Tongue Squamous Cell Carcinoma Cell Lines Research Articles

Capsaicin reverses cisplatin resistance in tongue squamous cell carcinoma by inhibiting the Warburg effect and facilitating mitochondrial-dependent apoptosis via the AMPK/AKT/mTOR axis.

Cisplatin is commonly used for the chemotherapy of tongue squamous cell carcinoma (TSCC); however, adverse side effects and drug resistance impact its therapeutic efficacy. Capsaicin is an active ingredient in chili peppers that exerts antitumor effects, whether it exerts antitumor effects on cisplatin-resistant cells remains unknown. Therefore, in this study, we investigated the effect of capsaicin on cisplatin resistance in TSCC cells and explored the underlying mechanisms. A cisplatin-resistant TSCC cell line was established by treated with increasing cisplatin concentrations. Combined treatment with cisplatin and capsaicin decreased the glucose consumption and lactate dehydrogenase activity and increased the adenosine triphosphate production both in vitro and in vivo, suggesting the inhibition of the Warburg effect. Moreover, this combined treatment induced cell apoptosis and significantly upregulated the levels of proapoptotic proteins, such as Bax, cleaved caspase-3, -7, and -9, and apoptosis-inducing factor. In contrast, levels of the antiapoptotic protein, Bcl-2, were downregulated. Additionally, LKB1 and AMPK activities were stimulated, whereas those of AKT and mTOR were suppressed. Notably, AMPK knockdown abolished the inhibitory effects of capsaicin and cisplatin on the AKT/mTOR signaling pathway and Warburg effect. Overall, combined treatment with capsaicin and cisplatin reversed cisplatin resistance by inhibiting the Warburg effect and facilitating mitochondrial-dependent apoptosis via the AMPK/AKT/mTOR axis. Our findings suggest combination therapy with capsaicin and cisplatin as a potentially novel strategy and highlight capsaicin as a promising adjuvant drug for TSCC treatment.

Anti-cancer effects of green synthesized gold nanoparticles using leaf extract of Annona muricata. L against squamous cell carcinoma cell line 15 through apoptotic pathway

Background: Oral cancer remains one of the most dreadful diseases in developing nations. Currently, there has been a rise in the prevalence of tongue squamous cell carcinoma (SCC), with a poor prognosis. The use of standard treatment approaches against oral cancer patients brings about several side effects. In recent years, nanomedicine has provided a versatile platform for developing new targeted therapeutic modalities. However, safety remains a concern in the synthesis of nanoparticles (NPs). Therefore, the present study aims to synthesize safer phytoconstituent-mediated gold NPs (AuNPs) utilizing leaf extracts of Annona muricata, where the biochemical components of the plant leaf act as the reducing and capping agents in the synthesis of NPs, and to evaluate its anti-cancer activity against SCC. Materials and Methods: In this in vitro experimental study, AuNPs were synthesized through an effective, simple, and ecologically sound green synthesis method. After characterization of these synthesized AuNPs, in vitro assays such as 3-(4, 5-dimethylthiazole2-yl)-2, 5-biphenyl tetrazolium bromide, wound healing, and clonogenic assays were carried out to investigate the anti-cancer potential of green synthesized AuNPs in the human tongue SCC cell line (SCC-15), and the possible mechanism of action was evaluated through gene and protein expression analysis of Bax, Bcl-2, and p53 genes. The results were expressed as mean ± standard deviation using Statistical Package for Social Sciences (SPSS) 20.0 software and Student's t-test was performed for experimental data. P ≤0.05 were considered statistically significant. Results: The in vitro assays demonstrated that the synthesized AuNPs are exhibiting anti-cancer activity by apoptosis of SCC-15 cells in a dose-dependent manner. Further, it also revealed a highly significant decrease in anti-apoptotic Bcl-2 gene expression, whereas pro-apoptotic genes p53 and Bax revealed a highly significant increase, which is statistically significant compared to the control (P < 0.05). Conclusion: Our findings demonstrated that the AuNPs synthesized from A. muricata leaf extract could act as a novel anticancer agent, particularly against SCC, after further scrutiny.

Actin filament-associated protein 1-antisense RNA1 promotes the development and invasion of tongue squamous cell carcinoma via the AFAP1-AS1/miR-133a-5p/ZIC2 axis.

