_0006232, EZH2, PTEN or FUS were subcloned into pcDNA3.1 vector, generating the overexpression vectors, pcDNA3.1-hsa_circ_0006232, pcDNA3.1-EZH2, pcDNA3.1-PTEN and pcDNA3.1-FUS. PcDNA3.1-NC served as negative control (NC). Small interference RNA (siRNA) specifically targeting hsa_circ_0006232 (Si-hsa_circ_0006232), EZH2 (Si-EZH2) or FUS (Si-FUS) were used to silence the corresponding genes. Non-silencing siRNA (Si-NC) oligonucleotide served as control (Ctrl). Lentivirus harboring Si-hsa_circ_0006232 and non-targeting plasmids (LV-Ctrl) were synthesized to knock down hsa_circ_0006232. These plasmids were designed and generated by GenePharma (Shanghai, China). HiPerFect Transfection Reagent (Qiagen, Hilden, Germany) was used to transfect plasmids into AMC-HN-8 or TU686 cells. The transfected cells were incubated at 37°C and 5% CO2 for 48 h.

Tumor xenograft experiments

Male BALB/c nude mice (4–6 weeks old, weighting 15–20 g) (SLAC, Shanghai, China) were housed under specific pathogen-free conditions. LSCC mouse model was established by subcutaneous injection of the LV-si-hsa_circ_0006232 or LV-Ctrl transfected AMC-HN-8 cells (1 × 106 cells/200 μL) through the right armpit. BALB/c nude mice injected with normal AMC-HN-8 cells (1 × 106 cells/200 μL) were used as control.

During the growth of mice, the tumor volume of LSCC was measured every 3 d. Tumor volume was calculated as the following formula: Tumor volume (mm3) = (length/width2)/2. Thirty days after inoculation, the mice were euthanized by cervical dislocation. The tumor tissues were separated from the mice, and the tumor tissues were utilized for weight measurement and western blot (WB) analysis. All protocols were authorized by the Ethics Committee of Henan Provincial People’s Hospital.

Quantitative real-time PCR (qRT-PCR)

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from AMC-HN-8 and TU686 cells and tumor tissues. RNA integrity was examined on 1.5% agarose gel electrophoresis. Complementary DNA was generated applying PrimeScript™ RT reagent Kit (Takara, Tokyo, Japan). The relative expression of genes was detected by performing quantitative real-time PCR (qRT-PCR) applying TB Green® Premix Ex Taq™ II (Tli RNase H Plus) (Takara). β-actin was used as a reference gene. Data were analyzed using 2−∆∆CT method.

Cell proliferation

MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Shanghai, China) was used to assess cell proliferation of AMC-HN-8 and TU686 cells following the instructions of manufacturer. Briefly, cells were seeded into 96-well plates at the concentration of 2000 cells/well. 10 µL of MTT reagent was added into each well, and incubated with 100 µL cells at 37°C for 4 h. Then, cells were incubated with 100 μL Formazan solubilization solution at 37°C for 4 h. The spectropho-tometrical absorbance of each well was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm.

Cell migration and invasion

A 24-well transwell insert system (Corning, NY, USA) was used to estimate migration and invasion of AMC-HN-8 and TU686 cells. For cell migration, cells (200 μL; 1.5 × 106 cells/mL) were seeded into the upper chamber. The lower chamber contained DMEM and 10% FBS. The up-migrated cells were removed from the upper side of chamber after 24 h of culture. The migrated cells were stained using 1% crystal violet (Solarbio) for 5 min. For cell invasion, the basolateral transwell chambers were covered with 1 mg/mL matrigel (Becton Dickinson Biosciences, San Diego, CA, USA). Except for this step, cell invasion assay had the same steps with cell migration assay. Finally, images of cell migration and invasion were obtained using an inverted microscope (Olympus, Tokyo, Japan) and analyzed using Image J software.

Protein expression

WB analysis was performed to assess protein expression. Total Protein Extraction Kit (Solarbio) was used to extract proteins from AMC-HN-8 and TU686 cells and tumor tissues as the protocol described. Protein samples were electrophoresed on 10% SDS-PAGE gels to separate the protein samples. The separated proteins were transferred onto PVDF membranes (Merck Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked with 5% skim milk, and then incubated with primary antibodies, PTEN (1:2000; Proteintech, Wuhan, China), EZH2 (1:1000; Proteintech) or H3K27m3 (1:5000; Thermo Fisher Scientific) at 4°C for 12 h. Then, horseradish peroxidase-conjugated secondary antibody (1:5000; Proteintech) was incubated with the membranes. β-actin antibody (1:5000; Proteintech) served as reference protein. The bands of protein were analyzed by Image J software.

