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. 2025 Jan 14;25:84. doi: 10.1186/s12885-025-13464-x

CircCCT2/miR-146a-5p/IRAK1 axis promotes the development of head and neck squamous cell carcinoma

Long He 1,2,✉, Lanruo Li 3, Liting Zhao 1,2, Xiaoya Guan 1,2, Yujia Guo 1,2, Qi Han 1,2, Huina Guo 1,2,✉, Hongliang Liu 1,2,4,5,✉, Chunming Zhang 1,2,4,✉
PMCID: PMC11734332  PMID: 39810134

Abstract

Background

Head and neck squamous cell carcinoma (HNSCC), a highly invasive malignancy with a poor prognosis, is one of the most common cancers globally. Circular RNAs (circRNAs) have become key regulators of human malignancies, but further studies are necessary to fully understand their functions and possible causes in HNSCC.

Methods

CircCCT2 expression levels in HNSCC tissues and cells were measured via qPCR. CircCCT2 was characterized by Sanger sequencing, qRT-PCR, RNase R & Actinomycin D treatment, nucleoplasmic separation and FISH experiments. CCK-8 and colony formation assays were performed to determine cell proliferation, and Transwell assays were used to determine migration and invasion. A xenograft tumor model was used to study the influence of circCCT2 on HNSCC in vivo. Dual-luciferase gene reporter, RIP, western blotting, and rescue experiments, were used to explore target-binding relationships and regulatory mechanisms.

Results

CircCCT2 was significantly upregulated in HNSCC tissues and cells. High circCCT2 levels were associated with advanced T stage, N stage, clinical stage and poor prognosis. Functionally, we verified that circCCT2 promotes HNSCC development in vitro and in vivo. Mechanistically, functioning as a competitive endogenous RNA (ceRNA) or miRNA sponge, circCCT2 binds directly to miR-146a-5p and increases interleukin-1 receptor-associated kinase 1 (IRAK1) levels, which enhances the malignant development of HNSCC by driving epithelial-mesenchymal transition (EMT).

Conclusion

CircCCT2 promotes HNSCC development through the miR-146a-5p/IRAK1 axis, revealing that circCCT2 is a potential biomarker and target for HNSCC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-025-13464-x.

Keywords: Head and neck squamous cell carcinoma, circCCT2, miR-146a-5p, IRAK1, EMT

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent carcinoma globally, which comprises a heterogeneous group of tumors that develop from the mucosa of the oral cavity, pharynx, larynx, along with sinonasal tract [1, 2]. HNSCC located in the oral cavity and larynx is usually associated with smoking and/or alcohol abuse, while human papillomavirus (HPV) infection, especially HPV16 infection, is increasingly recognized as a cause of oropharyngeal HNSCC [1–3]. Despite advances in comprehensive therapies including surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy alone or in combination, HNSCC still has an inadequate prognosis, with a less than 50% 5-year survival rate [4, 5]. The two primary risk elements for adverse outcomes in patients with HNSCC include lymph nodes metastasis and local invasion [6]. Nonetheless, additional research is still warranted to fully comprehend the molecular pathways underlying HNSCC proliferation and metastasis. Thus, it is essential to know the process behind the onset and progression of HNSCC, to find more trustworthy biomarkers, and to develop novel treatment targets. Currently, circular RNAs (circRNAs) as an emerging biomarker are providing new ideas for clinical diagnosis and treatment of HNSCC.

CircRNAs, a class of noncoding RNAs, are distinguished through their covalent closed-loop circular structure constructed by cyclizing exons, lacking 5′ caps & 3′ poly tails, which have been recently found to be the latest cancer research hotspot [7–9]. CircRNAs have been shown in a growing amount of research to have multiple biological contributions to cancer development. For example, circRNAs can act as an competitive endogenous RNA (ceRNA) or miRNA sponge to bind miRNA and remove miRNA inhibitory effects on target genes [10–12]. They also regulate alternative splicing or transcription, interact with proteins or translate into proteins or peptides or by incorporating with m6A ribonucleic acid in the untranslated region (UTR) on the 5’-end to perform their effects [12–14]. CircRNAs contribute to HNSCC tumorigenesis, progression and chemosensitivity. Some of these genes may act as prognostic biomarkers, such as circMTCL1 [15], circPARD3 [16], circFNDC3B [17]. Hsa_circ_0000418 is a newly discovered circRNA, whose host gene is chaperonin containing TCP1 subunit 2 (CCT2), hence the name circCCT2. Recent studies have reported that circCCT2 acts as an oncogene in glioma and hepatoblastoma [18, 19]. However, the functional roles and control processes of circCCT2 in HNSCC have not been reported.

It is well known that the main function of circRNA is to act as ceRNA [12, 18]. Therefore, we venture to hypothesise that circCCT2 is also a ceRNA in HNSCC, and it is a part of the circRNA-miRNA-mRNA pathway. Based on bioinformatics analysis, miR-146a-5p was identified as binding to cirCCT2. miR-146a-5p acts as a tumor suppressor in a variety of cancers, such as glioblastoma (GBM) [20] and lung cancer [21]. We further predicted that the interleukin-1 receptor-associated kinase 1 (IRAK1) was a downstream mRNA of miR-146a-5p through bioinformatics analysis.

IRAK1 is a member of the IRAK family, which contributes to inflammation and innate immunity pathways [22, 23]. According to a new study, the TRAF6-IRAK1 complex functions as both a switch and a brake to control the EMT characteristics of GBM cells [20]. The EMT has a key role in HNSCC metastasis [1]. Moreover, IRAK1 is upregulated and contributes to cancer progression in multiple cancers, for instance hepatocellular carcinoma [24, 25], breast cancer [26], colorectal cancer [27], endometrial carcinoma [28], melanoma [29] as well as glioma [30]. However, the expression of IRAK1 and its role in HNSCC need more research.