The present study aimed to explore the biological role and underlying mechanism of the long non-coding RNA actin filament-associated protein 1-antisense RNA1 (lncRNA AFAP1-AS1) in the progression of tongue squamous cell carcinoma (TSCC). A quantitative reverse transcriptase-PCR (RT-qPCR) was conducted to assess relative levels of the miR-133a-5p, lncRNAs AFAP1-AS1 and zinc finger family member 2 (ZIC2) in TSCC cell lines and specimens, whereas ZIC2 protein levels were measured using western blotting. After modifying the levels of expression of lncRNA AFP1-AS1, miR-133a-5p and ZIC2 using lentivirus or plasmid transfection, we examined AKT/epithelial-mesenchymal transition signaling pathway alterations, in vivo carcinogenesis of TSCC in nude mice and in vitro malignant phenotypes. A dual-luciferase reporter assay was conducted to confirm the targeting relationship between ZIC2 and miR-133a-5p, as well as between miR-133a-5p and lncRNA AFAP1-AS1. Based on The Cancer Genome Atlas (TCGA) database, we additionally validated AFP1-AS1. The potential biological pathway for AFP1-AS1 was investigated using gene set enrichment analysis (GSEA). We also evaluated the clinical diagnostic capacities of AFP1-AS1 and clustered the most potential biomarkers with the Mfuzz expression pattern. Finally, we also made relevant drug predictions for AFP1-AS1. In TSCC cell lines and specimens, lncRNA AFAP1-AS1 was upregulated. ZIC2 was upregulated in TSCC cells as a result of lncRNA AFAP1-AS1 overexpression, which also promoted TSCC cell migration, invasion, viability, and proliferation. Via the microRNA sponge effect, it was found that lncRNA AFAP1-AS1 could upregulate ZIC2 by competitively inhibiting miR-133a-5p. Interestingly, knockdown of ZIC2 reversed the biological roles of lncRNA AFAP1-AS1 with respect to inducing malignant phenotypes in TSCC cells. In addition, in vivo overexpression of lncRNA AFAP1-AS1 triggered subcutaneous tumor growth in nude mice implanted with TSCC cells and upregulated ZIC2 in the tumors. The TCGA database findings revealed that AFAP1-AS1 was significantly upregulated in TSCC specimens and had good clinical diagnostic value. The results of GSEA showed that peroxisome proliferator-activated receptor signaling pathway was significantly correlated with low expression of AFP1-AS1. Finally, the results of drug prediction indicated that the group with high AFAP1-AS1 expression was more sensitive to docetaxel, AZD4547, AZD7762 and nilotinib. The upregulation of lncRNA AFAP1-AS1, which increases TSCC cell viability, migration, proliferation and invasion via the AFAP1-AS1/miR-133a-5p/ZIC2 axis, aids in the progression of TSCC.

Knockdown of LRP5 Promotes Proliferation and Invasion of Tongue Squamous Cell Carcinoma through Compensatory Activation of Akt Signaling.

The role of LRP5, a critical receptor in the Wnt signaling pathway, remains unexplored in tongue squamous cell carcinoma (TSCC). This study investigates the impact of LRP5 knockdown on the biological behaviors of TSCC cell lines both in vitro and in vivo. Our findings indicate that LRP5 knockdown significantly enhances cell proliferation, migration, and invasion in CAL27 and SCC25 cell lines. RNA-seq analysis reveals compensatory activation of the Akt pathway, with 119 genes significantly upregulated post-LRP5 knockdown. Elevated MMP1 expression suggests its potential involvement in TSCC progression. Western blot analysis demonstrates increased Akt phosphorylation, upregulated proliferation-related PCNA, and downregulated apoptosis-related caspase-3 after LRP5 knockdown. Down-regulation of E-cadherin and β-Catenin, proteins associated with cell adhesion and invasion, further elucidates the molecular mechanism underlying increased cell migration and invasion. Our study concludes that compensatory Akt pathway activation is essential for the LRP5 knockdown-induced migration and proliferation of CAL27 and SCC25 cells. These results highlight LRP5 as a potential therapeutic target for TSCC. Simultaneous inhibition of Wnt and Akt signaling emerges as a promising approach for TSCC treatment.

Relative Biological Effectiveness Values of Spot-scanning Proton Beam Therapy at Shonan Kamakura General Hospital.

