Hsa_circ_0002162 has a critical role in malignant progression of tongue squamous cell carcinoma through targeting miR-33a-5p

The aim of this study was to explore the effect of hsa_circ_0002162 on regulating cell proliferation, apoptosis, and invasion, and investigate its potential target microRNA (miRNA) in tongue squamous cell carcinoma (TSCC). Hsa_circ_0002162 expression was detected in human TSCC cell lines and human oral keratinocytes (HOK) cell line. Cell proliferation, apoptosis, invasion, and candidate target miRNA expressions were detected in hsa_circ_0002162 knockdown-treated CAL-27 cells and hsa_circ_0002162 overexpression-treated SCC-9 cells. In the rescue experiment, miR-33a-5p knockdown plasmid was transfected into hsa_circ_0002162 knockdown-treated CAL-27 cells, while miR-33a-5p overexpression plasmid was transfected into hsa_circ_0002162 overexpression-treated SCC-9 cells. Subsequently, cell proliferation, apoptosis, and invasion were detected, and then luciferase reporter assay was performed. Hsa_circ_0002162 expression was increased in human TSCC cell lines SCC-9, CAL-27, HSC-4, and SCC-25 compared with HOK. In CAL-27 cells, hsa_circ_0002162 knockdown inhibited cell proliferation and invasion and promoted apoptosis. In SCC-9 cells, hsa_circ_0002162 overexpression enhanced cell proliferation and invasion and suppressed apoptosis. Furthermore, a negative regulation of hsa_circ_0002162 on miR-33a-5p (but not miR-302b-5p and miR-545-5p) was observed. In the rescue experiment, miR-33a-5p knockdown increased cell proliferation and invasion, and decreased apoptosis in hsa_circ_0002162 knockdown-treated CAL-27 cells, whereas miR-33a-5p overexpression decreased cell proliferation and invasion, but increased apoptosis in hsa_circ_0002162 overexpression-treated SCC-9 cells. The luciferase reporter assay showed the direct binding of hsa_circ_0002162 to miR-33a-5p. In conclusion, hsa_circ_0002162 had an important role in malignant progression of TSCC through targeting miR-33a-5p.


Introduction
Tongue squamous cell carcinoma (TSCC) is the most common malignancy of the oral cavity, and it is also one of the most lethal head and neck cancers worldwide (1,2). Due to its characteristic silence (progressing from a premalignant state into invasive carcinoma without any specific alarming symptoms), most TSCC patients are in an advanced stage when diagnosed (3). Even with improved treatments (including surgery resection, chemotherapy, and radiotherapy), the prognosis of TSCC patients is still unsatisfactory with the 5-year relative survival rate of 63% (4). Hence, investigation of molecular mechanisms underlying TSCC is necessary to aid in the development of novel therapy targets and improve the prognosis of TSCC patients.
Circular RNAs (circRNAs) are a novel type of endogenous RNAs featuring covalently closed continuous loops, which not only sponge microRNAs (miRNAs) but also interact with RNA-binding proteins (5). Previous studies reveal that multiple circRNAs are differentially expressed in several cancers, and their dysregulation contributes to tumor progression by promoting cell viability, cell mobility, epithelial mesenchymal transformation (EMT), and even cell stemness in various carcinomas (such as TSCC, hepatocellular cancer, and gastric cancer) (6)(7)(8). As one of the newly discovered circRNAs, hsa_circ_0002162 has been reported to be highly expressed in TSCC tumor tissues compared to adjacent tissues according to a recent study with highthroughput sequencing (in three TSCC tissues and adjacent tissues). Further reverse transcription quantitative polymerase chain reaction (RT-qPCR) also showed higher expression of hsa_circ_0002162 in TSCC tumor tissues than adjacent tissues, consistent with the high-throughput data (9). No more evidence regarding the role of hsa_ circ_0002162 in TSCC was found. In addition, miR-33a-5p, miR-302b-5p, and miR-545-5p are reported to be potential targets of hsa_circ_0002162 in TSCC (9), and these three miRNAs have been observed to exert regulatory effects on cell proliferation and cell invasion in multiple cancers (10)(11)(12). Thus, we hypothesized that hsa_circ_0002162 might target miR-33a-5p, miR-302b-5p, and/or miR-545-5p to promote TSCC tumorigenesis. Therefore, in the current study, the aim was to explore the effect of hsa_circ_0002162 on regulating cell proliferation, apoptosis, and invasion, and to investigate its potential target miRNA in TSCC cell lines.
Hsa_circ_0002162 plasmid construction and transfection pGPH1 vector was applied to construct hsa_circ_000 2162 knock-down plasmid and circRNA control knockdown plasmid by GenePharma Co., Ltd (China). pCD5-ciR vector was applied to construct hsa_circ_0002162 overexpression plasmid and circRNA control overexpression plasmid by Geneseed Biotech Co., Ltd. (China). Hsa_ circ_0002162 knock-down plasmid and circRNA control knock-down plasmid were transfected into CAL-27 cells using HilyMax (Dojindo, Japan), and the cells were divided into Circ(-) cells and NC(-) cells, accordingly. Hsa_circ_ 0002162 overexpression plasmid and circRNA control overexpression plasmid were transfected into SCC-9 cells using HilyMax (Dojindo), and the cells were divided into Circ(+) cells and NC(+) cells, accordingly. The expression of hsa_circ_0002162 in the four cell lines was evaluated by RT-qPCR at 24 h post transfection.