In the current research, we screened a circRNA named circCCT2 (circBase ID: hsa_circ_0000418), which is significantly upregulated in HNSCC. Clinically, upregulated circCCT2 expression was strongly associated with advanced T stage, lymph node metastasis, and clinical stage of HNSCC, and even indicated a worse prognosis of patients with HNSCC. Mechanistically, the circCCT2/miR-146a-5p/IRAK1 axis enhances the malignant development of HNSCC by driving EMT. This highlights that circCCT2 is a potential biomarker for HNSCC treatment.

Materials and methods

Clinical tissue

Clinical specimens removed from HNSCC patients during surgery were acquired from the First Hospital of Shanxi Medical University. Prior to surgery, all participants completed an informed consent form. All the tissue samples were used to obtain total RNA. Table S1 in the Additional file 1 displays the clinicopathologic characteristics of individuals with HNSCC.

Cell lines and transfection

Human laryngeal squamous cell carcinoma (LSCC) cell line FD-LSC-1 (kindly provided by Professor Liang Zhou [16, 31]) was cultured in Bronchial Epithelial Cell Growth Medium (BEGM) with 10% fetal bovine serum (FBS) (ExCell, China). Human tongue squamous cell carcinoma cell line CAL-27, human cell line HEK293T cells and human embryo lung fibroblast diploid cell line 2BS, purchased from the China Center for Type Culture Collection (CCTCC) were maintained in DMEM or MEM with 10% FBS. Human popharyngeal squamous cell carcinoma (PSCC) cell lines FaDu and Detroit 562, obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), were maintained in MEM with 10% FBS. Another human PSCC cell line HN-30 (kindly provided by Professor Qiancheng Shen) was grown in DMEM with 10% FBS. In addition, human LSCC cell line AMC-HN-8 purchased from the Huzhen Biotechnology (Shanghai, China), was cultured in RPMI-1640 with 10% FBS. The culture conditions being 37 °C, 5% CO2. The cell lines were tested for mycoplasma contamination using the TransDetect PCR Mycoplasma Detection Kit (TransGen, China). Cells were transfected according to the manufacturer’s guidelines using Lipofectamine 3000 (Invitrogen, MA) or PL Transfection Reagent (TransGen, China).

Plasmid construction

The p3×FLAG-CMV-10 plasmid was used to clone the IRAK1 coding sequence (CDS), creating the IRAK1 overexpression plasmid (OE-IRAK1). The pCD2.1-ciR plasmid was used to clone the circCCT2 sequence, resulting in the circCCT2 overexpression plasmid (OE-circCCT2). To obtain dual-luciferase reporter gene plasmids, the sequences of the circCCT2 and IRAK1 3′-untranslated region (3′ UTR) with wild-type (WT) and mutated (Mut) miR-146a-5p binding sites were cloned into the psiCHECK-2 plasmid (Promega, WI). These plasmids were named circCCT2-WT, circCCT2-Mut, IRAK1 3′ UTR-WT and IRAK1 3′ UTR-Mut, respectively.

miRNA mimics/inhibitor and siRNAs

The siRNAs used to silence circCCT2 (si-circ-1 & si-circ-2), and IRAK1 (si-IRAK1), miR-146a-5p mimics/inhibitor and NC siRNAs (si-NC) were generated by GenePharma (China). A 2’-OME modification is added to siRNA molecules to increase RNA stability. The siRNAs used are detailed in the Additional file 1: Table S2.

Nucleic acid preparation

Total RNA, nuclear and cytoplasmic RNA, and total gDNA were obtained from experimental samples according to the manufacturer’s guidelines using TRIzol reagent (Thermo Fisher Scientific, USA), a PARIS kit (Beyotime, China), and genomic DNA of blood/cell/tissue extraction kit (Tiangen, China), respectively.

RT‒PCR and qPCR

Reverse transcription of the mRNAs and circRNAs via cDNA Synthesis SuperMix (TransGen) to obtain cDNA. CDNA of miRNA was synthesized applying All-in-One™ miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia, USA). qPCR was conducted utilizing qPCR SuperMix (TransGen). U6 or 18 S rRNA was chosen as the housekeeping gene. The 2(−△△CT) technique was utilized to analyze the data [16]. The primer information is listed in the Additional file 1: Table S3.

RNase R and actinomycin D tests

To conduct the RNase R experiment, 3 µg of total RNA was incubated with or without 9 U of RNase R (Geneseed, China) for 15 min at 37 °C. To conduct an Actinomycin D test, incubate cells with Actinomycin D (3 µg/mL) for 0, 1, 2, 4, 8 & 10 h. Afterwards, the transcript levels of circCCT2 and CCT2 were assessed by qPCR.

Cell counting kit-8 (CCK8) assay

Following a 24-hour transfection period, 96-well plates were planted with 5000 cells/well in five replicates. Afterwards, cells got treated with 10 µL of CCK-8 (Yeasen, China) at 0, 24, 48, 72 and 96 h. The data were measured after one hour of incubation at 37 °C with 5% CO2.

Colony formation experiment

After a 24-hour transfection period, 6-well plates were planted with 800 cells/well. Change the medium every 2–3 days. Briefly, after approximately 10 days of culture, cell clones were paraformaldehyde fixed, stained with crystal violet, photographed, and counted. Refer to previous reports for a detailed description [16].

Transwell assay

Transwell experimental manipulations were performed according to previous studies [16]. The migration and invasion ability of FD-LSC-1 and CAL-27 cells was assessed using a 24-well Transwell assay. Cells were digested, washed twice with PBS and resuspended in serum-free medium. Transwell chambers for the invasion assay were precoated with matrix gel (ABW, USA). 200 µL of serum-free medium containing cells (1 × 105 cells/well for invasion assay, 8 × 104 cells/well for migration assay) was seeded into the upper chamber (8 μm pore size, NEST Biotechnology, Wuxi, China). The bottom chamber contained 500 µL of medium supplemented with 20% FBS. After 24 h, the cells in the upper chamber were removed and the bottom chamber was gently washed with PBS and fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and images were captured by microscope.

