A total of 50 pairs of tongue cancer specimens were collected from patients who underwent surgery at the First Affiliated Hospital of Fujian Medical University (Fuzhou, Fujian, China) between January 2017 and January 2020. The School and Hospital of Stomatology and the First Affiliated Hospital are cooperative units affiliated with Fujian Medical University. All samples were immediately snap frozen in liquid nitrogen and stored at −80°C until needed. Two pathologists pathologically confirmed each sample by hematoxylin-eosin (HE) staining. Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki and the study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University [approval number: No. 159 (2020) of the First Affiliated Hospital of Fujian Medical University] The clinicopathological characteristics of the patients are summarized in Table I.

TSCC cell lines CAL-27 and SCC-9 were purchased from the American Type Culture Collection and normal human oral keratinocyte (HOK) cells were generously donated by the research group of Professor Cheng Hui, School of Stomatology, Fujian Medical University. The primary HOK cells were purchased from ScienCell Research Laboratories Inc. In order to acquire adequate cells for the subsequent study, the immortalization of HOK was conducted. HOK were maintained in oral keratinocyte medium (OKM; ScienCell Research Laboratories Inc.) before proceeding with immortalization. The primary HOK were infected by hTERT retrovirus expressing simian virus 40 large T antigen (SV40-T). Once the infection of HOK was performed, the immortalized cells were subsequently selected and passaged with OKM changed every 3 days (25). CAL-27 cells and HOK cells were grown in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). SCC-9 cells were cultured in DMEM/F-12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 0.4 µg/ml hydrocortisone. The pH of the cell culture medium was 7.0 to 7.4. All cells were maintained at 37°C in an incubator supplied with 5% CO₂.

Total RNA was extracted from tissues or cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The cell density for RNA extraction was 1×10⁶. The concentration and purity of RNA were evaluated using a Quawell spectrophotometer (Q5000; Quawell Technology, Inc.). When the 260/280 ratio of RNA was >1.8, total RNA was reverse transcribed into cDNA using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. RT-qPCR analyses were performed using SYBR Green Master Mix (Takara Biotechnology Co., Ltd.) in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. PCR cycling conditions were: Pre-denaturation: 95°C, 1 min; denaturation: 95°C, 15 sec; annealing: 60°C, 30 sec; extension: 72°C, 1 min; 72°C, 10 min for a total of 40 cycles. Target mRNA expression levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative expression levels were calculated by the 2^{−∆∆ Cq} method (26). All experiments were repeated three times. MiR-mimics NC, hsa-miR-3180 mimics, miR-inhibitor NC and hsa-miR-3180 inhibitor were designed and synthesized by Shanghai GenePharma Co., Ltd. and the primers were provided by Fuzhou Sunya Biotechnology, Co., Ltd. and are listed in Table SI.

siRNAs targeting lncKRT16P6 and GATAD2A were designed and synthesized by Shanghai GenePharma Co., Ltd. (sequences listed in Table SII). The two siRNAs with the best knockdown efficiency were used in subsequent functional studies. The cell density of CAL-27 and SCC-9 cells for transfection was 2×10⁵. The mass of mimic, vector and si-NC used for transfection were 50 pmol. The transfection reagent was Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.) and the dosage for each well of a 6-well plate was 5 µl. Cell transfection steps were performed according to the product manual. Diluted Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.) in Opti-MEM Medium (Gibco; Thermo Fisher Scientific, Inc.) and mixed well. The master mix of DNA was prepared by diluting DNA in Opti-MEM Medium and mixing well. Diluted DNA was added to each tube of diluted Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.; 1:1 ratio). After incubation for 20 min at room temperature DNA-lipid complex was added to the cells. Cells were incubated for 48 h at 37°C before subsequent experiments. For lentiviral transduction, virus-containing supernatant was collected 48 h after cotransfection of the packaging plasmids pGag/Pol and pRev, the envelope plasmid pVSV-G (Shanghai GenePharma Co., Ltd.) and the short hairpin RNA (shRNA; shlncKRT16P6) vector or empty vector as control into 293(T) cells and the lentiviral vectors were then transduced into the target cells. The lentiviral vector pLV3/H1/GFP&Puro was constructed by Shanghai GenePharma Co., Ltd. To establish stable cell lines, tongue cancer cells were transduced with the above lentiviral vectors in the presence of polybrene (5 µg/ml, Shanghai GenePharma Co., Ltd.). After incubation for 72 h, cells were selected with 2 µg/ml puromycin (GeneChem) for 3 days. Information on the viral vectors and verification of the transduction efficiency are shown in Fig. S1.

