Human laryngeal cancer cells TU212 were purchased from Department of Otolaryngology-Head and Neck Surgery School of Medicine, Changsha University (Changsha, China), and human pharyngeal epithelial cells NP69 were kindly donated by Institute of Oncology, The First Affiliated Hospital of Guangxi Medical University (Nanning, China). The cell lines were authenticated by the original providers via Short Tandem Repeat (STR) profiling prior to our acquisition, and all experiments were performed using low-passage cultures (≤ 15 passages) to maintain the biological characteristics and avoid cross-contamination. Expression of GL1 gene stable strain and interference with GL1 gene stable strain were purchased from IEMed Guangzhou Biomedical Technology Co., Ltd. TU212 and NP69 cells were cultured in RPMI1640 complete culture medium containing 10% FBS (Gibco, USA) + 1% double antibody (Solarbio, Beijing, China) (Gibco, USA), placed in a cell culture incubator (37 °C, 5% CO2), every 1–2 days to observe the growth morphology of the cells, the condition of the culture medium, half or full replacement of the culture medium. When the cell culture coverage reached 80%∼90%, the superannuate was discarded, washed 2–3 times with PBS (Gibco, USA), digested with 0.25% trypsin (Gibco, USA) for 2–4 minutes, observed by inverted microscope, and then transferred to a sterile centrifuge tube for 4-minutes, and then diluted with 5 mL of medium and transferred to another sterile culture flask for further culture.

Transcript expression profiles of Head and Neck Squamous Cell Carcinoma (HNSCC) patients and their clinical data (n = 565) were obtained from the UCSC Xena database (https://tcga.xenahubs.net/download/TCGA.HNSC.sampleMap/HiSeqV2.gz, Full metadata). Among the 565 HNSCC patients, 126 had an “anatomic_neoplasm_subdivision” (tumor anatomical site) of “Larynx” (larynx), which included 114 primary tumors and 12 neoplastic tissues. Gene expression profiling of 109 primary laryngeal cancer samples was downloaded from Gene Expression Omnibus (GEO, GSE27020). To identify possible target genes of GLI1, ChIP-seq data of GLI1 was obtained from GEO (GSE42132). To construct a conscription regulatory network, a list of 1469 genes encoding transcription factors was downloaded from (http://bioinfo.life.hust.edu.cn/static/AnimalTFDB3/download/Homo_sapiens_TF).

Using the SAM method with R.fold > 1.5 and a mean expression value of regulated subgroups > 3, screen for C2 expression-regulated and downregulated miRNAs in TCGA's miRNA data (TCGA_HNSC_miRNA_HiSeq-2015-02-24). Querying the database of miRNA target genes mirDIP (http:// ophid.utoronto.ca/mirDIP/index.jsp) to screen the genes regulated by candidate miRNAs.

For GLI1 expression, full-length GLI1 cDNA was amplified and cloned into the vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro. Expression mimic and interference inhibitor were designed and prepared against hsa-mir-1293, using hsa-mir-1293 mimic instead of mir-1293, and inhibitor to inhibit the expression of mir-1293, using mimic nc and inhibitor nc as control. For transfection, cells were cultured in 6-well plates until 70% confluence, plaids were transacted using trifecta CP Transfection kit (RuiBo Bio, Guangzhou, China), and hsa-mir-1293 mimic/inhibitor was added to the cells and incubated for 48 h after transfection.

Total RNA was extracted from the nontransferable cells using Trizol reagent, cDNA was synthesized by reverse transcription, and the expression levels of mRNA and miRNA were quantified using SYBER Premix Ex TaqTM. GAPDH or U6 was used as the internal reference gene.

Cells were inoculated and cultured in 12-well plates, and after transfection for 8 h, the original medium was discarded and replaced with complete medium to continue culturing for 16 h. Cells were scratched with the tip of a plastic 10 μL pipette gun tip, and washed with PBS for 3-times. Fresh growth medium without FBS was added to the plates. Subsequently, they were placed in a 37 °C 5% CO2 incubator for incubation. Images of the different stages of wound healing were taken at 0 and 24 h by microscope.

Cells were inoculated in Transwell plates with serum-free medium, and after transfection for 8 h, the original medium was discarded and replaced with complete medium to continue incubation for 16 h, and cell status and density were observed. Add 10% FBS medium into the lower chamber of Transwell. At the end of culture, the upper chamber was removed and put into a new 24-well plate, and PBS was washed gently for 3-times. Add 4% paraformaldehyde into the small chamber and fix it at room temperature for 15 min, add crystal violet staining solution to stain the cells, wash away the excess staining solution with PBS, remove the upper layer of cells from the upper chamber with a cotton swab, then dry it at room temperature and observe it under the microscope.