This study aimed to confirm the relative biological effectiveness (RBE) values of the proton beam therapy (PBT) system installed in Shonan Kamakura General Hospital. Clonogenic cell-survival assays were performed with a human salivary gland (HSG) cell line, a human tongue squamous-cell carcinoma cell line (SAS), and a human osteosarcoma cell line (MG-63). Cells were irradiated with proton beams and X-rays with different doses (1.8, 3.6, 5.5, and 7.3 Gy for proton beams, and 2, 4, 6, and 8 Gy for X-rays). Proton beam irradiation used spot-scanning methods and three different depths (at the proximal, center, and distal sides of the spread-out Bragg peak). RBE values were obtained from a comparison of the dose that resulted in a surviving fraction of 10% (D10). D10 of proton beams at the proximal, center, and distal sides and X-rays in HSG were 4.71, 4.71, 4.51, and 5.25 Gy, respectively; those in SAS were 5.08, 5.04, 5.01, and 5.59 Gy, respectively; and those in MG-63 were 5.36, 5.42, 5.12, and 6.06 Gy, respectively. The RBE10 values at the proximal, center, and distal sides in HSG were 1.11, 1.11, and 1.16 respectively; those in SAS were 1.10, 1.11, and 1.12, respectively; and those in MG-63 were 1.13, 1.12, and 1.18, respectively. RBE10 values of 1.10-1.18 were confirmed by in vitro experiments using the PBT system. These results are considered acceptable for clinical use in terms of therapeutic efficacy and safety.

Hsa_circ_0000231 affects the progression of tongue squamous cell carcinoma by activating Wnt/β-catenin signaling pathway

Objective: To explore the action mechanism of hsa_circ_0000231 in the occurrence and development of tongue squamous cell carcinoma (TSCC). Methods: Tissue samples of 60 TSCC patients were examined. The patients, including 32 males and 28 females, aged from 36 to 84 years old, underwent surgery in the Affiliated Hospital of Nantong University and Affiliated Tumor Hospital of Nantong University from December 2014 to December 2017. Saliva samples were obtained from healthy volunteers (5 males and 5 females, aged from 40 to 75 years old) and 10 TSCC patients. The TSCC cell lines (CAL-27, Tca-8113 and HN-4) were used. The expression levels of hsa_circ_0000231 in 60 pairs of freshly matched TSCC and para-carcinoma tissue samples, 10 pairs of saliva samples and 3 TSCC cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR). hsa_circ_0000231 gene interference and lentiviral transfection were constructed, hsa_circ_0000231 in TSCC cell lines CAL-27 and Tca-8113 was knocked down, and the expressions of hsa_circ_0000231 in hsa_circ_0000231 interference group (sh-circ) and no-load lentivirus group (negative control) were tested with qRT-PCR. Cells with the highest knock-down efficiency were selected for CCK-8 test, colony formation assay, transwell invasion assay and scratch assay. The expressions of EMT-related proteins including E-cadherin, snail protein, N-cadherin and vimentin and proteins related to Wnt/β-catenin signaling pathway including β-catenin, C-myc, Bcl-2, MMP-9 and Cyclin D1 were measured by western blot. After TSCC cells in the interference group were co-cultured with Wnt/β-catenin pathway activator LiCl, the expressions of above proteins were re-measured by western blot. TSCC cells in interference group and control group were subcutaneously injected into nude mice to compare the effect of hsa_circ_0000231 knockdown on the growths of the tumors grafted subcutaneously in the nude mice. Statistical analysis software 25.0 was used for data analysis, and t-test or chi-square test was used for comparison between groups. Results: hsa_circ_0000231 was highly expressed in the tissue and saliva samples of TSCC patients and cell lines CAL-27, Tca-8113 and HN-4, but lowly expressed in paired para-carcinoma tissues, saliva samples of healthy people and normal human oral keratinocytes (all P<0.05). Log-rank univariate analysis showed that hsa_circ_0000231 expression level, tumor differentiation degree and T stage were related to the survival of TSCC patients (all P<0.05). Multivariate Cox risk regression model analysis suggested that hsa_circ_0000231 expression level (χ2=5.77,P=0.016) and T stage (χ2=5.27,P=0.029) were independent factors for the poor prognosis of TSCC patients. Western blot showed the expressions of snail protein, N-cadherin and vimentin were down-regulated, but E-cadherin was up-regulated in interference group compared with control group. In interference group, the expressions of β-catenin, C-myc, Bcl-2, MMP-9 and CyclinD1 were down-regulated, which were reversed after TSCC cells were co-cultured with LiCl. The knockdown of hsa_circ_0000231 reduced the proliferation, invasion and metastasis abilities of CAL-27 and Tca-8113 cells, which were reversed after TSCC cells were co-cultured with LiCl. The growth rate and volume of the tumors grafted subcutaneously in interference group using LiCl were greater than those in negative control group. Conclusion: hsa_circ_0000231 is an independent prognostic factor of TSCC. Highly expressed hsa_circ_0000231 can promote the proliferation, invasion and metastasis of TSCC cells.

