OBJECTIVES: Tongue squamous cell carcinoma (TSCC) is a common cancer in the oral and maxillofacial region, which seriously endangers people's life and health.Heterogeneous nuclear ribonucleoprotein A2/B1(hnRNP A2/B1) is an RNA-binding protein that regulates the expression of a variety of genes and participates in the occurrence and development of a variety of cancers. This study aims to investigate the role of hnRNP A2/B1 in TSCC progression. METHODS: The differential expression of hnRNP A2/B1 in oral squamous cell carcinoma (OSCC) and normal oral mucosa cells and tissues was analyzed based on the gene expression profiles of GSE146483 and GSE85195 in the Gene Expression Omnibus (GEO) database. The correlation between hnRNP A2/B1 expression and disease-free survival of TSCC patients was analyzed based on TSCC related chip of GSE4676. TSCC cancer and paracancerous tissue samples of 30 patients were collected in Hunan Cancer Hospital from July to December 2021. Real-time RT-PCR and Western blotting were used to verify the mRNA and protein expression of hnRNP A2/B1 in TSCC patients'samples, respectively. Human TSCC Tca-8113 cells were transfected with hnRNP A2/B1 empty vector (a sh-NC group), knockdown plasmid (a sh-hnRNP A2/B1 group), empty vector overexpression plasmid (an OE-NC group) and overexpression plasmid (an OE-hnRNP A2/B1 group), respectively. The knockdown or overexpression efficiency of hnRNP A2/B1 was detected by Western blotting. The proliferation activity of Tca-8113 cells was detected by cell counting kit-8 (CCK-8), and the apoptosis rate of Tca-8113 cells was detected by flow cytometry. RESULTS: Based on the analysis of OSCC-related chips of GSE146483 and GSE85195 in the GEO database, it was found that hnRNP A2/B1 was differentially expressed in the OSCC and normal oral mucosa cells and tissues (all P<0.01). Meanwhile, the analysis of TSCC related chip GSE4676 confirmed that the expression of hnRNP A2/B1 was negatively correlated with the disease-free survival of TSCC patients (P=0.006). The results of real-time RT-PCR and Western blotting showed that the relative expression levels of hnRNP A2/B1 mRNA and protein in TSCC tissues were significantly up-regulated compared with those in adjacent tissues (all P<0.01). The results of Western blotting showed that the expression level of hnRNP A2/B1 in Tca-8113 cells was significantly inhibited or promoted after knockdown or overexpression of hnRNP A2/B1 (all P<0.01). The results of CCK-8 and flow cytometry showed that inhibition of hnRNP A2/B1 expression in Tca-8113 cells reduced cell proliferation activity (P<0.05) and increased cell apoptic rate (P<0.01). Overexpression of hnRNP A2/B1 in Tca-8113 cells significantly increased cell proliferation (P<0.05) and decreased cell apoptosis (P<0.01). CONCLUSIONS HnRNP A2/B1 is a key factor regulating the proliferation and apoptosis of TSCC cells. Inhibition of hnRNP A2/B1 expression can reduce the proliferation activity of TSCC cells and promote the apoptosis of TSCC cells.
Humans ; Carcinoma, Squamous Cell/genetics* ; Sincalide/metabolism* ; Tongue Neoplasms/genetics* ; Mouth Neoplasms ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism* ; RNA, Messenger ; Tongue/metabolism* ; Cell Line, Tumor

In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N⁶-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.
AlkB Homolog 1, Histone H2a Dioxygenase/metabolism* ; Carcinoma, Squamous Cell/pathology* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism* ; Tongue Neoplasms/metabolism*

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.
Male ; Animals ; Mice ; Female ; Humans ; Adult ; Middle Aged ; Aged ; Aged, 80 and over ; Tongue Neoplasms ; Wnt Signaling Pathway/genetics* ; Carcinoma, Squamous Cell/genetics* ; beta Catenin/metabolism* ; Mice, Nude ; Vimentin ; Matrix Metalloproteinase 9/metabolism* ; RNA, Circular ; Gene Expression Regulation, Neoplastic ; Cell Proliferation/genetics* ; Cadherins/genetics* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Tongue