Cell proliferation, apoptosis, and invasion measurements
At 0, 24, 48, and 72 h after transfection, Cell Counting kit-8 (Dojindo) was used to perform CCK-8 assay to detect cell proliferation. At 48 h after transfection, Annexin V-FITC apoptosis detection kit (R & D, USA) was used to perform annexin V/propidium iodide (AV/PI) assay to assess cell apoptosis. Furthermore, 24 h after transfection, Matrigel s basement membrane matrix coated chamber (Corning, USA) was used to perform Transwell assay to determine cell invasion ability.

RT-qPCR assay
Total RNA was extracted by TRIzolt reagent (Invitrogen, USA), and the concentration and purity of RNA were determined by spectrophotometry. For circRNA (but not for miRNA or mRNA), 1 mg total RNA was used for linear RNAs digestion using RNase R enzyme (Epicentre, USA), and subsequently, reverse transcription was performed using PrimeScriptt RT reagent kit (Takara), followed by qPCR using TB Green s Fast qPCR mix (Takara). For miRNA and mRNA detection, 1 mg total RNA was reversely transcribed to cDNA using PrimeScriptt RT reagent kit (Takara), and qPCR was carried out using TB Green s Fast qPCR mix (Takara). According to a previous study (9), hsa_circ_0002162 is identified in TSCC and detected by RT-qPCR. Therefore, we referred to the prior hsa_circ_ 0002162 primer in this study. Primers are listed in Table 1. Glyceraldehyde-phosphate dehydrogenase (GA PDH) was used as internal reference of circRNA, and U6 was used as internal reference of miRNA. Relative quantification of gene expression was performed by the 2 -WWCt method.

Statistical analysis
Data are reported as mean and standard deviation. GraphPad Prism software version 7.0 (GraphPad Software Inc., USA) was applied for data analysis and graph making. Comparison between two groups was determined by the unpaired t-test, and comparison among more than two groups was determined by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. A P value o0.05 was considered as statistically significant. Table 1. Primer sequences used in the study.

Gene
Forward primer

Hsa_circ_0002162 expression after transfection
In CAL-27 cells, hsa_circ_0002162 expression was decreased in the Circ(-) group compared to the NC(-) group (Po0.001) after transfection (Figure 2A). In SCC-9 cells, hsa_circ_0002162 expression was increased in the Circ(+) and NC(+) groups (Po0.001) after transfection ( Figure 2B). In addition, in CAL-27 cells, hsa_circ_ 0002162 expression was increased in the Circ(+)and NC (+) groups (Po0.001) after transfection ( Figure S1A). In SCC-9 cells, hsa_circ_0002162 expression was decreased in the Circ(-) group compared to the NC(-) group  In SCC-9 cells, cell proliferation was enhanced in the Circ (+) group compared to the NC(+) group at 72h (Po0.05) ( Figure 3F) and cell apoptosis was repressed in the Circ (+) group compared to the NC(+) group at 48 h (Po0.05) ( Figure 3G and H). In addition, cell invasion was increased in the Circ(+) group compared to the NC(+) group at 24 h (Po0.01) ( Figure 3I and J). In addition, the expression of proliferation protein ki-67 was decreased but the expression of apoptosis protein C-caspase 3 was increased in the Circ(-) group compared to the NC(-) group ( Figure 4A). However, the opposite trend was shown in the Circ(+) group compared to the NC(+) group ( Figure 4B).

