CircMAT2B Induced by TEAD1 Aggravates the Warburg Effect and Tumorigenesis of Oral Squamous Cell Carcinoma through the miR-942-5p/HSPD1 Axis

Oral squamous cell carcinoma (OSCC) is one of the most lethal cancers worldwide. The high morbidity and mortality of OSCC are a great burden to global health-care systems. Therefore it is important to understand the underlying molecular mechanisms of OSCC initiation and progression. This study aimed to investigate the role of circMAT2B in OSCC progression and its molecular mechanisms. First, the expression and circularization of circMAT2B in OSCC cells were verified. Subsequently, knockdown of circMAT2B was shown to inhibit OSCC cell proliferation, migration, invasion, and the Warburg effect. Bioinformatics prediction, RNA-pull down, and luciferase reporter gene assays led to the identification of a novel TEAD1/circMAT2B/miR-942-5p/HSPD1 axis in OSCC progression. In conclusion, the novel TEAD1/circMAT2B/miR-942-5p/HSPD1 axis is a potential target for OSCC.


Introduction
Oral squamous cell carcinoma (OSCC) is one of the most lethal cancer types globally, accounting for about 3% of overall malignant tumors, and 95% of head and neck cancers [1][2][3]. OSCC has a poor 5-year survival rate because of diffuse metastasis and frequent recurrence, so less than 50% of patients diagnosed with OSCC survive more than one year [4,5]. Despite advancements in diagnosis and treatments made in recent years, the high morbidity and mortality of OSCC still represent an enormous health burden worldwide, with OSCC patients having a poor quality of life. erefore, it is important to understand the underlying molecular mechanisms of OSCC to develop more effective therapies.
Circular RNAs (circRNAs) are a class of non-coding RNAs and products of back-splicing events from an exon to a covalently closed loop. A few circRNAs have been reported in the nucleus and regulate gene transcription. However most circRNAs are located in the cytoplasm, indicating the biological function of circRNAs in cellular behaviors [6,7]. Structurally, circRNAs have conserved, stable, tissue-specific, subcellular location and developmental stagespecific characteristics, suggesting a specific biological role in cellular behaviors [8]. Mechanically, circRNAs can interact with miRNAs and proteins and can be translated into proteins [9][10][11][12][13]. Biologically, circRNAs are involved in multiple biological or pathological events, especially in cancers [14][15][16][17][18]; therefore, there are promising therapeutic targets for various diseases and they demand further exploration.
In the current study, we aimed to investigate the role of circMAT2B in OSCC progression, showing that circMAT2B is highly expressed in OSCC cells. Furthermore, the dysregulation of circMAT2B in OSCC cells influences cell proliferation, migration, invasion, and Warburg effects. Also, a novel circMAT2B/miR-942-5p/HSPD1 axis in OSCC progression was identified and circMAT2B expression in OSCC cells was transcriptionally regulated by TEAD1. is study has partially demonstrated the role of the TEAD1/ circMAT2B/miR-942-5p/HSPD1 axis in OSCC development and might provide new insight for OSCC diagnosis and therapeutic research.

Western Blotting.
e OSCC cells were lysed in RIPA buffer with protein inhibitors and the protein content was quantified using a BCA kit. e proteins were separated on 10% SDS polyacrylamide gels and electrically transferred to PVDF membranes (Millipore). e membranes were blocked with no-fat milk before incubation with the primary and corresponding secondary antibodies. Antibodies used in this study were as follows: anti-HSPD1 (CST, # 12165S, 1 : 1000), GAPDH (CST, # 5174S, 1 : 1000).

Ethynyl-2-Deoxyuridine (EdU) Incorporation Assay.
e incorporation of EdU into DNA was applied to detect cells undergoing DNA replication. After cell fixation and permeabilization, cells were incubated with 50 μM EdU solution for 3 hours and stained with 1 μg/m DAPI for 10 mins. Fluorescence microscopy was used to visualize and count the EdU-positive cells.