OBJECTIVES: A study was conducted to investigate the molecular mechanism of chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) influencing the invasion and metastasis of tongue squamous cell carcinoma and to provide a new target for clinical inhibition of invasion and metastasis of tongue squamous cell carcinoma. METHODS: Ualcan website was used to analyze the expression of CHD1L in normal epithelial tissue and primary head and neck squamous cell carcinoma and to analyze the effect of lymph node metastasis on the expression of CHD1L in tissues with head and neck squamous cell carcinoma. The relationship between CHD1L expression and the survival rate of patients with head and neck squamous cell carcinoma was tested by the GEPIA website. Western blot was used to quantify the levels of CHD1L protein in human tongue squamous cell carcinoma CAL27 and immortalized human skin keratinocyte cell HaCaT. After knocking down CAL27 in human tongue squamous cell carcinoma cells with an RNA interference plasmid, the cells were designated as SiCHD1L/CAL27 and Scr/CAL27. Western blot was utilized to detect the expression of CHD1L in each group of cells. The change in CAL27 cell proliferation ability was tested by EdU proliferation test after CHD1L knockdown. The change of cell migration ability of each group cells was tested through the wound healing assay. Western blot was used to detect epithelial-mesenchymal transition (EMT) marker E-cadherin and Vimentin protein expression levels. RESULTS: Ualcan database showed that the expression of CHD1L in primary head and neck squamous cell carcinoma tissues was higher than in normal epithelial tissues and in head and neck squamous cell carcinoma tissues with lymph node metastasis. GEPIA website analysis showed that the overall survival rate of patients with head and neck squamous cell carcinoma with high expression of CHD1L was significantly lower than that of patients with low expression. Western blot results showed that CHD1L expression in human tongue squamous carcinoma cells CAL27 was higher than that of human normal skin cells HaCaT. CHD1L expression in SiCHD1L/CAL27 cells was much lower than that in Scr/CAL27 cells. Results of EdU proliferation experiments showed the significant reduction in the cell proliferation ability of the SiCHD1L/CAL27 cells. Results of the wound healing experiments showed the reduction in the migration capacity of the SiCHD1L/CAL27 cells. The expression of E-cadherin increased, whereas that of Vimentin decreased, in SiCHD1L/CAL27 cells. CONCLUSIONS CHD1L promoted the EMT, proliferation, migration, and invasion ability of tongue squamous cell carcinoma cells.
Adenosine Triphosphatases ; Carcinoma, Squamous Cell/genetics* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; DNA Helicases ; DNA-Binding Proteins ; Epithelial-Mesenchymal Transition ; Gene Expression Regulation, Neoplastic ; Head and Neck Neoplasms ; Humans ; Neoplasm Invasiveness/genetics* ; Tongue ; Tongue Neoplasms/genetics*

BACKGROUND: The association between miR-532-3p and tongue squamous cell carcinoma (TSCC) has been examined in the literature to improve the survival rate of patients with this tumor. However, further studies are needed to confirm the regulatory roles of this microRNA (miRNA) in TSCC. The objective of this study was to investigate the roles played by and the underlying mechanism used by the miR-532-3p/podoplanin (PDPN) axis in TSCC development. METHODS: Western blotting and quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) were performed to evaluate the PDPN expression level in TSCC tissues and cells. The proliferative, adhesive, and migratory capabilities of TSCC cells (CAL-27 and CTSC-3) were examined using cell counting kit-8 (CCK-8), cell adhesion, and wound-healing assays, respectively. The dual-luciferase reporter (DLR) assay was later conducted to confirm the relationship between miR-532-3p and PDPN. RESULTS: The results indicated that PDPN expression was enriched in TSCC tissues and cells, and that the expression of PDPN was associated with some clinicopathological parameters of TSCC, including lymph node metastasis (P = 0.001), tumor-node-metastasis (TNM) staging (P = 0.010), and grading (P = 0.010). Further analysis also showed that PDPN knockdown inhibited the viability, adhesive ability, and migratory capacity of CAL-27 and CTSC-3 cells, effects that could be reversed by the application of a miR-532-3p inhibitor. Additionally, PDPN was found to be a direct target of miR-532-3p. CONCLUSIONS This research suggested that by targeting PDPN, miR-532-3p could inhibit cell proliferation viability, adhesion, and migration in TSCC. Findings also revealed that the miR-532-3p/PDPN axis might provide more insights into the prognosis and treatment of TSCC.
Carcinoma, Squamous Cell/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Membrane Glycoproteins ; MicroRNAs/genetics* ; Tongue Neoplasms/genetics*

OBJECTIVES: This study aims to investigate the effect of RhoE expression on the migration and invasion of tongue squamous cell carcinoma (TSCC). METHODS: Forty-eight TSCC cases were selected from the Maxillofacial Surgery Center of Qingdao Municipal Hospital from 2017 to 2019. The expression of RhoE in the specimens (TSCC and adjacent tissues) was detected by immunohistochemistry, and RhoE mRNA and protein were extracted to further detect the expression of RhoE. SCC-4 and CAL-27 cells were selected for RESULTS: The expression level of RhoE in TSCC was significantly lower than that in adjacent tissues ( CONCLUSIONS RhoE expression is low in TSCC. Over expression RhoE in TSCC can significantly decrease its migration and invasion abilities. Hence, RhoE may play an important role in regulating the metastasis and invasion of TSCC and provide a new target for gene therapy.
Carcinoma, Squamous Cell ; Cell Line, Tumor ; Humans ; Matrix Metalloproteinase 2 ; Neoplasm Invasiveness ; Tongue ; Tongue Neoplasms ; rho GTP-Binding Proteins/genetics* ; rho-Associated Kinases

OBJECTIVETo study the effects of RhoA down-regulation by RNA interference on the invasion of tongue carcinoma Tca8113 and SCC-4.