Luciferase reporter assay

EZH2 promoter was cloned into pGL3-basic vector, generating the vector pGL3-basic-EZH2 (GenePharma). PGL3-basic-EZH2 was transfected into 293A cells together with pcDNA3.1-hsa_circ_0006232, pcDNA3.1-NC, Si-hsa_circ_0006232 or Si-NC. After 48 h of transfection, the luciferase activity of cells was examined using luciferase assay system (Promega, Madison, WI, USA).

RNA binding protein immunoprecipitation (RIP)

RIP assay was performed to examine the relationship among hsa_circ_0006232, FUS and EZH2 in the AMC-HN-8 and TU686 cells using Millipore Magna RIP Kit (Merck Millipore). Cells were lysed by lysis buffer, and then cell lysate was incubated with anti-FUS (1:200, Proteintech) or anti-IgG (1:200, Proteintech) at 4°C overnight. The intracellular protein-RNA complex was obtained through capturing antibody specifically targeting proteins. The protein-RNA complex was digested by proteinase K, and then washed repeatedly with RIP washing buffer to remove the nonspecific adsorption. Finally, qRT-PCR was performed to quantify the immunoprecipitant RNAs (hsa_circ_0006232 and EZH2 mRNA).

AMC-HN-8 cells were transfected with pcDNA3.1-hsa_circ_0006232 or pcDNA3.1-NC. TU686 cells were transfected with Si-hsa_circ_0006232 or Si-NC. The modified AMC-HN-8 and TU686 cells were lysed and incubated with anti-FUS antibody to pull-down EZH2. Finally, the effect of hsa_circ_0006232 on EZH2 mRNA was assessed by qRT-PCR.

Statistical analysis

Each assay was performed for three times. All data reported as mean ± standard deviation. SPSS 22.0 statistical software (IBM, Armonk, NY, USA) was used for statistical analysis. Two-tailed Student’s t test (for two groups), one-way ANOVA (for multiple groups) were used to analyze the statistical difference. P < 0.05 was considered as a significant difference.

Results

Hsa_circ_0006232 promoted proliferation, migration and invasion of LSCC cells

In order to investigate the biological role of hsa_circ_0006232 in LSCC, we compared the expression of hsa_circ_0006232 between HBECs and LSCC cells by qRT-PCR. We found that the expression of hsa_circ_0006232 was up-regulated in both AMC-HN-8 and TU686 cells as compared with HBECs (Figure 1(a)). Subsequently, to induce hsa_circ_0006232 up-regulation or down-regulation, AMC-HN-8 cells were transfected with pcDNA3.1-hsa_circ_0006232 and TU686 cells were transfected with Si-hsa_circ_0006232. QRT-PCR data revealed that hsa_circ_0006232 overexpression caused an up-regulation of hsa_circ_0006232 in AMC-HN-8 cells, and hsa_circ_0006232 expression was decreased in TU686 cells in the presence of Si-hsa_circ_0006232 (Figure 1(b,c)). We further assessed proliferation, migration and invasion of AMC-HN-8 and TU686 cells by performing MTT and transwell assays. The proliferation, migration and invasion of AMC-HN-8 cells were significantly enhanced in the presence of pcDNA3.1-hsa_circ_0006232 (Figure 2(a) and c, Supplementary Figure 1 a). However, hsa_circ_0006232 silencing notably repressed proliferation, migration and invasion of TU686 cells (Figure 2(b and d) Supplementary Figure 1 b). Thus, these data showed that hsa_circ_0006232 promoted proliferation, migration and invasion of LSCC cells.

Figure 1.

Figure 1.

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Hsa_circ_0006232 was up-regulated in AMC-HN-8 and TU686 cells

(a) QRT-PCR was performed to assess the expression of hsa_circ_0006232 in HBECs, AMC-HN-8 and TU686 cells. AMC-HN-8 cells were transfected with pcDNA3.1-hsa_circ_0006232 or pcDNA3.1-NC. TU686 cells were transfected with Si-hsa_circ_0006232 or Si-NC. (b-c) QRT-PCR was performed to assess the expression of hsa_circ_0006232 in the AMC-HN-8 and TU686 cells. **P < 0.01 vs. HBECs; ##P < 0.01 vs. Vector; $$P < 0.01 vs. Si-NC.