Genome-wide profiling of dysregulated piRNAs and their target genes implicated in oncogenicity of tongue squamous cell carcinoma

PIWI-interacting RNAs (piRNAs) are single-stranded, 23–36 nucleotide long RNAs that regulate gene expression in the germline but are also detected in some cancers. However, there are no reports yet on piRNA expression in tongue squamous cell carcinoma (TSCC), the most common oral cancer (80–90% percent of all oral cancers). We performed small RNA and whole transcriptome sequencing in H357 tongue cancer and HOK cells (GEO database accession numbers: GSE196674 and GSE196688). We also examined nine published sets of gene expression array data of TSCC tissues from the GEO database to decode piRNAs and their putative targets that may be involved in tumorigenesis. We identified a pool of 16,058 and 25,677 piRNAs in H357 and HOK, respectively, among which 406 are differentially expressed. We also found that 2094 protein-coding genes are differentially expressed in either TSCC tissues or cell lines. We performed target predictions for these piRNAs, pathway and disease function (DF) analyses, as well as qRT-PCR validation of piRNA-target pairs. These experiments revealed one up-regulated (FDFT1) and four down-regulated (OGA, BDH1, TAT, HYAL4) target genes that are enriched in 11 canonical pathways (CPs), with postulated roles in the initiation and progression of TSCC. Downregulation of piR-33422 is predicted to upregulate the FDFT1 gene, which encodes a mevalonate/cholesterol-pathway related farnesyl-diphosphate farnesyltransferase. The FDFT1 appears to be involved in the largest number of oncogenesis-related processes and is interacting with statins, which is a classical cancer drug. This study provides the first evidence of the piRNome of TSCC, which could be investigated further to decode piRNA-mediated gene regulations in malignancy and potential drug targets, such as FDFT1.

M6A modification-mediated BATF2 suppresses metastasis and angiogenesis of tongue squamous cell carcinoma through inhibiting VEGFA

ABSTRACT The aim is to explore the underlying mechanism of basic leucine zipper ATF-like transcription factor 2 (BATF2) in tongue squamous cell carcinoma (TSCC). The expression of BATF2 in TSCC tissues and corresponding adjacent normal TSCC tissues, human TSCC cell lines (SCC-15 and CAL-27) and human normal tongue epithelial cells NTEC was detected. Then, SCC-15 cells with stable BATF2 knockdown and CAL-27 cells with BATF2 overexpression were established to investigate the functional effect of BATF2 on TSCC. Thereafter, the effect of BATF2 on TSCC angiogenesis and BATF2 m6A methylation was also examined. BATF2 was significantly downregulated in TSCC tissues and cell lines, and BATF2 overexpression could suppress growth, metastasis and angiogenesis of TSCC. Mechanistically, vascular endothelial growth factor A (VEGFA) was identified as a downstream gene of BATF2, and it was confirmed that BATF2 suppressed growth, metastasis and angiogenesis of TSCC via inhibiting VEGFA. In addition, the N6-methyladenosine (m6A) modification of BATF2 mRNA mediated by METTL14 suppressed its expression in TSCC. METTL14/BATF2 axis could serve as a novel promising therapeutic candidate against angiogenesis for TSCC.

LncKRT16P6 promotes tongue squamous cell carcinoma progression by sponging miR‑3180 and regulating GATAD2A expression.

Tongue squamous cell carcinoma (TSCC) is characterized by a poor prognosis and its 5-year overall survival rate has not improved significantly. However, the precise molecular mechanisms underlying TSCC remain largely unknown. Through RNA screening, the present study identified a novel long noncoding RNA (lncRNA), keratin 16 pseudogene 6 (lncKRT16P6), which was upregulated in TSCC tissues and cell lines and associated with TSCC tumor stage and differentiation grade. Inhibition of lncKRT16P6 expression reduced TSCC cell migration, invasion and proliferation. lncKRT16P6 sponged microRNA (miR)-3180 and upregulated GATA zinc finger domain containing 2A (GATAD2A) expression. miR-3180 inhibition reversed the lncKRT16P6 depletion-induced attenuation of TSCC malignancy and GATAD2A depletion reversed the miR-3180 silencing-induced enhancement of TSCC malignancy. In summary, the present study revealed a potential competitive endogenous RNA (ceRNA) regulatory pathway in which lncKRT16P6 modulates GATAD2A expression by binding miR-3180, ultimately promoting tumorigenesis and metastasis in TSCC. Therefore, lncKRT16P6 may be used as a prognostic biomarker and therapeutic target for clinical intervention in TSCC.