For protein blot analysis, total protein samples were extracted from transfected shGLI1−1, shGLI1−2, shGLI1−3, TU212-shPTCH1, TU212-PTCH1 cells using RIPA lysate. Equal amounts of proteins were up sampled onto 8% SDS-polyacrylamide gels and then transferred to PVDF membranes. The membranes were next closed in in 5% BSA solution for 1 h and then incubated with primary antibodies against GLI1 (Abcam, Cat# ab134906), GLUL (Abcam, Cat# ab176562), MAP1B (Abcam, Cat# ab154333), PTCH1 (Abcam, Cat# ab53715), GAPDH, E-cadherin (Abcam, Cat# ab40772), N-cadherin (Abcam, Cat# ab245117), Snail (Abcam, Cat# ab216347), Vimentin (Abcam, Cat# ab92547) primary antibodies at 4 °C overnight. The membrane was then incubated with secondary antibodies Hrp-Goat Anti-Rabbit IgG (Jackson, Cat# 111-035-003), Hrp-Goat Anti-Mouse IgG (Jackson, Cat# 115-035-003). Finally, immunoreactivity was detected using a chemiluminescent imaging system (Tanon, China).

Cells were inoculated into 6-well plates and transacted for 8 h. After transfection, complete medium was replaced, and incubation continued for 40 h. The medium was discarded, and the cells were dialyses. Firefly luciferase substrate (50×) and sea kidney luciferase substrate (50×) were diluted to 1× working solution with corresponding buffers. Firefly luciferase reaction solution and sea kidney luciferase reaction solution were added to the stately to detect the luciferase activity.

Statistical analysis was performed using SPSS 19.0 software. All data were presented as mean ± standard deviation from at least three independent biological replicates. Comparisons between two groups were conducted using Student's t-test. For experiments involving more than two groups, one-way ANOVA followed by Dunnett’s post-hoc test was applied for multiple comparisons. Differences were considered statistically significant when p < 0.05.

In the TCGA miRNA data (TCGA_HNSC_miRNA_HiSeq-2015-02-24), consistent with the C1 and C2 subtypes, there were 103 cases, split between 60 cases and 43 cases. Using the SAM method, with R.fold > 1.5 and the mean of the expression values of the upregulated miRNAs > 3, a total of 2 miRNAs were identified as upregulated, while a total of 10 miRNAs were identified as downregulated for the C2 subtype (Table 1). As shown in Table 1, the down-regulated miRNAs in C2 are most significantly down-regulated in hsa-miR-1293, with C2 showing only 0.36-fold of C1 and a q-value of 0.

In the previous study,1 in order to identify the possible target genes of GLI1, ChIP-seq data for GLI1 was obtained from GEO (GSE42132). Since GLI1 is a transcription factor, three up-regulated genes (PTCH1, GLUL, and MAP1B) associated with laryngeal cancer C2 subtype were screened for their tendency to be recognized and bound by GLI1 based on the ChIP-seq data (GSE42132).

Querying the miRNA target gene database mirDIP, hsa-mir-1293 was found to co-regulate GLI1 and its downstream signaling molecules GLI2 and PTCH1 (Table 2). In the data of 103 laryngeal cancers with both mRNA and miRNA detected in TCGA, the expression (log2) scatter plot of has-mir-1293 versus GLI1 was plotted (Fig. 1a). The results revealed that has-mir-1293 was significantly negatively correlated with GLI1 expression (Pearson correlation coefficient r = −0.23, p = 0.021).

Fig. 1.

Hsa-miR-1293 targets GLI1 and regulates cell cycle and proliferation in TU212 cells. (a) Scatter plot of hsa-miR-1293 versus GLI1 expression (log2) in TCGA laryngeal cancer data shows a significant negative correlation (Pearson r = −0.23, p = 0.021). (b) Western blot confirms hsa-miR-1293 negatively regulates GLI1 expression (*p < 0.05). (c) Dual luciferase assay demonstrates hsa-miR-1293 directly targets GLI1, reducing luciferase activity (* p < 0.05). (d) CCK8 assay shows hsa-miR-1293 overexpression reduces cell viability (**** p < 0.0001). ns, not significant.

In the TCGA laryngeal cancer mRNA and miRNA data, hsa-miR-1293 was significantly negatively correlated with GLI1 expression, suggesting that hsa-miR-1293 is a candidate upstream miRNA for GLI1. We transfected TU212 cells with hsa-mir-1293 mimic or inhibitor. RT-qPCR (Fig. S1C) and western blot assays showed that the expression of hsa-miR-1293 was negatively related to the expression of GLI1 (p < 0.05; Fig. 1b). To investigate the effect of hsa-miR-1293 on GLI1, we transfected TU212 cells with a dual luciferase reporter gene plasmid containing hsa-miR-1293 and GLI1. The luciferase activity of the transfected hsa-miR-1293 mimic GLI1 reporter gene vector was significantly reduced compared to the control vector, indicating that hsa-miR-1293 could target the GLI1 gene (Fig. 1c). The results of the cell cycle assay showed that in TU212 cells overexpressing hsa-miR-1293, the proportion of cells in the G1 phase was higher than that in the control group. Conversely, when hsa-miR-1293 was interfered with, the proportion of cells in the G1 phase was lower than that in the control group (p < 0.05, Fig. S2A). To investigate the biological function of hsa-mir-1293, CCK8 cell proliferation assay showed that overexpression of hsa-mir-1293 in TU212 cells decreased cell viability (p < 0.0001) (Fig. 1d). Clone formation assay showed that overexpression of hsa-mir-1293 resulted in diminished cell clone formation (p < 0.05, Fig. 2a). In addition, Transwell assay showed that hsa-mir-1293 mimic transfection of TU212 cells resulted in no significant change in cell invasion ability, but inhibition of hsa-mir-1293 expression increased cell invasion ability (p < 0.05, Fig. 2b). Cell scratch assay showed that overexpression of hsa-mir-1293 decreased cell migration ability (p < 0.05, Fig. 2c).