Figure 2.

Figure 2.

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Hsa_circ_0006232 overexpression promoted proliferation, migration and invasion of AMC-HN-8 cells, and hsa_circ_0006232 knockdown inhibited proliferation, migration and invasion of TU686 cells

AMC-HN-8 cells were transfected with pcDNA3.1-hsa_circ_0006232 or pcDNA3.1-NC. TU686 cells were transfected with Si-hsa_circ_0006232 or Si-NC. (a-b) MTT assay was performed to examine proliferation of AMC-HN-8 and TU686 cells. (c-d) Transwell assay was performed to detect migration and invasion of AMC-HN-8 and TU686 cells. *P < 0.05, **P < 0.01 vs. Vector; #P < 0.05, ##P < 0.01 vs. Si-NC.

Hsa_circ_0006232 promoted proliferation, migration and invasion of LSCC cells by regulating PTEN/EZH2 axis

We further explored the molecular mechanism of hsa_circ_0006232 in regulating proliferation, migration and invasion of LSCC cells. We first examined the expression of PTEN, EZH2 and H3K27m3 in AMC-HN-8 and TU686 cells. Hsa_circ_0006232 overexpression repressed PTEN expression, and led to an up-regulation of EZH2 and H3K27m3 in AMC-HN-8 cells (Figure 3(a)). However, the expression of PTEN was significantly enhanced in TU686 cells in the presence of Si-hsa_circ_0006232. Hsa_circ_0006232 silencing reduced the expression of EZH2 and H3K27m3 in TU686 cells (Figure 3(b)). Moreover, AMC-HN-8 cells were co-transfected with pcDNA3.1-hsa_circ_0006232 and Si-EZH2. TU686 cells were transfected with Si-hsa_circ_0006232 and pcDNA3.1-EZH2. WB data revealed that hsa_circ_0006232 overexpression repressed PTEN expression, and enhanced H3K27m3 expression in AMC-HN-8 cells. EZH2 silencing caused an up-regulation of PTEN, and led to a decrease of H3K27m3 expression in AMC-HN-8 cells. The influence conferred by EZH2 knockdown was rescued by hsa_circ_0006232 overexpression (Figure 3(c)). Furthermore, PTEN expression was enhanced in TU686 cells in the presence of Si-hsa_circ_0006232, and H3K27m3 expression was suppressed by hsa_circ_0006232 deficiency in TU686 cells. EZH2 overexpression suppressed PTEN expression and enhanced H3K27m3 expression in TU686 cells. Hsa_circ_0006232 silencing reversed the impact of EZH2 overexpression on PTEN and H3K27m3 expression in TU686 cells (Figure 3(d)).

Figure 3.

Figure 3.

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Hsa_circ_0006232 inhibited PTEN expression by regulating EZH2 expression

AMC-HN-8 cells were transfected with pcDNA3.1-hsa_circ_0006232 or pcDNA3.1-NC. TU686 cells were transfected with Si-hsa_circ_0006232 or Si-NC. (a-b) WB was performed to examine the expression of PTEN, EZH2 and H3K27m3 in AMC-HN-8 and TU686 cells. AMC-HN-8 cells were co-transfected with pcDNA3.1-hsa_circ_0006232 or pcDNA3.1-NC and Si-EZH2 or Si-NC. TU686 cells were transfected with Si-hsa_circ_0006232 or Si-NC and pcDNA3.1-EZH2 or pcDNA3.1-NC. (c-d) WB was performed to examine the expression of PTEN and H3K27m3 in AMC-HN-8 and TU686 cells. **P < 0.01 vs. Vector; ##P < 0.01 vs. Si-NC; $$P < 0.01 vs. Vector + Si-NC; &&P < 0.01 vs. Vector + Si-EZH2; @@P < 0.01 vs. EZH2 + Si-NC.