Luciferase reporter assay

In HEK293T cells, the dual-luciferase gene reporter plasmid was transfected together with NC or miR-146a-5p mimics. Luciferase activity was measured using a Dual Luciferase Reporter Assay System (Promega) following 48 h of transfection.

Western blotting

Western blotting was performed as described in previous studies [32]. Cell samples were collected and lysed in RIPA lysis buffer containing protease inhibitor cocktail (ThermoFisher Scientific) ice bath for half an hour, followed by centrifugation at 12,000 g for 15 min at 4 °C. The supernatant was collected, and the protein concentration was determined by the Bradford method. Equal amounts of proteins were separated by SDS-PAGE electrophoresis, transferred to PVDF membrane (Millipore, MA), sealed by 10% skimmed milk for 2 h at room temperature, primary antibody was incubated overnight at 4 °C, and washed three times with TBST. The secondary antibody was then incubated for 2 h at room temperature, washed three times with TBST, and ECL chemiluminescence imaging. The antibodies used were as follows: IRAK1 antibody (#10478-2-AP; 1:1000 dilution, Proteintech, China), N-cadherin antibody (#22018-1-AP; 1:2000 dilution, Proteintech), SNAI2 antibody (#12129-1-AP; 1:5000 dilution, Proteintech), secondary antibodies rabbit IgG (#A0208; 1:1000 dilution, Beyotime, China) and mouse IgG (#A0216; 1:1000 dilution, Beyotime), and GAPDH antibody (#HC301; 1:1000 dilution, TransGen).

Fluorescence in situ hybridization (FISH)

A Cy3-marked probe for circCCT2 (5’-TCATCGGAACTTTATTTTGTCTGTA-3’) was generated via GenePharma. The FISH assay was done according to the guidelines of a FISH Kit (GenePharma). DAPI was utilized to stain cell nuclei. The pictures were obtained using a fluorescence microscope (Leica, Germany).

RNA immunoprecipitation (RIP)

Employing a RIP Kit (Millipore, MA), RIP investigations were conducted in FD-LSC-1 & CAL-27 cells following the guidelines. The following antibodies were used: anti-AGO2 antibody (#2897; CST, MA) and negative control rabbit IgG (#A7016; Beyotime).

In vivo research

Four-week-old female BALB/c nude mice (Vitonix, China) were preserved in an SPF-grade animal laboratory. For in vivo tumor growth, 4 × 106 CAL-27-miRFP709 cells, which were a screened stable CAL-27 cell line transfected with the miRFP709 NIR protein, were injected bilaterally subcutaneously into nude mice. Tumors lengh and width were measured every 2–3 days [33]. Tumor volume = (length × width2)/2 [34]. When the tumor size approached about 60 mm3, 5 µg of siRNA mixed with 3 µL of Entranster (Engreen Biosystem, China) was injected into the tumors every 3–4 days (left injection of NC, right injection of si-circCCT2). When the length of some tumors exceeded 10 mm, a total of eight doses of siRNAs were given to the nude mice for about one month, and we placed the mice in the induction box of a small anesthesia machine (RWD, Shenzhen, China), anesthetized them by inhalation with 2% isoflurane (RWD), and then, after the mice had lost their consciousness, they were put to death by neck-breaking, then the xenografts were excised, weighed, and photographed.

Immunohistochemical (IHC) staining

IHC was performed as described in previous studies [32]. In brief, after formalin fixation, tissue samples were paraffin-embedded and cut into 4 μm-sized sections. Sections underwent dewaxing, re-hydration, antigen retrieval, and blocking, overnight incubation at 4 ◦C with primary antibody, and washed three times with PBST. Incubated with secondary antibody for half an hour at room temperature, washed three times with PBST, and then stained with DAB and hematoxylin re-staining, then dehydrated and transparent, and neutral gum sealing. Images were acquired using the Panoramicscan II. The antibodies or kits used were as follows: Ki67 (#RMA-0731; MXB, China) and the secondary antibody Universal Kit (#PV-6000; ZSGB, China); IRAK1, N-cadherin & SNAI2 antibodies were the same as above.

Statistical analysis

The values, on the basis of a minimum of three separate experiments, are the mean ± standard deviation (SD), and were analyzed by SPSS 20.0 (Armonk, USA). Two-tailed t-test was employed to analyze difference. P < 0.05 was considered significant.

Results

Characterization of circCCT2 in HNSCC

To study the role of circRNAs in HNSCC progression, we analyzed the differentially expressed circRNAs from the RNA-seq results in 57 pairs of LSCC and matched adjacent normal mucosa (ANM) tissues (Additional file 1: Table S4) [34]. We subsequently selected 23 circRNAs with a fold change ≥ 1.5 and P < 0.01 (Fig. 1A). The bioinformatics analysis focused on the circular RNA hg19_circ_0012581. Its sequence is available from circBase (circBase ID: hsa_circ_0000418), with 304 nucleotides (nt), which consists of exons 7 and 8 of the human CCT2 mRNA; hence, we named it circCCT2. The junction site and the existence of circCCT2 were proved using sequencing results (Fig. 1B).

Fig. 1.