Transwell Assay.
Transwell assays were performed using Transwell chambers (Corning, USA). Infected cells were added to the upper chamber, and a culture medium with 20% FBS was added to the lower chamber. e migrated and invaded cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet, then visualised with an Olympus microscope.

Detection of Warburg Effect OSCC Cells.
e Warburg effects relative to glucose uptake, lactate production and ATP consumption were evaluated to assess the effect of the circMAT2B/miR-942-5p/HSPD1 axis in OSCC cells using commercial kits (Glucose uptake (#K924), lactate production (#K627), ATP generation (#K345), BioVision, USA) according to previous studies.

RNA Pull-Down.
e biotinylated RNA pull-down assay was conducted using MyOne ™ Dynabeads ® Streptavidin C1 beads. e beads were incubated with cell lysates for 60 mins at 4°C for preclearance. e RNA-probes were synthesized and commercially procured by Tsingke Biotech (Beijing, China). Subsequently, probes were incubated with beads for 10 min at room temperature for immobilization, and then incubated with the cell lysates for 12 hours overnight. e beads were magnetically separated and washed five times before qRT-PCR.

Luciferase Reporter
Assay. e luciferase assay was performed using the dual-luciferase reporter system psi-CHECKTM (Fisher Scientific). Cells (4 × 10 4 cells per well) were cultured in 24-well plates overnight and transfected with Wide Type (WT) or Mutant Type (MUT) vectors together with the plasmid for Renilla luciferase expression. Lipofectamine 2000 (Invitrogen) was used to perform transfections. After 24 hours, the cells were lysed, and their luciferase activities were measured using the dual-luciferase reporter assay system (Promega).

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Journal of Oncology

Statistical Analysis.
e data were analysed using SPSS 20.0 (IBM, USA) and presented as the mean ± standard deviation (mean ± SD). One-way ANOVA test or Student's t-test was used to determine the statistical significance and P < 0.05 was considered statistically significant. All experiments were repeated three times.

Characterization of circMAT2B in OSCC.
e expression of circMAT2B (Figure 1(a)) in OSCC cell lines (HOK, SCC-9, SCC-15, Cal-27, HSC-3, and HSC-6) was generally elevated, especially in Cal-27 and HSC-6 cells (Figure 1(b)). Next, Actinomycin D and RNase R were applied to evaluate the stability of circMAT2B in OSCC cells, showing that circMAT2B was more stable than its liner form in Cal-27 and HSC-6 cells (Figures 1(c) and 1(d)) and more resistant to Rnase R digestion than its liner form in Cal-27 and HSC-6 cells (Figures 1(e) and 1(f )). Subsequently, circMAT2B was found to be mainly distributed in OSCC cell cytoplasm (Figures 1(g) and 1(h)), indicating its potential for biological function. It was hypothesized that circMAT2B is involved in OSCC progression. ere is emerging evidence that circRNAs exert their biological function in cancer progression via modulating the Warburg effect, [26,27]. We examined the Warburg effect relative to glucose uptake, lactate production, and ATP level showing that circMAT2B knockdown suppressed the Warburg effects (Figures 2(i)-2(k)).

CircMAT2B Expression Is Transcriptionally Regulated by TEAD1.
e JASPAR dataset (https://jaspar.genereg.net/) was used to investigate whether circMAT2B expression in OSCC cells is transcriptionally regulated. ree potential transcriptional regulators were identified (TEAD1, TEAD2, and ZNF384) and as shown in Figures 3(a) and 3(b), the expression of circMAT2B was significantly decreased upon si-TEAD1 infection in Cal-27 and HSC-6 cells indicating that TEAD1 might regulate circMAT2B expression. e predicted binding sites of TEAD1 on MAT2B promoter sequences are presented in Figure 3(c). Subsequently, the interaction between TEAD1 and MAT2B promoter regions P2 and P3 was confirmed by conducting luciferase reporter gene assays in Cal-27 and HSC-6 cells as indicated (Figures 3(d) and 3(e)).
ese results suggest that circMAT2B sponges to miR-942-5p and negatively regulates miR-942-5p expression in OSCC cells. To verify the biological role of miR-942-5p in circMAT2B modulated OSCC cellular behaviors, we conducted rescue experiments, including EdU, Transwell migration assay, Transwell invasion assay, and Warburg effects detection. e results (presented in supplementary Figure S) suggest that downregulation of miR-492-5p reversed the inhibitory impacts of circMAT2B knockdown on OSCC cell proliferation, migration, invasion, and the Warburg effects.