Next, we examined proliferation, migration and invasion of AMC-HN-8 cells by MTT and transwell assays. Hsa_circ_0006232 overexpression promoted proliferation, migration and invasion of AMC-HN-8 cells. EZH2 deficiency suppressed proliferation, migration and invasion of AMC-HN-8 cells, which was partly rescued by hsa_circ_0006232 up-regulation (Figure 4(a-c) and Supplementary Figure 2a-b). In addition, AMC-HN-8 cells were co-transfected with pcDNA3.1-hsa_circ_0006232 and pcDNA3.1-PTEN. MTT and transwell assays revealed that hsa_circ_0006232 overexpression enhanced proliferation, migration and invasion of AMC-HN-8 cells. However, PTEN overexpression caused a decrease in proliferation, migration and invasion of AMC-HN-8 cells. The inhibiting effect of PTEN up-regulation on proliferation, migration and invasion of AMC-HN-8 cells was abolished by hsa_circ_0006232 overexpression (Figure 5(a-c) and Supplementary Figure 3a-b).

Figure 4.

Figure 4.

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Hsa_circ_0006232 promoted proliferation, migration and invasion of AMC-HN-8 cells by enhancing EZH2 expression

AMC-HN-8 cells were co-transfected with pcDNA3.1-hsa_circ_0006232 or pcDNA3.1-NC and Si-EZH2 or Si-NC. MTT assay (a) and transwell assay (b-c) were performed to examine proliferation, migration and invasion of AMC-HN-8 cells. **P < 0.01 vs. Vector + Si-NC; &&P < 0.01 vs. Vector + Si-EZH2.

Figure 5.

Figure 5.

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Hsa_circ_0006232 promoted proliferation, migration and invasion of AMC-HN-8 cells by repressing PTEN expression

AMC-HN-8 cells were co-transfected with pcDNA3.1-hsa_circ_0006232 or pcDNA3.1-NC and pcDNA3.1-PTEN. MTT assay (a) and transwell assay (b-c) were performed to examine proliferation, migration and invasion of AMC-HN-8 cells. **P < 0.01 vs. Vector; &&P < 0.01 vs. PTEN.

Taken together, these data demonstrated that hsa_circ_0006232 promoted proliferation, migration and invasion of AMC-HN-8 cells by regulating PTEN and EZH2 expression.

Hsa_circ_0006232 promoted EZH2 expression by interacting with FUS

We performed luciferase reporter assay to verify the effect of hsa_circ_0006232 on EZH2 promoter. The data showed that hsa_circ_0006232 overexpression enhanced the activity of EZH2 promoter, whereas hsa_circ_0006232 silencing repressed the activity of EZH2 promoter (Figure 6(a,b)). It indicated that hsa_circ_0006232 promoted transcription of EZH2. Then, RIP assay was used to examine the relationship among hsa_circ_0006232, FUS and EZH2 mRNA in the AMC-HN-8 and TU686 cells. Figure 6(c,d) showed that hsa_circ_0006232 interacted with FUS in AMC-HN-8 and TU686 cells, and FUS interacted with EZH2 mRNA in the AMC-HN-8 and TU686 cells. We further verified the impact of hsa_circ_0006232 on the FUS-interacted EZH2 mRNA expression in AMC-HN-8 cells and TU686 cells. In the presence of pcDNA3.1-hsa_circ_0006232, EZH2 mRNA was significantly enhanced in AMC-HN-8 cells (Figure 6(e)). EZH2 mRNA was severely down-regulated in TU686 cells following transfection of Si-hsa_circ_0006232 (Figure 6(f)).

Figure 6.

Figure 6.

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Hsa_circ_0006232 promoted EZH2 expression by interacting with FUS

(a-b) Luciferase reporter assay was performed to examine the effect of hsa_circ_0006232 on EZH2 promoter. (c-d) RIP assay was performed to estimate the relationship among FUS, hsa_circ_0006232 and EZH2 in AMC-HN-8 and TU686 cells. AMC-HN-8 cells were transfected with pcDNA3.1-hsa_circ_0006232 or pcDNA3.1-NC. TU686 cells were transfected with Si-hsa_circ_0006232 or Si-NC. (e-f) RIP assay was performed to estimate the relationship between hsa_circ_0006232 and EZH2 in AMC-HN-8 and TU686 cells. **P < 0.01 vs. Vector; &&P < 0.01 vs. Si-NC; $$P < 0.01 vs. IgG.