Fig. 1

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Characterization of circCCT2 in HNSCC. (A) Heatmap showing 23 upregulated circRNAs identified from the RNA-seq results. (B) Diagram showing the circularization structure and back-splicing junctions of circCCT2. (C) The circCCT2 expression level in 50 sets of HNSCC samples using qPCR. (D–F) CircCCT2 expression was strongly correlated with T stage (D), N stage (E), and clinical stage (F). (G) Kaplan-Meier curve study of the prognosis of 50 HNSCC patients with different expression levels of circCCT2. (H) The expression of circCCT2 in cells was assessed by qPCR. (I) Divergent primers amplified when circCCT2 existed. (J) After RNase R treatment and qPCR detected circCCT2 expression. (K) The expression level of circCCT2 and CCT2 after being treated with Actinomycin D via qPCR analysis. The values are the means ± standard deviation (SD) of three separate experiments, *P < 0.05, **P < 0.01, the same as below

More importantly, the expression level of circCCT2 was significantly higher in HNSCC tissues than in ANM tissues by qPCR (Fig. 1C). A substantial correlation was observed between highly expressed circCCT2 and clinicopathological features include pathological T3 + T4 stage, N+ (cervical lymph node metastasis) stage along with clinical III+IV stage in individuals with HNSCC (Fig. 1D-F). Furthermore, the high expression of circCCT2 indicates poor overall survival in HNSCC patients, based on a Kaplan-Meier curve study of the prognosis of 50 HNSCC patients with different expression levels of circCCT2 (Fig. 1G).

Next, we analyzed circCCT2 expression in HNSCC cell lines (CAL-27, FD-LSC-1, HN-30, AMC-HN-8, FaDu and Detroit 562) compared with normal cell lines (HEK293T and 2BS). As compared to the other types of cells, FD-LSC-1 and CAL-27 cells expressed higher levels of circCCT2 (Fig. 1H). To further confirm the existence of circCCT2, we synthesized divergent/convergent primers for circular/linear RNA. Only when the cDNA acts as a template can circCCT2 be amplified using divergent primers (Fig. 1I). A key characteristic of circRNAs is their high stability [16]. The transcript level of circCCT2 can’t be influenced by RNase R treatment because of its circular structure (Fig. 1J). Furthermore, circCCT2 had a much longer half-life than linear CCT2 in an Actinomycin D experiment (Fig. 1K).

In a word, circCCT2 is a circular RNA, has a much longer half-life and confers resistance to exonucleases, thereby increasing their stability compared to linear RNAs. Clinically, high expression of circCCT2 was correlated with HNSCC advanced stage, cervical lymph node metastasis and poor overall survival. This suggests that circCCT2 is a potential biomarker for HNSCC. What is the biological role of circCCT2 in HNSCC cells? This is the question we will be investigating further.

What is the biological role of circCCT2 in HNSCC cells?

To investigate the biological role of circCCT2 in HNSCC cells, first, two siRNAs (si-circ-1, si-circ-2) were designed and produced to precisely silence circCCT2. Subsequently, FD-LSC-1 & CAL-27 cells were transfected with them, resulting in significantly effective interference with circCCT2 but did not alter linear CCT2 mRNA expression (Fig. 2A). Cell proliferation was significantly reduced by circCCT2 silencing, as determined through CCK-8 and colony formation tests (Fig. 2B, C). HNSCC cell migration and invasion were significantly impeded through knockdown of circCCT2 in the Transwell assay (Fig. 2D).

Fig. 2.

Fig. 2

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CircCCT2 may be involved in the carcinogenic process of HNSCC. (A) Knockdown efficiency of FD-LSC-1 & CAL-27 cells transfected with siRNA. (B, C) CCK-8 (B) and colony formation (C) experiments examined the influence of circCCT2 silencing on the growth of HNSCC cells. (D) The ability to migrate and invade was evaluated using the Transwell invasion and migration assays. (E) Use of a small animal live imager to show mouse xenograft tumors. (F) Images of tumors in nude mice. (G) A tumor growth curve graph was drawn to demonstrate the tumor’s growth. (H) The tumor weight of nude mice was calculated. (I) H&E staining was used to visualize tumor structures of si-NC and si-circCCT2 (×10). The inset shows that knockdown of circCCT2 resulted in a significant reduction in the number of lesions (×40). (J) IHC staining was applied to find variations in the expression of Ki67, N-cadherin & SNAI2 in tumors

Furthermore, we investigated how circCCT2 affects HNSCC in vivo through a xenograft tumor study. A total of 10 nude mice participated in this experiment. Two of them did not develop tumors, and the other two had ruptured tumors when injected with siRNA and were discarded. The xenograft tumors formed in the si-circCCT2-injected group had a significantly lower size than those in the other group (Fig. 2E-G), and the weight of the tumor was also significantly less than those in the other group (Fig. 2H). A significant decrease in the quantity of lesions was found with the knockdown of circCCT2, as shown by H&E staining (Fig. 2I). Si-circCCT2 xenograft tumors expressed lower levels of Ki67, and the EMT markers N-cadherin & SNAI2 than controls, as determined by IHC staining (Fig. 2J). Overall, circCCT2 may act as an oncogenic gene in the progression of HNSCC in vivo and in vitro.

CircCCT2 sponged miR-146a-5p in HNSCC

Our study investigated whether circCCT2 could be used as a miRNA sponge to combine with miRNAs to regulate downstream gene expression. This depends on whether it is distributed in the cytoplasm. First, circCCT2 was predominantly present in the cytoplasm, as shown by nucleoplasmic separation and FISH assays (Fig. 3A, B). We then used two databases (circBank and starBase) to predict circCCT2 binding miRNAs (Additional file 1: Table S5), and intersected the results with miRNAs downregulated in RNA sequencing databases (GSE133632). Notably, miR-146a-5p was the only miRNA found (Fig. 3C). Subsequently, the RIP test was done to further confirm this association. In contrast to IgG, circCCT2 was specifically enriched by anti-AGO2 antibodies (Fig. 3D). And miR-146a-5p mimics suppressed WT circCCT2 luciferase action, but it did not affect MUT circCCT2 in HEK293T cells, as shown by the dual-luciferase gene reporter experiment (Fig. 3E).