Fig. 2.

Hsa-miR-1293 regulates clone formation, invasion, and migration in TU212 cells. (a) Clone formation assay shows hsa-miR-1293 overexpression reduces cell colony formation (NC; Scale bar = 200 μm). (b) Transwell assay reveals hsa-miR-1293 inhibition increases cell invasion ability, while mimic transfection has no significant effect (NC; Scale bar = 500 μm). (c) Cell scratch assay demonstrates hsa-miR-1293 overexpression decreases cell migration ability (NC; Scale bar = 200 μm).

To investigate the effect of silencing GLI1 on GLUL, MAP1B, and PTCH1 genes, three interfering siRNAs (shGLI1−1, shGLI1−2, shGLI1−3) were synthesized, and control con and NC groups were set up. The results showed that shGLI1−2 had the highest silencing efficiency, and its GLI1 mRNA level was significantly lower than that of the Control (Con) and NC groups (Fig. 3a); the different sequences of siRNAs had no significant effect on the GLUL (Fig. 3b) and MAP1B genes (p > 0.05, Fig. 3c), but could significantly reduce the expression of the PTCH1 gene (p < 0.05, Fig. 3d), suggesting that GLI1 could affect the PTCH1 expression. Western blot assay results were consistent with the above description (Fig. 3e).

Fig. 3.

Silencing GLI1 regulates PTCH1 expression. (a) TU212 cells were transfected with siRNAs of different sequences, and the mRNA expression levels of GLI1, * p < 0.05, ** p < 0.01, *** p < 0.001. (b) GLUL, ns, not significant. (c) MAP1B, ns, not significant. (d) PTCH1 genes were detected by RT-qPCR. shGLI1-2 shows the highest silencing efficiency, significantly reducing GLI1 mRNA levels compared to control (con) and nc groups (* p < 0.05). (e) Western blot results confirm reduced GLI1 and PTCH1 protein levels. (f) Morphology of stably transfected cell lines TU212-shPTCH1 and TU212-PTCH1(Scale bar = 100 μm). PTCH1 promotes cell proliferation and regulates cell cycle progression in TU212 cells.

Next, we transfected PTCH1 overexpression vectors into TU212 cells to determine the effects of PTCH1 on EMT, cell migration and invasion (Fig. 3f). The results of flow assay cell cycle showed that silencing PTCH1 induced cell cycle arrest in S phase, while G1 phase was increased and S phase and G2 were decreased in TU212-PTCH1 cells compared with TU212 cells (p < 0.05) (Fig. 4a). Thus, overexpression of PTCH1 could promote cell proliferation. In addition, CCK8 cell proliferation assay showed that PTCH1 promoted cell proliferation (Fig. 4b).

Fig. 4.

PTCH1 modulates protein expression, cell cycle progression, and proliferation in laryngeal cancer cells. (a) Flow cytometry cell cycle analysis shows PTCH1 overexpression increases G1 phase and decreases S and G2 phases compared to controls. * p < 0.05, while PTCH1 silencing induces S phase arrest. (b) CCK8 assay confirms PTCH1 enhances cell proliferation.

Clone formation assay showed that the number of clone formation in TU212-shPTCH1 cells was significantly lower than that in TU212 cells, whereas the number of clone formation in TU212-PTCH1 cells was significantly higher than that in TU212 (p < 0.05), thus, PTCH1 could promote cell clone formation (Fig. 5a). Invasion assay in TU212 cells showed that compared with empty vector transfection, PTCH1 promoted cell invasion (Fig. 5b). Cell scratch assay showed that PTCH1 overexpression promoted cell migration (Fig. 5c). In addition, we found that in TU212 cells, PTCH1 overexpression led to downregulation of E-cadherin and upregulation of N-cadherin, Snail, and Vimentin, suggesting that PTCH1 may promote EMT (Fig. 5d).

Fig. 5.

PTCH1 promotes clone formation, invasion, migration, and EMT in TU212 cells. (a) Clone formation assay shows PTCH1 overexpression increases colony formation, while PTCH1 silencing reduces it compared to controls (Scale bar = 200 μm). (b) Invasion assay reveals PTCH1 enhances cell invasion (Scale bar = 500 μm). (c) Cell scratch assay demonstrates PTCH1 overexpression promotes cell migration (Scale bar = 200 μm). (d) Western blot detection of EMT-related protein expression levels of TU212, TU212-shPTCH1 and TU212-PTCH1.