Insufficiency of hepatocyte growth factor activator inhibitor-1 confers lymphatic invasion of tongue carcinoma cells.

Hepatocyte growth factor (HGF) activator inhibitor type‐1 (HAI‐1), encoded by the SPINT1 gene, is a transmembrane protease inhibitor that regulates membrane‐anchored serine proteases, particularly matriptase. Here, we explored the role of HAI‐1 in tongue squamous cell carcinoma (TSCC) cells. An immunohistochemical study of HAI‐1 in surgically resected TSCC revealed the cell surface immunoreactivity of HAI‐1 in the main portion of the tumor. The immunoreactivity decreased in the infiltrative front, and this decrease correlated with enhanced lymphatic invasion as judged by podoplanin immunostaining. In vitro homozygous deletion of SPINT1 (HAI‐1KO) in TSCC cell lines (HSC3 and SAS) suppressed the cell growth rate but significantly enhanced invasion in vitro. The loss of HAI‐1 resulted in enhanced pericellular activities of proteases, such as matriptase and urokinase‐type plasminogen activator, which induced activation of HGF/MET signaling in the co‐culture with pro‐HGF‐expressing fibroblasts and plasminogen‐dependent plasmin generation, respectively. The enhanced plasminogen‐dependent plasmin generation was abrogated partly by matriptase silencing. Culture supernatants of HAI‐1KO cells had enhanced potency for converting the proform of vascular endothelial growth factor‐C (VEGF‐C), a lymphangiogenesis factor, into the mature form in a plasminogen‐dependent manner. Furthermore, HGF significantly stimulated VEGF‐C expression in TSCC cells. Orthotopic xenotransplantation into nude mouse tongue revealed enhanced lymphatic invasion of HAI‐1KO TSCC cells compared to control cells. Our results suggest that HAI‐1 insufficiency leads to dysregulated pericellular protease activity, which eventually orchestrates robust activation of protease‐dependent growth factors, such as HGF and VEGF‐C, in a tumor microenvironment to contribute to TSCC progression.

Yes-associated protein 1 exerts its tumor-promoting effects and increases cisplatin resistance in tongue squamous cell carcinoma cells by dysregulating Hippo signal pathway.

Tongue squamous cell carcinoma (TSCC) has been well-known for its high metastasis and poor prognosis, but the molecular mechanisms of TSCC pathogenesis and chemoresistance are still largely unknown. Thus, the present study aimed to identify the involvement of a classic Hippo/Yes-associated protein 1 (YAP1) pathway in regulating TSCC progression and cisplatin (DDP) resistance. DDP-resistant TSCC cell lines were established by gradual exposure to DDP. Through western blot analysis, the protein expression of Hippo/YAP1 axis in TSCC tissues and cell lines was detected separately. Then, YAP1 was inhibited or overexpressed in TSCC cells. Cell viability and drug resistance were evaluated by cell counting kit-8 method, colony formation assay and Trypan blue staining assay. Cell migration ability was measured by Transwell assay. The Hippo pathway was dysregulated, and YAP1 was upregulated and dephosphorylated in the TSCC tissues or DDP-resistant cell lines, compared with normal tissues or DDP-sensitive cells. YAP1 knockdown inhibited cell proliferation, colony formation ability and migration, whereas overexpression of YAP1 exacerbated these malignant characteristics. YAP1 knockdown increased DDP-sensitivity by reducing the RAD51-mediated DNA damage repair behavior under DDP intervention in the DDP-resistant TSCC cells. Conversely, YAP1 overexpression significantly increased DDP-resistance by enhancing the RAD51-mediated DNA damage repair behavior under DDP intervention in the DDP-sensitive TSCC cells. In a word, upregulation and dephosphorylation of YAP1 caused dysregulation of the tumor-inhibiting Hippo pathway, resulting in the aggressiveness and DDP resistance in TSCC.