Fig. 3.

Fig. 3

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CircCCT2 sponged miR-146a-5p in HNSCC. (A) CircCCT2 & 18 S RNA were abundantly present in the cytoplasm, but U6 was abundantly present in the nucleus via qPCR. U6 & 18 S RNA serve as markers in the nucleus and cytoplasm, correspondingly. (B) FISH was used to detect circCCT2 localization. (C) Probable targeted miRNAs of circCCT2 were displayed via Venn diagram. (D) RIP assays were performed to detect circCCT2 enrichment in HNSCC cells with an anti-AGO2 antibody. (E) Based on a dual-luciferase reporter test, circCCT2 may bind directly to miR-146a-5p. (F) miR-146a-5p transcript levels were assessed by qPCR after circCCT2 knockdown. (G) miR-146a-5p transcript levels in 23 sets of HNSCC samples were detected using qPCR. (H) Pearson correlation analysis of miR-146a-5p and circCCT2 in HNSCC

In addition, the miR-146a-5p level was upregulated following circCCT2 silencing in FD-LSC-1 and CAL-27 cells (Fig. 3F). Moreover, miR-146a-5p expression was downregulated in HNSCC samples (Fig. 3G). MiR-146a-5p expression was also found to be negatively correlated with circCCT2 in 23 sets of HNSCC clinical samples (Fig. 3H). Based on these results, it suggests that circCCT2 may bind competitively to miR-146a-5p in HNSCC. Is miR-146a-5p a tumor-suppressor gene for HNSCC? For an answer, we will conduct further research.

Is miR-146a-5p a tumor-suppressor gene for HNSCC?

First, we designed and synthesized miR-146a-5p mimics. A qPCR test revealed that FD-LSC-1 & CAL-27 cells added with miR-146a-5p mimics had overexpressed miR-146a-5p (Additional file 2: Fig. S1A). Overexpression of miR-146a-5p repressed the proliferation and metastasis capacity of HNSCC cells through CCK-8, colony formation along with Transwell experiments (Additional file 2: Fig. S1B-D).

Moreover, we carried out a rescue experiment to determine whether circCCT2 promotes HNSCC cell progression via interacting with miR-146a-5p. The si-circCCT2 (si-circ-2) and miR-146a-5p inhibitor were co-transfected into CAL-27 and FD-LSC-1 cells. qPCR experiments showed that knockdown of circCCT2 inhibited circCCT2 expression and promoted miR-146a-5p expression, but this effect was reversed by miR-146a-5p inhibitor transfection; miR-146a-5p inhibitor inhibited miR-146a-5p expression and promoted circCCT2 expression but this effect was reversed by knockdown of circCCT2 (Fig. 4A). HNSCC cell proliferation and colony formation ability were greatly enhanced via miR-146a-5p inhibitor transfection using CCK-8 and colony formation experiments (Fig. 4B, C). Additionally, the circCCT2 silencing-induced reduction in cell proliferative capacity was restored via the addition of the miR-146a-5p inhibitor (Fig. 4B, C). miR-146a-5p inhibition rescued the reduced migration and invasion ability caused by si-circCCT2 transfection through Transwell assays (Fig. 4D). Overall, miR-146a-5p works as a tumor-suppressor gene for HNSCC, and circCCT2 can enhance the malignant development of HNSCC via interacting with miR-146a-5p.

Fig. 4.

Fig. 4

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miR-146a-5p reverses circCCT2 pro-cancer impact in HNSCC cells. (A–D) Transfection of si-NC, si-circ-2, miR-146a-5p inhibitor, or si-circ-2 + miR-146a-5p inhibitor toward FD-LSC-1 & CAL-27 cells. (A) CircCCT2 and miR-146a-5p expression was measured via qPCR. (B, C) CCK8 (B) & colony formation (C) experiments were conducted to examine the cell growth capacity of each group. (D) Transwell tests were performed to assess the migration & invasion ability of each group

IRAK1 represents a downstream candidate of miR-146a-5p

To ascertain prospective downstream mRNAs of miR-146a-5p, a cross-analysis was conducted via the miRDB, miRpathDB, miRWalk, TargetScan and starBase databases, which identified 29 potential downstream targets (Fig. 5A; Additional file 1: Table S6). Subsequently, we crossed these 29 targets with mRNAs with high expression in LSCC clinical samples from the RNA-seq database (GSE127165), resulting in 3 intersecting targets (Fig. 5B; Additional file 1: Table S7). Only IRAK1 mRNA levels showed a significant decrease in FD-LSC-1 cells owing to the addition of miR-146a-5p mimics via qPCR (Fig. 5C). We further examined IRAK1 expression in HNSCC clinical samples by qPCR. The findings confirmed that IRAK1 expression was upregulated in HNSCC samples (Fig. 5D).

Fig. 5.

Fig. 5

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IRAK1 represents a downstream candidate of miR-146a-5p in HNSCC. (A) Predicted downstream targets of miR-146a-5p. (B) Analysis of miR-146a-5p targets on the basis of bioinformatics predictions and the RNA sequencing database GSE127165. (C) After miR-146a-5p overexpression, downstream target gene expression was assessed via qPCR. (D) IRAK1 expression was measured in 50 pairs of HNSCC tissues via qPCR. (E) Dual-luciferase gene reporter assays confirmed that miR-146a-5p binds to IRAK1. (F, G) IRAK1 expression was observed through western blotting. (H) Pearson correlation analysis of miR-146a-5p and IRAK1 in HNSCC. (I) Pearson correlation analysis of IRAK1 and circCCT2 in HNSCC. (J–L) Infection of FD-LSC-1 & CAL-27 cells with OE-NC, OE-IRAK1, OE-IRAK1 + mimics-NC, or OE-IRAK1 + miR-146a-5p mimics. (J) IRAK1 expression was determined via qPCR. (K) Cell colony formation assays were conducted to examine the cell growth capacity of each group. (L) The ability to migrate & invade were investigated using Transwell migration and invasion assays

Afterwards, miR-146a-5p overexpression only weakened the luciferase activity of IRAK1-WT through a dual-luciferase gene reporter test in HEK293T cells (Fig. 5E). The results of western blotting demonstrated that circCCT2 knockdown or miR-146a-5p overexpression markedly lowered the translation level of IRAK1 (Fig. 5F, G), but IRAK1 overexpression reversed the inhibition of IRAK1 expression after circCCT2 knockdown (Fig. 5F). Moreover, IRAK1 expression and miR-146a-5p had a negative association, however IRAK1 expression and circCCT2 had a positive correlation, according to a Pearson correlation study (Fig. 5H, I).