AMC-HN-8 cells were co-transfected with pcDNA3.1-hsa_circ_0006232 and Si-FUS. TU686 cells were transfected with Si-hsa_circ_0006232 and pcDNA3.1-FUS. We examined the expression of PTEN in AMC-HN-8 and TU686 cells. WB data showed that hsa_circ_0006232 overexpression significantly repressed PTEN expression in AMC-HN-8 cells. PTEN was highly expressed in AMC-HN-8 cells after transfected with Si-FUS, which was rescued by hsa_circ_0006232 up-regulation (Figure 7(a)). Moreover, hsa_circ_0006232 deficiency caused an up-regulation of PTEN in TU686 cells. PTEN was down-regulated in TU686 cells in the presence of pcDNA3.1-FUS. FUS overexpression rescued the influence conferred by hsa_circ_0006232 knockdown (Figure 7(b)).

Figure 7.

Figure 7.

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Hsa_circ_0006232 repressed PTEN expression by regulating FUS expression

AMC-HN-8 cells were co-transfected with pcDNA3.1-hsa_circ_0006232 or pcDNA3.1-NC and Si-FUS and Si-NC. TU686 cells were transfected with Si-hsa_circ_0006232 or Si-NC and pcDNA3.1-FUS or pcDNA3.1-NC. (a-b) WB was performed to examine the expression of PTEN in AMC-HN-8 and TU686 cells. **P < 0.01 vs. Vector + Si-NC; ##P < 0.01 vs. Vector + Si-FUS; &&P < 0.01 vs. FUS + Si-NC.

Thus, these findings taken together revealed that hsa_circ_0006232 promoted EZH2 expression by interacting with FUS.

Hsa_circ_0006232 deficiency inhibited tumor growth of LSCC in mice

Finally, we verified the role of hsa_circ_0006232 on tumor growth of LSCC in vivo. Tumor xenograft mouse model was constructed by subcutaneous injection of the LV-si-hsa_circ_0006232 or LV-Ctrl transfected AMC-HN-8 cells. The tumor growth of LSCC was observed, revealing that hsa_circ_0006232 silencing significantly repressed tumor growth of LSCC in mice (Figure 8(a,b)). WB data showed that hsa_circ_0006232 deficiency notably suppressed EZH2 expression and enhanced PTEN expression in the tumor tissues of LSCC (Figure 8(c)). Thus, in vivo data confirmed that inhibition of hsa_circ_0006232 inhibited tumor growth of LSCC in mice.

Figure 8.

Figure 8.

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Hsa_circ_0006232 deficiency inhibited tumor growth of LSCC in mice

Tumor xenograft mouse model was constructed by subcutaneous injection of the LV-si-hsa_circ_0006232 or LV-Ctrl transfected AMC-HN-8 cells. Mice injected with normal AMC-HN-8 cells were served as control. (a-b) The weight and volume of tumor tissues were measured. (c) WB was performed to examine the expression of EZH2 and PTEN in tumor tissues. **P < 0.01 vs. LV-Ctrl.

Discussion

The biological role of circRNA in the tumor progression has attracted more and more attention. Many researchers have confirmed the functional role of circRNA in the progression of various tumors, including LSCC. Lu et al. have screened the abnormal expression of circRNA between LSCC tissues and normal tissues, showing that there are 29 circRNAs are up-regulated and 19 circRNAs are down-regulated in the LSCC tissues [20]. The abnormal expression of these circRNAs may be associated with the occurrence, development and prognosis of LSCC. Guo et al. have found that hsa_circ_0036722 is down-regulated in LSCC tissues and correlated with the differentiation level of LSCC, suggesting that hsa_circ_0036722 may be a potential marker for diagnosis of LSCC [21]. Circ_0067934 is highly expressed in LSCC tissues and cells, and circ_0067934 plays a vital role in promoting tumor growth, lymph node status, and distant metastasis of LSCC [22]. In our work, we first revealed the biological role of a novel circRNA, hsa_circ_0006232, in the progression of LSCC. Compared with HBECs, the expression of hsa_circ_0006232 was significantly enhanced in AMC-HN-8 and TU686 cells. Moreover, hsa_circ_0006232 promoted proliferation, migration and invasion of LSCC cells. These data suggested that hsa_circ_0006232 participated in promoting proliferation, migration and invasion of LSCC cells.