Discussion
Head and neck squamous cell carcinoma (HNSCC) accounts for more than 800,000 new cases and causes about 450,000 cancer-related deaths in 2018. Of these, OSCC accounts for approximately 95% of HNSCC and is responsible for 350,000 new cases and 170,000 cancer-related deaths worldwide in 2018 [28]. OSCC generally originates in the tongue, gingiva, mouth floor, lips, alveolar ridge, buccal mucosa, hard palate, and retromolar trigone, with surgical resection with or without lymph nodes removal being the primary clinical treatment. However, the high recurrence and metastasis formation of OSCC lead to poor survival and life quality of OSCC patients [29,30]. erefore, novel diagnostic strategies and therapeutic targets for OSCC are necessary.
With the advent of high-through putting sequencing, multiple circRNAs in various cancers have been investigated and verified, including OSCC. In the current study, we investigated circMAT2B function in OSCC progression.
CircMAT2B was highly expressed in OSCC tumor tissues and mainly expressed in the cell cytoplasm. e downregulation of circMAT2B in OSCC cells inhibited cell proliferation, migration, invasion and the Warburg effect. e Warburg effect is an important phenomenon of aerobic glycolysis which maintains tumor cell proliferation and aggressive characteristics [31].
Subsequently, circMAT2B expression in OSCC could be regulated by transcriptional regular TEAD1. Furthermore, by conducting bioinformatic analysis, RNA pull-down, and luciferase reporter gene assays, a novel circMAT2B/miR-942-5p/HSPD1 axis was identified in OSCC cells. A previous study reported that miR-942-5p expression is upregulated in OSCC tumor tissues and contributes to OSCC progression (i)-(k) warburg effect relative glucose uptake (i), lactate production (j), and ATP levels (k) were detected. Each experiment was performed three times. Data are presented as mean ± SD. * P < 0.05, * * P < 0.01, * * * P < 0.001. 6 Journal of Oncology [35], whereas the role of HSPD1 in OSCC progression is still unclear. e present study found that circMAT2B upregulated HSPD1 expression to modulate OSCC cell proliferation, migration, invasion, and Warburg effect level through sponging miR-942-5p.
e upstream and downstream molecular mechanisms of circMAT2B in OSCC cells have been explored, providing a new diagnostic or therapeutic target for OSCC.
Data Availability e data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Disclosure
Chen Zou, Dahong Huang, and Haigang Wei are the co first authors.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
XL and YLA designed the study. CZ, DHH, and HGW performed the experiments. SYW analysed the data and made the figures. JS and ZT provided experimental material support. CZ, XL, and YLA wrote the paper. All authors contributed to the article and approved the submitted version.

Supplementary Materials
Supplementary Figure S MiR-492-5p reversed the inhibitory effects of circMAT2B on OSCC cellular process. A-B: cell models were constructed by transfecting Sh-NC, Sh-circMAT2B#1, Sh-circMAT2B#1 + si-NC, and Sh-circ-MAT2B#1 + si-miR-492-5p into Cal-27 or HSC-6 cells as indicated, transfection efficiency was assessed by qRT-PCR (A) and Western blot (B). C-D: cell proliferation level was detected by EDU assay (C) and results were statistically analyzed (D). E-F: cell migration level was assessed by Transwwell migration assay (E) and results were calculated (F). G-H: cell invasion level was measured by Transwwell invasion assay (F) and results were analyzed (H). I-K: Warburg effect relative glucose uptake (I), lactate production (J), and ATP levels (K) in OSCC cells were detected. Each experiment was performed three times. Data is presented as the Mean ± SD. * P < 0.05, * * P < 0.01, * * * P < 0.001. (Supplementary Materials)