The different groups of treated CAL-27 and SCC-9 cells (1,000) were seeded into each well of a 96-well plate. Cell viability was determined every 24 h using a CCK-8 assay (Dojindo Laboratories, Inc.) by measuring the absorbance of the different cell cultures at 450 nm (BioTek Instruments, Inc.), in accordance with the manufacturer's instructions. The proliferation of CAL-27 and SCC-9 cells expressing control or lncKRT16P6 siRNA was detected by CCK-8 assays, CCK-8 assays were performed in CAL-27 and SCC-9 cells stably transfected with sh-lncKRT16P6 with or without the miR-3180 inhibitor and CCK-8 assays were performed in CAL-27 and SCC-9 cells transfected with the miR-3180 inhibitor with or without GATAD2A siRNAs.

The invasive and migratory capacities of cells were evaluated using Transwell chambers with 8-µm pore filters. After 48 h of transfection, cells in serum-free medium (5×10⁴ cells/100 µl) were plated in the upper chambers, which contained membranes coated with or without 50 µl of Matrigel (BD Biosciences) at 37°C for 2 h. DMEM containing 10% FBS was added to the lower chambers as a chemoattractant. After incubation for 12 h at 37°C, cells that had migrated to the bottom surface of the membrane were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with a crystal violet solution at room temperature for 10 min. Cells in five random fields were counted and the numbers were averaged.

Transfected cells (1×10³) were uniformly seeded in 6-well plates and cultured for 2 weeks at 37°C in an incubator supplied with 5% CO₂, then the colony formation of the cells was observed under a low magnification (×10) microscope. The number of cells >10 were counted as a colony, and then the number of colonies in different treatments counted. The cells were then washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde at room temperature for 30 min, stained with 0.5% crystal violet at room temperature for 10 min and images captured.

Cells were routinely transfected and cultured for 48 h and were then digested with EDTA-free trypsin. An Annexin V-fluorescein isothiocyanate (FITC) apoptosis assay kit (BD Biosciences) was used to estimate the apoptosis rate according to the manufacturer's instructions. Cells were suspended in 1X Annexin binding buffer and 5 µl of Annexin V reagent and 3 µl of propidium iodide (PI) reagent were then added to 100 µl of the cell suspension and mixed. The mixture was incubated in the dark for 30 min at room temperature and 400 µl of 1X Annexin binding buffer was then added to each sample to terminate staining.

For cell cycle analysis, a PI/RNase staining kit (BD Biosciences) was used according to the manufacturer's instructions. After treatment, tongue cancer cells were harvested, washed with ice-cold PBS and fixed with 70% ethanol for 24 h at 4°C. After staining with PI in the dark for 30 min at 4°C, the apoptosis rate (the percentage of late apoptotic cells) and cell cycle distribution were evaluated using a FACSVerse flow cytometer (BD Biosciences).

Cy3-labeled probes specific for lncKRT16P6 were designed and synthesized by Shanghai GenePharma Co., Ltd. Probe signals were detected with a FISH kit (Shanghai GenePharma Co., Ltd.) according to the manufacturer's instructions. In brief, cells were fixed, permeabilized (1X PBS/0.5% Triton X-100) and prehybridized. Then, the cells were hybridized in hybridization buffer with Cy3-labeled probes specific for lncKRT16P6 at 37°C overnight. Cells were sequentially rinsed with 4X SSC (containing 0.1% Tween 20), 2X SSC and 1X SSC at 42°C and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Institute of Biotechnology). Probe signals were observed via fluorescence microscopy, the (magnification, ×630). The probe sequences are listed in Table SIII.