EZH2 and PTEN play a crucial role in LSCC. Up-regulation of EZH2 promotes proliferation and cell-cycle progression of LSCC cells, thereby enhancing tumorigenicity in LSCC [23]. Moreover, EZH2 enhances the resistance of LSCC cells to chemotherapy drugs, which may lead to failure of LSCC treatment. Long non-coding RNA XIST regulates EZH2 expression by sponging miR-124, thereby accelerating tumor growth of LSCC [24]. MiR-340 inhibits the expression of tumor suppressor gene p27 and inactivates PI3K/Akt signaling pathway by interacting with EZH2. Thus, MiR-340/EZH2/p27 axis inhibits the progression of LSCC [25]. In addition, the abnormal expression of PTEN in LSCC tissues may be related to the dedifferentiation of LSCC [26]. MiR-744-3p represses AKT/mTOR/NF-κB signaling pathway by promoting PTEN expression, thereby inhibiting MMP-9-mediated metastasis in LSCC [27]. Tai et al. have confirmed that EZH2 promotes LSCC progression by promoting PTEN methylation and inhibiting PTEN expression [19]. Our work confirmed that the regulatory mechanism among hsa_circ_0006232, EZH2 and PTEN in LSCC. Hsa_circ_0006232 overexpression enhanced EZH2 and H3K27m3 expression and repressed PTEN expression in LSCC cells. EZH2 silencing enhanced PTEN expression and repressed H3K27m3 expression in LSCC cells, which was reversed by hsa_circ_0006232 overexpression. Thus, hsa_circ_0006232 inhibited PTEN expression by promoting EZH2-mediated methylation of PTEN in LSCC cells. Furthermore, hsa_circ_0006232 promoted proliferation, migration and invasion of LSCC cells by repressing PTEN expression.

FUS takes part in regulating RNA metabolism, including transcription, pre-mRNA splicing, mRNA transport and translation. FUS also maintains DNA integrity [28]. Previous study has confirmed that LINC00205 facilitates malignant phenotypes in lung cancer by recruiting FUS to stabilize CSDE1 [29]. LINC00893 maintains the stabilization of PTEN mRNA by recruiting FUS, and thus inhibits proliferation and migration of papillary thyroid cancer cells [30]. We verified that FUS had the function of stabilizing EZH2. FUS interacted with hsa_circ_0006232 and EZH2 mRNA. Hsa_circ_0006232 enhanced the activity of EZH2 promoter. Thus, we speculated that hsa_circ_000623 interacted with FUS to promote EZH2 expression. EZH2 up-regulation enhanced methylation of PTEN and inhibited PTEN expression. Our in vivo experiments showed that hsa_circ_0006232 silencing reduced tumor growth of LSCC. Hsa_circ_0006232 deficiency repressed EZH2 expression and enhanced PTEN expression in tumor tissues of LSCC. These data suggested that hsa_circ_0006232 knockdown repressed tumor growth of LSCC by regulating EZH2/PTEN expression.

Most circRNAs are located in the cytoplasm. Some circRNAs containing protein-binding sequences can interact with RNA binding proteins. The specific mechanism of the interaction between the two molecules has not been fully reported, but it can be confirmed by experimental methods such as RNA pull down, RNA immunoprecipitation, and laser confocal microscopy to analyze the co-localization of circRNA and protein [11,12]. CircRNA combines with proteins either in the cytoplasm or in the nucleus. CircRNA can function as a protein transporter to promote the transport of functional proteins from the cytoplasm to the nucleus, or as a “reservoir” for functional proteins to inhibit the biological functions of these proteins [11]. Hsa_circ_0006232 interacted with RNA binding protein FUS, indicating that hsa_circ_0006232 regulated FUS through protein interactions. Whether hsa_circ_0006232 plays its role in the cytoplasm or the nucleus is still unclear, and further research is needed.

Additionally, this article also has certain limitations. This work only initially determined the effect of hsa_circ_0006232 on proliferation, migration and invasion of LSCC cells and tumor growth of LSCC through in vivo and in vitro experiments. EMT and immune escape are important processes involved in the progression of LSCC [31,32]. Whether hsa_circ_0006232 can affect LSCC progression by regulating EMT and immune escape still needs further research. We will conduct research on this topic. At the same time, circRNA as competing endogenous RNA, plays an important role in tumor progression. We will continue to explore the miRNA combined with hsa_circ_0006232 and explore its role in the progression of LSCC.

In conclusion, this work demonstrates that hsa_circ_0006232 promotes proliferation, migration and invasion of LSCC cells, and accelerates tumor growth of LSCC through FUS-mediated EZH2 stabilization. Thus, hsa_circ_0006232 may be a novel therapeutic target in LSCC treatment.

Acknowledgments

Not applicable.

Disclosure statement

All authors declare no competing interest.

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