Role of fibulin-1 gene promoter methylation in the carcinogenesis and development of tongue squamous cell carcinoma

Recently, fibulin-1 (FBLN1) has been shown to be downregulated in various cancers via promoter hypermethylation. Our study aimed to determine the expression and methylation status of FBLN1 in tongue squamous cell carcinoma (TSCC) tissues and cells. Methylation-specific polymerase chain reaction was implemented to detect the methylation status of the FBLN1 gene in TSCC tissues and Western blot analysis was used to detect the expression of FBLN1 protein. The human TSCC cell lines CAL27 and SCC9 were cultured in vitro and treated with 5-aza-deoxycytidine (5-Aza-dC). CCK-8, colony formation, and Transwell assays were performed to test TSCC cell proliferation, migration, and invasion following 5-Aza-dC treatment or overexpression of FBLN1, which was further verified in in vivo experiments. FBLN1 was hypermethylated and the protein expression was reduced in TSCC tissues. After human TSCC cell lines (CAL27 and SCC9) were treated with 5-Aza-dC or overexpressed FBLN1, FBLN1 expression was upregulated and the TSCC cell proliferation, migration, and invasion abilities were suppressed. In vivo experiments further showed that demethylation or overexpression of FBLN1 slowed tumor growth in nude mice. This study demonstrated that 5-Aza-dC treatment or overexpression of FBLN1 inhibited the growth of human TSCC.

LncRNA GACAT1 induces tongue squamous cell carcinoma migration and proliferation via miR-149.

Recent studies have observed that lncRNAs (long non‐coding RNAs) are involved in the progression of various tumours including tongue squamous cell carcinoma (TSCC). Recently, a new lnRNA, GACAT1, has been firstly identified in gastric cancer. However, its potential role in TSCC remains unknown. In this reference, we observed that GACAT1 was overexpressed in TSCC samples and cell lines. Of 25 TSCC specimens, GACAT1 expression was overexpressed in 18 patients (18/25, 72%) compared to non‐tumour specimens. Ectopic expression of GACAT1 induced cell growth and migration and promoted epithelial to mesenchymal transition in TSCC. In addition, ectopic expression of GACAT1 decreased miR‐149 expression in SCC1 cell. We observed that miR‐149 expression was down‐regulated in TSCC cell lines. Moreover, we observed that GACAT1 expression was negatively correlated with miR‐149 expression. GACAT1 overexpression induced TSCC cell growth and migration via regulating miR‐149 expression. These data provided that GACAT1 played an oncogenic role in the progression of TSCC partly through modulating miR‐149 expression.

CDH11 Regulates Adhesion and Transcellular Migration of Tongue Squamous Cell Carcinoma.

PurposeCDH11, as a member of cadherins, mediates homotypic cell adhesion. Some studies have shown that CDH11 plays an important role in the development of tumors, especially in the processes of tumor invasion and metastasis. While features of CDH11 in tongue squamous cell carcinoma (TSCC) are still indeterminate, the purpose of the present study is to explore the role of CDH11 in TSCC.MethodsThe expression of cadherin gene in a TSCC cell line with high metastatic potential (LN4) and the parental CAL27 were examined both in the TCGA database and in collected clinical samples, further verified by quantitative real-time PCR. The effects of CDH11 on the proliferation, apoptosis, migration, invasion and adhesion were tested in appropriate ways after CDH11 was overexpressed in TSCC cells.ResultsAmong the 22 cadherin genes, CDH11 was one of the most obviously inhibited genes in LN4 cells as compared with the parental cells. Overexpression of CDH11 did not show a significant effect on cell proliferation, apoptosis, stemness, migration and invasion ability of TSCC cells themselves, but it increased the adhesion of TSCC cells with human oral epithelial cells and decreased their ability to pass through human oral epithelial cells (HOECs) for migration.ConclusionThe results indicated that CDH11 plays as a tumor suppressor in tongue squamous cell carcinoma by inhibiting the invasion and migration of tongue cancer cells. CDH11 may serve as an effective clinical target for new tongue cancer treatments.

Expression profile and bioinformatics analysis of circular RNAs in tongue squamous cell carcinoma.