Cell nucleocytoplasmic fractionation was performed with a Nuclear and Cytoplasmic Protein Extraction kit and RNase Inhibitor (Beyotime Institute of Biotechnology,) according to the manufacturer's instructions. Cytoplasmic and nuclear RNA was separated from tongue cancer cells and reverse transcribed. The lncKRT16P6 level was evaluated by RT-qPCR as aforementioned and its percentages in the nuclear and cytoplasmic fractions were determined.

The Animal Experimentation Ethics Committee of Fujian Medical University approved the present study and ensured that all experiments conformed to all relevant regulatory standards (approval number: FJMU IACUC NO. 2020-0039 of Fujian Medical University). In the present study, 4-week-old BALB/c athymic male nude mice (Shanghai SLAC Laboratory Animal Co., Ltd.) were used. The weight of nude mice was ~20±5 g. All mice were maintained under controlled temperature (22±1°C) and humidity (50±5%) in a 12 h light/dark cycle with food and water available ad libitum. CAL-27 cells (5×10⁶ cells/100 µl) transfected with different constructs were subcutaneously injected into the nude mice. A total of eight mice were randomly divided into two groups: i) sh-NC group (control group, mice were injected subcutaneously with CAL-27 cells transfected with empty vector, n=4); ii) sh-lncKRT16P6 group (mice were injected subcutaneously with CAL-27 cells transfected with sh-lncKRT16P6, n=4). Tumors were measured weekly and the tumor volume (V) was calculated using the equation V=(LxW²)/2, where L and W were the length and width of the tumor, respectively. After 24 days, the mice were sacrificed; 1% pentobarbital sodium 40 mg/kg was injected intraperitoneally and the animals were sacrificed by acute exsanguination after they became unconscious. The tumors were excised for hematoxylin and eosin (HE) and immunohistochemical (IHC) staining.

For HE staining, in brief, paraffin-embedded tumor tissues were sliced into 4-µm-thick sections. Deparaffinized and rehydrated sections were stained with HE (Beyotime Institute of Biotechnology,) at room temperature for 5 min and the stained tissues were evaluated under a light microscope (magnification, ×100 and ×200). For IHC staining, the Ready-to-use Immunohistochemical UltraSensitive SP Detection kit (Mouse/Rabbit; Fuzhou Maixin Biotech Co., Ltd.) and Enhanced DAB Plus kit (Fuzhou Maixin Biotech Co., Ltd.) were used according to the manufacturer's instructions. Paraffin-embedded tumor sections (4-µm-thick) were deparaffinized and blocked with 5% goat non-immune serum (UltraSensitive SP kit; Fuzhou Maixin Biotech Co., Ltd.) and incubated at room temperature for 10 min. Primary antibodies were diluted in bovine serum albumin and the sections were then incubated with the anti-Ki67 antibody (1:500; Fuzhou Maixin Biotech Co., Ltd.) overnight at 4°C in a wet chamber. Then the tumor sections were incubated with secondary antibodies (UltraSensitive SP kit; Fuzhou Maixin Biotech Co., Ltd.) at room temperature for 10 min, diaminobenzidine reagent was added and hematoxylin counterstaining was performed to visualize nuclei.

Interaction probabilities between lncRNAs and miRNAs were predicted using the online in silico tool MicroRNA Target Prediction Database (miRDB 6.0; http://mirdb.org/). Interaction probabilities between miRNAs and mRNAs were analyzed using online in silico tools, including miRDB, the Encyclopedia of RNA Interactomes (ENCORI 3.0, also known as StarBase; https://starbase.sysu.edu.cn/index.php), Prediction of MicroRNA Targets (TargetScan 3.1; http://www.targetscan.org/mamm_31/) and MicroRNA-Target Interactions (miRTarBase 9.0 beta; https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTar-Base_2022/php/index.php). Ensembl database (Ensembl 104; http://asia.ensembl.org/index.html) was used to identify the location of lncKRT16P6 and its sequence was predicted to exhibit lower protein-coding potential by Coding Potential Calculator 2 (CPC 2.0; http://cpc2.gao-lab.org/).