Additionally, we conducted rescue experiments to test whether miR-146a-5p could specifically target IRAK1 and affect HNSCC cell growth, migration and invasion. FD-LSC-1 & CAL-27 cells were transfected with both miR-146a-5p mimics and the OE-IRAK1 plasmid, qPCR experiments showed that overexpression of IRAK1 promoted IRAK1 expression, but this effect was reversed by miR-146a-5p mimics transfection (Fig. 5J). IRAK1 overexpression reversed the decrease in proliferative ability owing to miR-146a-5p mimics addition, as determined via colony formation experiments (Fig. 5K). Because of the overexpression of miR-146a-5p, Transwell tests demonstrated that IRAK1 overexpression restored the ability to migrate and invade (Fig. 5L). Collectively, IRAK1 represents a downstream candidate of miR-146a-5p and the miR-146a-5p/IRAK1 axis is able to suppress HNSCC cells growth and metastasis.

IRAK1 can reduce the inhibitory effect of circCCT2 knockdown on HNSCC cells

To examine whether circCCT2 enhances the progression of HNSCC through IRAK1, we restored the IRAK1 transcription level (Fig. 6A) and translation level (Fig. 6B) caused by circCCT2 knockdown in FD-LSC-1 & CAL-27 cells. CCK-8 along with colony formation tests revealed that IRAK1 overexpression reversed the reduction in proliferative and colony formation capacity caused by circCCT2 knockdown (Fig. 6C, D). Transwell experiments showed that IRAK1 overexpression reversed the reduction in cell migration and invasion caused by cirCCT2 knockdown (Fig. 6E).

Fig. 6.

Fig. 6

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IRAK1 can reduce the inhibitory effect of circCCT2 knockdown on HNSCC cells. (A–E) The following siRNAs or plasmids were transfected into FD-LSC-1 and CAL-27 cells: si-NC + OE-NC, si-NC + OE-IRAK1, si-circ-2 + OE-NC, or si-circ-2 + OE-IRAK1. (A) IRAK1 expression was measured using qPCR. (B) The expression of IRAK1 and EMT markers (N-cadherin & SNAI2) was evaluated under different conditions using western blotting. (C, D) CCK-8 (C) along with colony formation (D) experiments were conducted to examine each group of cells growth capacity. (E) The invasion and migration capacity of each cell group was examined via Transwell migration & invasion tests

Moreover, EMT marker expression was assessed via western blotting. IRAK1 overexpression rescued the inhibition of the EMT markers N-cadherin and SNAI2 after circCCT2 silencing (Fig. 6B). Overall, circCCT2 promoted HNSCC proliferation and metastasis by upregulating IRAK1 expression and influencing EMT at the same time. It is worth noting that all of the above research work was a series of experiments carried out by knocking down circCCT2, but overexpression of circCCT2 is also very important, what is the effect of overexpression of circCCT2 on HNSCC cells? We are continuing with the following studies.

circCCT2 facilitates malignant behavior of HNSCC cells by regulating the miR-146a-5p/IRAK1 axis

The role of circCCT2/miR-146a-5p/IRAK1 axis in the regulation of HNSCC was analyzed through cell rescue experiments. First, circCCT2 overexpression plasmid (OE-circCCT2) and siRNA (si-IRAK1) were designed and produced to overexpress circCCT2 and knock down IRAK1, respectively. Subsequently, FD-LSC-1 & CAL-27 cells were transfected with both OE-circCCT2 plasmid and miR-146a-5p mimics or si-IRAK1, respectively. qPCR experiments showed that overexpression of circCCT2 promoted circCCT2 and IRAK1 expression, and inhibited miR-146a-5p expression, but this effect was reversed by miR-146a-5p mimics or si-IRAK1 transfection, respectively (Fig. 7A). CCK-8 and colony formation assays showed that overexpression of miR-146a-5p or IRAK1 knockdown reversed the promoting effect of circCCT2 on the growth capacity of HNSCC cells caused by overexpression of circCCT2 (Fig. 7B, C). Transwell assays showed that overexpression of circCCT2 could promote the invasion and migration ability of HNSCC cells, but this effect was reversed by miR-146a-5p mimics or si-IRAK1 transfection, respectively (Fig. 7D).

Fig. 7.

Fig. 7

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circCCT2 facilitates malignant behavior of HNSCC cells by regulating the miR-146a-5p/IRAK1 axis. (A-D) The following siRNAs or plasmids were transfected into FD-LSC-1 and CAL-27 cells: OE-NC + si-NC, OE-circCCT2 + si-NC, OE-circCCT2 + miR-146a-5p mimics, or OE-circCCT2 + si-IRAK1. (A) circCCT2, miR-146a-5p and IRAK1 expression were measured using qPCR. (B and C) CCK-8 (B) together with colony formation (C) experiments were performed to examine the growth capacity of each cell group. (D) The invasion and migration capacity of each cell group was examined via Transwell migration & invasion assays. (E) The expression of circCCT2, miR-146a-5p and IRAK1 in tumors was assessed by qPCR. (F) IHC staining was applied to find variations in the expression of IRAK1 in tumors. (G) Diagram showing how the circCCT2/miR-146a-5p/IRAK1 axis regulates the malignant progression of HNSCC

In addition, through a xenograft tumor study we conducted previously, qPCR experiments showed that circCCT2 and IRAK1 expression decreased in xenograft tumors with circCCT2 knockdown, yet there was an increase in miR-146a-5p expression (Fig. 7E). And si-circCCT2 xenograft tumors expressed lower expression levels of IRAK1 than controls, as determined by IHC staining (Fig. 7F). Collectively, circCCT2 was derived from CCT2 mRNA, and circCCT2 competitively combined with miR-146a-5p to upregulate IRAK1 expression, thereby increasing proliferation, migration and invasion of HNSCC (Fig. 7G).