Luciferase reporter assay
Hsa_circ_0002162 had a strong potential to bind miR-33a-5p, and the designed sequences between hsa_ circ_0002162 WT/MT and miR-33a-5p are shown ( Figure  9A). Relative luciferase activity was reduced in the WT & OE-miR-33a-5p group compared to the WT & OE-NC group (Po0.01), but there was no difference in the relative luciferase activity between the MT & OE-miR-33a-5p group and the MT & OE-NC group (P40.05) ( Figure 9B).

Discussion
In the present study, hsa_circ_0002162 was highly expressed in human TSCC cell lines SCC-9, CAL-27, HSC-4, and SCC-25 compared with HOK, and it promoted cell proliferation and invasion and repressed cell apoptosis in TSCC cell lines. Interestingly, hsa_circ_ 0002162 contributed to TSCC tumorigenesis through targeting miR-33a-5p.
CircRNAs are a type of RNA featured by covalent closed loops formed by back-splicing, which play key regulatory roles in pathological processes of various carcinomas, including digestive cancers (7,13,14). For instance, an interesting study reveals that silencing of circ-DONSON (circbase ID: hsa_circ_0004339) represses cell proliferation, migration, and invasion, and enhances apoptosis via recruiting the nucleosome remodeling factor (NURF) complex to initiate SOX4 expression in gastric cancer (7). In addition, silencing of hsa_circ_0067 934 represses cell proliferation, inhibits migration, and blocks cell cycle in esophageal squamous cell carcinoma (ESCC) (13). Furthermore, circ_001569 facilitates cell growth, migration, and invasion through sponging miR-411-5p and miR-432-5p in hepatocellular carcinoma (14).
In TSCC, limited information was found in only two previous studies. One interesting study shows that knockdown of circ_0001742 serves as a competing endogenous RNA (ceRNA) to mediate the miR-431-5p/activating transcription factor 3 (ATF3) axis, thereby suppressing cell proliferation, cell migration, cell invasion, and EMT in TSCC cells (15). The other study showed that hsa_circ_ 0001742 inhibition suppresses cell proliferation, invasion, and EMT processes through targeting the miR-634/rasrelated protein Rab-1A (RAB1A) pathway in TSCC (8). Taken together, multiple circRNAs play important roles in the pathology of various carcinomas, including TSCC.
Hsa_circ_0002162 is one of the newly discovered circRNAs. One recent study performed the high-throughput sequencing to screen circRNA expression profiles from three TSCC tissues and adjacent tissues, and then carried out RT-qPCR for validation of several circRNAs expression profiles, which revealed that hsa_circ_0002 162 is highly expressed in TSCC tumor tissues compared to adjacent tissues (9). Based on above information, we hypothesized that has-circ_0002162 might contribute to tumor progression in TSCC. The possible explanations for our results were as follows: i) hsa_circ_0002162 might mediate multiple genes or pathways (such as miR-33a-5p, miR-634/RAB1A pathway (seen as the role of hsa_circ_ 0001742 in TSCC) [8], or miR-431-5p/ATF3 axis (seen as the role of circ_0001742 in TSCC) [15]) to promote tumorigenesis in TSCC; hence, TSCC cell lines were  characterized by increased expression of hsa_circ_0002 162. ii) hsa_circ_0002162 might negatively regulate several miRNAs (including miR-33a-5p, miR-411-5p, and miR-432-5p (seen as the role of circ_001569 in hepatocellular carcinoma) [14]) to accelerate cell proliferation and invasion while repressing cell apoptosis in TSCC.
In conclusion, hsa_circ_0002162 induced apoptosis and decreased cell viability by suppressing miR-33a-5p. The findings may provide a novel perspective on circRNA and lay a foundation for future research of potential roles of circRNA in TSCC.