This experiment used the Dual-Luciferase reporter gene detection system kit (Promega Corporation), which was used according to the manufacturer's instructions. GP-transfect-Mate transfection reagent (Shanghai GenePharma Co., Ltd.) was used for transfection. Cells were seeded in 12-well plates at a density of 5×10⁵ cells per well for 24 h before transfection. Reporter plasmids [pGP-miRGLO-Firefly luciferase-Renilla luciferase, containing lncKRT16P6, the GATAD2A 3′ untranslated region (UTR), or the corresponding mutant (MUT) sequences] were designed by Shanghai GenePharma Co., Ltd. 293(T) cells were cotransfected with a reporter plasmid and either miR-3180 mimic or the NC mimic (Shanghai GenePharma Co., Ltd.). After 48 h, luciferase activity was measured using a dual-luciferase reporter assay system (Promega Corporation) according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

Cells were prepared at 4°C in radio-immunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF; Beijing Solarbio Science & Technology Co., Ltd.). Then, the protein concentration was determined using the BCA kit (Beyotime Institute of Biotechnology,). Proteins (25 µg per lane) were electrophoretically separated on 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes. The membrane was placed in 5% non-fat milk (Hangzhou Fude Biological Technology Co., Ltd.) and blocked for 3 h at room temperature to remove nonspecific binding sites. Membranes were then incubated with the appropriate primary antibodies overnight at 4°C, and incubated with the appropriate secondary antibodies at room temperature for 30 min. Primary antibodies specific for the following proteins were used: E-cadherin (1:1,000; cat. no. 3195; CST Biological Reagents Co., Ltd.), N-cadherin (1:1,000; cat. no. 13116; CST Biological Reagents Co., Ltd.), Vimentin (1:1,000; #5741; CST Biological Reagents Co., Ltd.), GATAD2A (1:2,000; cat. no. ab188472, Abcam), Cyclin A (1:1,000; cat. no. WL01841, Wanleibio Co., Ltd.), Cyclin D1 (1:1,000; cat. no. WL01435a, Wanleibio Co., Ltd.), Cyclin E (1:500; cat. no. WL01072, Wanleibio Co., Ltd.), Cyclin-dependent kinase 2 (CDK2; 1:1,000; cat. no. WL01543, Wanleibio Co., Ltd.), GAPDH (1:10,000; cat. no. 60004-1-Ig, ProteinTech Group, Inc.), β-Actin (1:1,000; cat. no. 3700; CST Biological Reagents Co., Ltd.) and β-Tubulin (1:10,000; cat. no. BS1482M, Bioworld Technology). Since each target protein had a different molecular weight distribution, in order not to overlap with the experimental protein in each case, three different loading controls were used (GAPDH, β-tubulin, β-actin) in this experiment. Horseradish peroxidase-conjugated secondary antibodies, including goat anti-mouse (1:5,000; cat. no. SA00001-1) and goat anti-rabbit (1:5,000; cat. no. SA00001-2) antibodies were purchased from ProteinTech Group, Inc. Immunoreactions were visualized by electrochemiluminescence using a BeyoECL Moon kit (Beyotime Institute of Biotechnology,). Then western blot analysis was performed with the ImageJ software (v1.44p; National Institutes of Health.).

Statistical analyses were performed using SPSS 22 software (IBM Corp.). For all continuous variables, Shapiro Wilk tests were used to test the normality. Experimental data are presented as the mean ± standard error of the mean of a minimum of three biological replicates or are representative of three independent experiments with similar results. Unpaired Student's t test was used for two-group comparisons. To test for statistical significance between > two groups, one-way ANOVA coupled with Dunnett T3′s post-hoc test was used. The chi-square test was used to determine the statistical significance of differences in clinicopathological data. Paired Student's t test was used to determine the statistical significance of differences in the expression levels in clinical tissue samples. All statistical tests were two-sided and P<0.05 was considered to indicate a statistically significant difference.