This study was designed to identify the role of circRNAs in tongue squamous cell carcinoma (TSCC) and discover novel circRNAs as potential diagnostic or therapeutic targets for TSCC. The circRNA expression profiles in TSCC were evaluated by high-throughput sequencing in 6 TSCC patients. Our data showed that 69 circRNAs were downregulated and 208 were upregulated significantly (fold change ≥2.0; p<0.05). GO and KEGG analysis demonstrated that the parental genes of differentially expressed circRNAs were potentially implicated in TSCC pathogenesis. After bioinformatics analysis, 4 circRNAs (hsa_circ_0005035, hsa_circ_0002360, hsa_circ_0066251, and hsa_circ_0003161) were selected and successfully validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in 40 patients and 3 TSCC cell lines. The establishment and analysis of the circRNA-miRNA-mRNA network revealed the potential function and mechanism of these candidate circRNAs. Our study provided a comprehensive circRNAs expression profile of TSCC by RNA-seq and discovered 4 novel circRNAs with potential great diagnostic and therapeutic value. These findings provide new insights into the development of potential biomarkers and targets for TSCC treatment.

Model of Human Tongue Squamous Cell Lines Stably Transfected with Human Papillomavirus (HPV)16 E6 and E7 Genes and Biological Characteristic Analysis.

Background Human tongue squamous cell carcinoma (TSCC) is the most common oral cancer with the highest human papillomavirus (HPV) infection rate in oral cancer. The purpose of this study was to research the correlation between HPV and TSCC. Method Plasmid pEGFP/HPV16 E6E7 and plasmid pEGFP/no HPV16 E6E7 were constructed. TSCC cell lines SCC9 and SCC15 were infected by liposome transfection and would be highly selected by antibiotic. Fluorescence imaging, PCR, and Western blot were used to detect the expression of HPV16 E6E7 in cells. The biological characteristics were detected by CCK-8, wound healing assay, qRT-PCR, and Western blot. Result TSCC cell lines transfected with HPV16 E6E7 gene were successfully established and identified. And the proliferation and migration ability of the TSCC cell lines infected with HPV16 E6E7 gene were significantly stronger than that of the blank group. Conclusion TSCC cell lines infected with HPV16 E6E7 with significantly higher ability of proliferation and migration were more malignant than those not infected with HPV16 E6E7.

Silencing SHMT2 inhibits the progression of tongue squamous cell carcinoma through cell cycle regulation

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) is a vital metabolic enzyme in one carbon metabolism catalyzing the conversion of serine to glycine, which has been reported to play a crucial role in the progression of tumors. However, its function in tongue squamous cell carcinoma (TSCC) remains unclear.MethodsSHMT2 expression was analyzed using samples in online databases, and was assessed through immunohistochemistry staining of collected clinical specimens. The correlation between SHMT2 expression and the cell cycle was predicted through bioinformatic analysis, including weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA). After transfection with siRNA, CCK8 assay, Edu staining, flow cytometry, trans-well assay, and wound healing experiments were performed to verify the functional role of SHMT2 in vitro. A stable cell line with SHMT2 silencing was established to detect the oncogenic function of SHMT2 in vivo.ResultsThe expression of SHMT2 was up-regulated in TSCC tissues and cell lines compared with normal groups, and highly expressed SHMT2 significantly indicated a poorer clinical outcome for TSCC patients. Bioinformatic analysis found that high expression of SHMT2 was closely related with biologic process including cell cycle and cell cycle G1/S transition. Down regulating of SHMT2 significantly suppressed the proliferation, invasive and migrative ability of TSCC cells, and induced the prolongation of the G1 phase of the cell cycle in vitro. Furthermore, western blot showed that cell cycle-related regulators such as cyclin-dependent kinase 4 (CDK4) and cyclinD1 expression levels were decreased, while the expression levels of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 were increased after SHMT2 knockdown. Silencing SHMT2 in the HN6 cell line using short hairpin RNA also impeded tumor growth in vivo.ConclusionsOverexpression of SHMT2 in TSCC indicated low survival rates, and was associated with aggressive behaviors of TSCC. It was also found to be involved in cell cycle regulation of TSCC cells. SHMT2 may serve as a novel prognostic indicator of TSCC.

Circular RNA circCLK3 promotes the progression of tongue squamous cell carcinoma via miR-455-5p/PARVA axis.