Discussion

CircRNAs are widely distributed, highly stable, conserved, and have unique closed-loop structures, and circRNAs can be considered as potential biomarkers for various diseases [12, 35, 36]. They have been shown in numerous studies to be significant in many different kinds of bioprocesses, including those related to cancer onset, progression, metastasis and chemoradiotherapy sensitivity [37–41]. The expression profile of circRNAs in different cancers is distinct, and the expression of a given circRNA across cancer types is significantly different; these findings, taken together with the stability of circRNAs, suggest that they can be used as potential cancer biomarkers [42]. A number of circRNAs with differential expression have been identified in HNSCC tissues via circRNA expression profiling [15, 33, 34, 43–45], indicating that circRNAs may play a crucial role in the onset and development of HNSCC. For example, the level of hsa-circ-0013561 is increased in HNSCC tissues, and its downregulation prevents HNSCC cells from proliferating and migrating, which leads to apoptosis [43]. CircGNG7 is a potent tumor suppressor, and circGNG7 overexpression significantly reduces HNSCC xenograft development in vivo, migration, colony formation, and cell proliferation in vitro [44]. Qiu et al. reported that circSCMH1 knockdown suppresses DDP-resistant oral squamous cell carcinoma chemoresistance and progression [45]. Researchers have shown that circMTCL1, circCORO1C and circRASSF2 can enhance LSCC cell invasion, migration, and growth [15, 33, 34]. In this study, we screened for a circular RNA, circCCT2, consisting of exons 7 and 8 of CCT2. Recent studies have reported that circCCT2 is significantly upregulated in glioma and hepatoblastoma and functions as an oncogene, suggesting a potential therapeutic target [18, 19]. Here, we reported the biological role of highly expressed circCCT2 in HNSCC for the first time. Actinomycin D and RNase R treatments showed that circCCT2 had a much longer half-life and was more resistant to RNase R than linear CCT2. Clinically, we discovered that high expression of circCCT2 was positively correlated with advanced T stage, lymph node metastasis, and clinical stage of HNSCC, and that individuals with HNSCC who express more circCCT2 have a worse overall prognosis. In vitro, overexpression of circCCT2 can enhance HNSCC cell proliferation, migration, and invasion, while knockdown of circCCT2 had the opposite effect. In vivo, knockdown of circCCT2 inhibited both EMT and the development of HNSCC cell xenograft tumors. These results indicate that circCCT2, which has a longer half-life and higher stability, acts as a critical oncogene to promote HNSCC malignant progression, suggesting a potential biomarker for HNSCC.

There is more research to be done on the precise regulating mechanism of circRNAs in HNSCC development. CirRNAs, protein-coding mRNAs, lncRNAs and pseudogenes are examples of RNA transcripts that function as endogenous competitive RNA (ceRNA) [46]. These transcripts interact with one another by competitively binding miRNAs, a class of non-coding RNAs that are significant gene expression regulators post-transcriptionally [46]. CircRNAs, as key members of the ceRNA network, are part of the circRNA‒miRNA‒mRNA pathway, and play a crucial role in cancer development and progression [47–49]. This is one of the major circRNA mechanisms of action [31]. In our research, circCCT2 was shown to be localized in the cytoplasm, and miR-146a-5p might act as a downstream miRNA of circCCT2, based on a bioinformatics analysis, dual-luciferase gene reporter along with RIP experiments. Researchers have discovered that miR-146a-5p weakens EMT markers, migration as well as invasion abilities in GBM [20]. Another study showed that miR-146a-5p inhibited the proliferation, migration and invasion through focusing on EIF4G2 in lung cancer [21]. However, it has been shown that enhancing miR-146a-5p decreased apoptosis and increased the growth of mice spinal cord neurons [50]. In the current work, circCCT2 silencing enhanced miR-146a-5p expression, while overexpression of circCCT2 produced the opposite effect. HNSCC tissues expressed considerably lower levels of miR-146a-5p, and miR-146a-5p inhibited the proliferation, migration and invasion of HNSCC cells. Rescue assays showed that miR-146a-5p inhibitor or mimics rescued the reduced or promoted proliferation, migration and invasion ability owing to circCCT2 silencing or overexpression in HNSCC cells. The above outcomes revealed that circCCT2 may promote HNSCC development through interacting with miR-146a-5p.

To determine potential downstream mRNAs of miR-146a-5p, applying bioinformatics method, we identified 3 intersecting downstream targets, then qPCR experiments showed that overexpression of miR-146a-5p only inhibited IRAK1 expression, so IRAK1 was selected. IRAK1 contributes to cancer progression because IRAK1 levels are increased in more than 20 different forms of cancer, and IRAK1 expression is associated with poor outcomes [23]. It’s worth noting that IRAK1 transcript levels are significantly higher in HNSCC than in similar tissue types, and IRAK1 expression is linked to bad overall survival in HNSCC [23]. In our study, we also confirmed that IRAK1 was highly expressed in HNSCC tumor tissues, and that the malignant progression of HNSCC cells may be enhanced through IRAK1 overexpression. Consistently, a dual-luciferase reporter experiment found that IRAK1 was a downstream mRNA of miR-146a-5p. Based on rescue tests, miR-146a-5p rescued HNSCC cell growth, migration and invasion caused by IRAK1 overexpression. Taken together, the miR-146a-5p/IRAK1 axis is able to suppress HNSCC cells growth and metastasis. It’s reported that miR-146a-5p damages NF-κB activity and EMT behavior by binding to IRAK1 and TRAF6 in GBM [20]. This suggests that the miR-146a-5p/IRAK1 signaling axis is not HNSCC specific.