Previously, we screened for differentially expressed lncRNAs by analyzing six pairs of TSCC and healthy adjacent normal tissues through high-throughput RNA sequencing (Kangchen BioTech Co., Ltd.) (24) and identified the significantly overexpressed lncRNA lncKRT16P6. RT-qPCR was applied to detect the expression of differentially expressed lncRNAs. The expression level of lncKRT16P6 was determined in 50 paired human TSCC and normal tissues and lncKRT16P6 expression was consistently upregulated in the cancerous samples compared with their corresponding normal tissues (P<0.001; Fig. 1A). Notably, high lncKRT16P6 expression was significantly correlated with stage I-II disease (P=0.0209) and advanced tumor grade (P=0.0414; Table I). Consistent with the above results, the expression of lncKRT16P6 was dramatically increased in the human tongue cancer cell lines CAL-27 and SCC-9 compared with the normal human oral keratinocyte cell line HOK (Fig. 1B). In addition, Ensembl database analysis indicated that lncKRT16P6 is located on chromosome 17 with a total length of 715 bp and its sequence was predicted to exhibit lower protein-coding potential (coding probability, 0.04 by Coding Potential Calculator 2; http://cpc2.gao-lab.org/) compared with those of the classical lncRNA H19, XIST and coding genes (TP53 and MYC; Fig. 1).

To explore the role of lncKRT16P6 in TSCC cells, two specific siRNA oligonucleotides to target lncKRT16P6 in CAL-27 and SCC-9 cells were designed (Fig. 2A-B). The CCK-8 assay showed that knockdown of lncKRT16P6 significantly reduced the proliferation ability of CAL-27 and SCC-9 cells (Fig. 2C-D) and the Transwell and colony formation assay results showed that knockdown of lncKRT16P6 significantly reduced the invasion, migration and colony-forming abilities of CAL-27 and SCC-9 cells (Fig. 3). Moreover, the flow cytometry results indicated that knockdown of lncKRT16P6 induced apoptosis (Fig. 4) and affected cell cycle progression in CAL-27 and SCC-9 cells; most of the cells arrested in G₀/G₁ phase (Fig. 5).

To further investigate the role of lncKRT16P6, CAL-27 cells with stable knockdown of lncKRT16P6 were generated (Supplementary Fig. 1B-C). Moreover, to further determine the potential role of lncKRT16P6 in the progression of TSCC, an in vivo model of subcutaneous tumorigenesis was established in nude mice. CAL-27 cells with stable lncKRT16P6 knockdown or CAL-27 cells transfected with the control vector were inoculated into the right flanks of nude mice (n=4 mice per group). Markedly, 24 days after injection, lncKRT16P6 knockdown was found to suppress the tumorigenic and proliferation abilities of tongue cancer cells (Fig. 6A). The volumes of the subcutaneous tumors were significantly smaller in the lncKRT16P6 knockdown group compared with the control group (Fig. 6B). HE and IHC staining indicated that the model of subcutaneous tumorigenesis had been successfully established in nude mice and that the tumor tissue was Ki-67-positive (Fig. 6). Collectively, the findings suggested that lncKRT16P6 may promote the progression of tongue cancer.

Next, the molecular mechanism of lncKRT16P6 in TSCC was investigated and FISH and cell nucleocytoplasmic fractionation used to determine its localization. lncKRT16P6 was localized mainly in the cytoplasm (Fig. 7), suggesting that it may function by acting as a sponge for various miRNAs. MiRDB was used to predict potential target miRNAs (Fig. 8A) and miR-3180 was selected for further investigation. To confirm the predicted interactions, dual-luciferase reporter assays were performed. Bioinformatic analysis revealed the shared miRNA response elements (MREs) between lncKRT16P6 and hsa-miR-3180. Therefore, these MREs were mutated (MUT) and cloned into the luciferase reporter gene in place of the wild-type (WT) lncKRT16P6 3′ UTR (Fig. 8B). miR-3180 directly bound lncKRT16P6 (Fig. 8C). RT-qPCR was performed to detect miR-3180 expression in tongue cancer cell lines. The expression of miR-3180 was significantly reduced in tongue cancer cells compared with HOK cells (Fig. 8D).