A previous study has elucidated that circular RNA circCLK3 acts as an oncogenic gene in cervical cancer. However, the role and regulatory mechanism of circCLK3 in tongue squamous cell carcinoma (TSCC) remain unknown. Quantitative real-time PCR was used to examine targeted gene expression in different groups. Cell viability and proliferation were investigated by MTT and 5-ethynyl-2'-deoxyuridine assays. Cell migration and invasion were detected by Transwell assays, and cell apoptosis was measured by flow cytometry analysis. The interaction among genes was investigated using luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation assay. In the present study, our findings revealed the upregulated expression of circCLK3 in TSCC tissues and cell lines. CircCLK3 knockdown suppressed cell proliferation, migration invasion, and induced cell cycle arrest at G0/G1 phase in TSCC. Moreover, circCLK3 acted as a molecular sponge for miR-455-5p. PARVA was the target gene of miR-455-5p. Furthermore, the negative correlation between expression of miR-455-5p and circCLK3 or PARVA in TSCC tissues was discovered. Rescue assays indicated that PARVA overexpression reversed the circCLK3 knockdown-mediated inhibitory effects on the progression of TSCC. In summary, circCLK3 exerts its carcinogenic effects on TSCC progression via absorbing miR-455-5p to upregulate PARVA, which expands our knowledge on the underlying mechanism of TSCC.

LUCAT1 as an oncogene in tongue squamous cell carcinoma by targeting miR-375 expression.

Emerging studies suggested that lncRNAs play a crucial molecular role in cancer development and progression. LncRNA LUCAT1 has been proved as oncogenic molecular in lung cancer, glioma, osteosarcoma, renal carcinoma and oesophageal squamous cell carcinoma. However, its roles and function mechanisms in tongue squamous cell carcinoma (TSCC) are still unknown. We showed that the expression of LUCAT1 was up‐regulated in the TSCC cells and tissues and the higher LUCAT1 expression was associated with the poor overall survival (OS). Knockdown expression of LUCAT1 suppressed TSCC cell proliferation, cycle and migration. In addition, we demonstrated that miR‐375 overexpression inhibited the luciferase activity of LUCAT1 wild‐type and knockdown LUCAT1 promoted the miR‐375 expression in TSCC cell. Furthermore, we indicated that miR‐375 expression was down‐regulated in the TSCC cell lines and tissues and the lower expression of miR‐375 was associated with poor OS. The expression of miR‐375 was inversely correlated with LUCAT1 expression in the TSCC tissues. Knockdown LUCAT1 promoted TSCC cell proliferation, cell cycle and migration partly through regulating miR‐375 expression. In summary, this study suggested the tumorigenic effect of lncRNA LUCAT1 in TSCC cells by targeting miR‐375 expression.

LncRNA NOP14-AS1 Promotes Tongue Squamous Cell Carcinoma Progression by Targeting MicroRNA-665/HMGB3 Axis.

PurposeThe expression profile, clinical effects, and detailed roles of NOP14 antisense RNA 1 (NOP14-AS1) in tongue squamous cell carcinoma (TSCC) remain ambiguous and need to be further explored. Thus, this work was initiated to offer further solid evidence regarding the expression and roles of NOP14-AS1 in TSCC. Furthermore, additional efforts were exerted to reveal the molecular events by which NOP14-AS1 affects the malignant behaviours of TSCC.MethodsNOP14-AS1 expression was detected in TSCC tissues and cell lines using quantitative reverse transcription-polymerase chain reaction. Cell Counting Kit-8 assay, flow cytometric analysis, Transwell migration and invasion assays, and xenograft tumor model analysis were performed to assess the malignant biological behaviors of TSCC cells after NOP14-AS1 depletion. Mechanistic studies were performed using bioinformatics analysis, luciferase reporter assay, RNA immunoprecipitation, and rescue experiments.ResultsNOP14-AS1 upregulation was identified in TSCC tissues and cell lines. Patients with TSCC exhibiting a high NOP14-AS1 expression faced shorter overall survival than those with a low NOP14-AS1 expression. Functionally, NOP14-AS1 depletion facilitated apoptosis and impeded cell proliferation, migration, and invasion in TSCC. In vivo, the growth of TSCC cells was hindered by NOP14-AS1 depletion. Mechanically, NOP14-AS1 functioned as a competing endogenous RNA by sponging microRNA-665 (miR-665), thereby overexpressing the target high mobility group box 3 (HMGB3) of miR-665. Lastly, rescue experiments confirmed that the introduction of HMGB3 overexpression plasmid or miR-665 inhibitor could abrogate the inhibition of aggressive phenotypes triggered by NOP14-AS1 knockdown.ConclusionNOP14-AS1 executed pro-oncogenic activities in TSCC cells by targeting the miR-665/HMGB3 axis. The NOP14-AS1/miR-665/HMGB pathway may be a valuable prognostic indicator and therapeutic target for preventing TSCC.