Moreover, rescue experiments confirmed that in HNSCC cells, the overexpression of IRAK1 can restore si-circCCT2 inhibitory effects upon proliferation, migration and invasion, whereas knockdown of IRAK1 had the opposite effect. Many features of cancer development, such as invasion and metastasis, have been linked to EMT [51]. miR-146a-5p damages NF-κB activities and EMT behavior by binding to IRAK1 and TRAF6, was deficient in M2-EVs, suggesting that miR-146a-5p could be an encouraging GBM treatment candidate. One essential EMT network regulating protein in GBM is IRAK1 [20]. Our data showed that changes in circCCT2 or IRAK1 expression levels influenced the expression of EMT indicators, suggesting that the circCCT2/miR-146a-5p/IRAK1 axis facilitates HNSCC cell metastasis via controlling EMT. Mechanistically, circCCT2 competitively combines with miR-146a-5p to upregulate IRAK1 expression, thereby promoting malignant progression in HNSCC. However, further investigation is still required to determine the underlying mechanisms and upstream regulation of circCCT2.

RNA therapeutics are promising due to their scalability, editability and potential for personalized medicine, however, linear RNA therapy is limited by factors such as low stability, limited expression time and potential immunogenicity [52]. In contrast, circular RNAs show unique advantages. Latest research reported that administration of vaccines consisting of tumour-specific circFAM53B or its encoded peptides in mice bearing breast cancer tumors or melanoma induced enhanced anti-tumor immune responses and inhibited tumor growth [53]. Liu et al. reported that downregulated circASH2 expression indicated a worse prognosis of hepatocellular carcinoma (HCC) patients, circASH2 remodeled tumor cytoskeleton via suppressing TPM4 and exerted a significant antimetastatic activity in vivo and in vitro in HCC [54]. Notably, circRNAs can also exert anti-tumor effects through non-coding functions. For example, in 2018, circRNAs synthesized in vitro containing multiple miR-21 protruding binding sites effectively inhibited miR-21 activity and gastric cancer cell proliferation compared to miR-21 inhibitors [35]. Thus, due to these features of higher stability, longer half-life and pro-cancer pathway circCCT2/miR-146a-5p/IRAK1 axis, circCCT2 could be a therapeutic target for HNSCC. It is worth noting that the same circRNA may have different functions in different cells or diseases, which may raise safety issues for the clinical application of circRNA-based vaccines or therapies [52].

Conclusion

In summary, we identified a novel circular RNA, circCCT2, functioned as an oncogene in HNSCC, which competitively combines with miR-146a-5p to upregulate IRAK1 expression, thereby increasing HNSCC cell proliferation, migration and invasion (Fig. 7G). This signaling axis is critical for controlling the occurrence and progression of HNSCC, and circCCT2 is a potential biomarker and therapeutic target for HNSCC.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (204.9KB, pdf)
Supplementary Material 2 (25.4MB, tif)
Supplementary Material 3 (18.2KB, docx)
Supplementary Material 4 (16.3KB, docx)
Supplementary Material 5 (16.4KB, docx)
Supplementary Material 6 (54.8KB, xlsx)
Supplementary Material 7 (11.8KB, xlsx)
Supplementary Material 8 (127.7KB, xlsx)
Supplementary Material 9 (10.3KB, xlsx)

Acknowledgements

Not applicable.

Author contributions

LH designed the research and wrote the first manuscript text. LH, HNG, QH and LRL contributed to the cell function experiments, western blotting, qPCR and so on. LTZ and XYG helped with the animal experiments and performed the IHC and H&E staining. YJG helped prepare the figures. The paper underwent editing by CMZ, HLL, and HNG. The final version has been reviewed and accepted by all the writers.

Funding

This study was funded by the Basic Research Program of Shanxi Province (Free Exploration) (No. 20210302124594, 202203021211015, 202203021212029, 202203021212036).

Data availability

The RNA-seq data presented in this study are available in [GEO database] at [https://www.ncbi.nlm.nih.gov/geo/], reference number [GSE133632; GSE127165].

Declarations

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Institutional review board

All patients with HNSCC completed an informed consent form. The study was approved by the Ethics Committee of the First Hospital of Shanxi Medical University (protocol code NO. K-K092). The animal study was approved by the Ethics Committee of the First Hospital of Shanxi Medical University for Research Involving Animals (protocol code NO. DWYJ-2023-023).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Long He, Email: hl@sxent.org.

Huina Guo, Email: guohuina@sxent.org.

Hongliang Liu, Email: liuhl2018@sxent.org.

Chunming Zhang, Email: zcmsxmu@sxent.org.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (204.9KB, pdf)
Supplementary Material 2 (25.4MB, tif)
Supplementary Material 3 (18.2KB, docx)
Supplementary Material 4 (16.3KB, docx)
Supplementary Material 5 (16.4KB, docx)
Supplementary Material 6 (54.8KB, xlsx)
Supplementary Material 7 (11.8KB, xlsx)
Supplementary Material 8 (127.7KB, xlsx)
Supplementary Material 9 (10.3KB, xlsx)

Data Availability Statement

The RNA-seq data presented in this study are available in [GEO database] at [https://www.ncbi.nlm.nih.gov/geo/], reference number [GSE133632; GSE127165].

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