Rescue experiments were next designed to explore whether lncKRT16P6 enhanced the malignant behavior of TSCC cells by interacting with miR-3180. CAL-27 and SCC-9 cells were stably cotransduced with the sh-lncKRT16P6 lentiviral vector and miR-3180 inhibitor. The inhibitory effect on miR-3180 partially attenuated the reduction in viability caused by sh-lncKRT16P6, as demonstrated by CCK-8 assays (Fig. 9A). Flow cytometry showed that the proportion of apoptotic cells was significantly decreased following sh-lncKRT16P6/miR-3180 inhibitor cotreatment (Fig. 9B). In addition, Transwell invasion and migration assays and colony formation assays demonstrated that miR-3180 partially reversed the decreases in the invasion, migration and colony formation abilities caused by lncKRT16P6 depletion (Fig. 10). Western blot analysis was used to detect the expression of invasion- and migration-related marker proteins, such as E-cadherin, N-cadherin and Vimentin and the results were consistent with the Transwell results (Fig. 11). These findings indicate that lncKRT16P6 promotes TSCC progression by eliminating the antitumor effects of miR-3180.

Then, the target genes of miR-3180 in TSCC were investigated. Four databases (ENCORI, miRDB, TargetScan and miRTarBase) were used to predict potential target genes (Fig. 12A) and GATAD2A was selected for further investigation. To confirm whether GATAD2A is a potential target gene of miR-3180, 3′ UTR sensors downstream of the luciferase reporter sequence were generated and cotransfected into 293(T) cells with miR-3180 mimics. When miR-3180 was overexpressed, GATAD2A showed reduced luciferase activity (Fig. 12B and C). The expression of GATAD2A in TSCC and head and neck squamous cell carcinoma (HNSC) tissues and cell lines was higher than that in healthy normal tissues and cells (Fig. 12D-F).

Furthermore, rescue experiments were performed to verify whether miR-3180 serves a role in TSCC by interacting with GATAD2A. CAL-27 and SCC-9 cells were stably cotransfected with the miR-3180 inhibitor and GATAD2A siRNAs. The transfection efficiency of miR-3180 inhibitor, miR-3180 mimics and GATAD2A siRNAs were verified in CAL-27 and SCC-9 cells (Fig. S2). One (si-2) of the two siRNAs against GATAD2A was used in the subsequent experiments after verifying the transfection efficiency. The inhibitory effect on GATAD2A may reverse the increase in cell viability induced by the miR-3180 inhibitor, as demonstrated by CCK-8 assays (Fig. 13A). Flow cytometric analysis showed that the proportion of apoptotic cells was significantly increased following si-GATAD2A/miR-3180 inhibitor cotreatment (Fig. 13B). In addition, Transwell invasion and migration assays and colony formation assays demonstrated that GATAD2A partially attenuated the increases in promotion of migration and invasion caused by miR-3180 depletion (Fig. 14). These findings indicated that miR-3180 inhibits TSCC progression by interacting with GATAD2A.

Further experiments were performed to confirm the regulatory relationship among lncKRT16P6, GATAD2A and miR-3180 and found that the miR-3180 mimic downregulated lncKRT16P6 expression, but knockdown of miR-3180 rescued lncKRT16P6 expression (Fig. 15A). In parallel, lncKRT16P6 expression was decreased after GATAD2A knockdown and was increased when miR-3180 was inhibited (Fig. 15B). Moreover, it was verified that the miR-3180 mimic downregulated GATAD2A expression, but the knockdown of miR-3180 rescued GATAD2A expression (Fig. 15C and D). GATAD2A expression was decreased after lncKRT16P6 knockdown and was increased when miR-3180 was inhibited (Fig. 15E and F). Moreover, in experiments to confirm the effect of lncKRT16P6 on the cell cycle in TSCC, it was found that the expression of CDK2, Cyclin A, Cyclin D1 and Cyclin E decreased following lncKRT16P6 knockdown (Fig. 16). Taken together, these data revealed that lncKRT16P6 serves as a ceRNA and sponges miR-3180, leading to enhanced GATAD2A expression to regulate tongue cancer progression.