METHODSDetermination of the human RhoA sequence as well as the design and constructionof a short specific small interfering RNAs (siRNA) were performed. The siRNA of RhoA gene was transfected into humantongue squamous cell carcinoma Tca8113 and SCC-4 cells line by Lipofectamine 2000. Quantitative real-time polymerasechain reaction was used to examine the mRNA expressionlevels of RhoA. Protein expressions of mRNA, galectin-3,and matrix metalloproteinase (MMP)-9 were evaluated byWestern blot. Transwell invasion assay was performed toassess the invasion ability of tongue carcinoma.

RESULTSRhoA expressions in Tca8113 and SCC-4 cells were reducedsignificantly after transfection of RhoA-siRNA. Protein levels f galectin-3 and MVP-9 were also down-regulated significantly. Invasion ability was inhibited as well.

CONCLUSIONRhoA-siRNA can effectively inhibit RhoA expression in Tca8113 and SCC-4 cells. The invasion ability of tongue carcinoma cells decreased with down-regulation of the protein expressions of galectin-3 and MMP-9, indicating that RhoA-siRNA can inhibit invasion of tongue carcinoma. Results show that RhoA may play an important role in the processes of invasion and metastasis of tongue carcinoma.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Down-Regulation ; Galectin 3 ; metabolism ; Gene Silencing ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Tongue Neoplasms ; genetics ; metabolism ; pathology ; Transfection

OBJECTIVETo explore the effect of MSH3 knock-down on sensitivity of tongue cancer cells to cisplatin.

METHODSThree small interfering RNA (siRNA) fragments targeting MSH3 CDS region were synthesized and transfected into CAL27 cells via Lipofectamine. Real-time PCR and Western blotting were used to assess the efficiency of MSH3 silencing. MTS, apoptosis staining and cell immunofluorescence assay were used to examine the cisplatin sensitivity, apoptosis and DNA repair of transfected CAL27 cells.

RESULTSs One of the 3 siRNAs was found to significantly reduce the expression of MSH3 protein in CAL27 cells (P<0.05). MTS assay showed that MSH3 silencing resulted in an significant reduction of IC50 of cisplatin from 21.32 to 13.95 µmol/L (P<0.05) and increased the apoptotic index of the exposed cells from 4.23∓1.27 to 11.32∓1.82 (P<0.05). Immunofluorescence assay demonstrated that silencing MSH3 markedly reduced the number of γ-H2AX foci.

CONCLUSIONSilencing MSH3 can significantly increase cisplatin sensitivity of tongue cancer cells, the mechanism of which involves mainly attenuation of repair of DNA double-strand damage in the cells.

Apoptosis ; Cell Line, Tumor ; Cisplatin ; pharmacology ; DNA-Binding Proteins ; genetics ; metabolism ; Gene Silencing ; Humans ; MutS Homolog 3 Protein ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Tongue Neoplasms ; drug therapy ; genetics ; Transfection

OBJECTIVETo investigate the expression of LRG-1 in clinical specimens and Tca8113 cell line of tongue carcinoma and analyze the relationship between LRG-1 expression and the clinicopathological parameters.

METHODSLRG-1 expression was detected in 40 tongue squamous cell carcinoma (TSCC) tissues and paired normal adjacent tissues, 20 atypical hyperplasia tissues of the tongue, and 20 tissues of tongue cancer in situ using immunohistochemical method. The expression of LRG-1 in Tca8113 cell line was detected using flow cytometry. The expression of LRG-1 was also detected in human TSCC tissues and Tca8113 cells with Western blotting. The effect of LRG-1 on the proliferation of HUVECs was determined using MTT assay, and its effect on angiogenesis was evaluated with Matrigel tube formation assays.

RESULTSHuman TSCC tissues had a significantly higher rate of positive expression for LRG-1 (85%, 34/40) than the adjacent tissues (10%, 4/40), invasive tongue cancer (30%, 6/20), and tongue cancer in situ (50%, 10/20) (P<0.05). LRG-1 expression was correlated with the degree of tumor differentiation, clinical stage and lymph node metastasis of the tumor (P<0.05) but not with the patients' age or gender. In the in vitro experiment, LRG-1 promoted HUVEC proliferation and angiogenesis.

CONCLUSIONAbnormal LRG-1 expression is present in the human TSCC tissue and Tca8113 cells. LRG-1 can promote HUVEC proliferation and angiogenesis in vitro, suggesting its possible role in promoting tumor angiogenesis.

Carcinoma, Squamous Cell ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Glycoproteins ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Lymphatic Metastasis ; Tongue ; metabolism ; pathology ; Tongue Neoplasms ; genetics ; metabolism

OBJECTIVETo study the expression of connexin 43 (Cx43) in various stages of oral carcinogenesis and explore the relation between Cx43 and oral mucous carcinogenesis.

METHODS4-nitroquinoline-1-oxide (4NQO) was used for inducing oral carcinogenesis in SD rats. Tissue samples were obtained from various stages of the disease including normal oral mucosa, precancerous lesions and oral squamous cell carcinoma. Immunohistochemical method was used to determine the expression of Cx43 in various stages of oral carcinogenesis.

RESULTSIn the normal rat lingual mucosa, immunohistochemical staining of Cx43 protein was mainly found in the cell membrane, weakly positive in the basal cell layer, increased in stratum spinosum and stratum granulosum, but was negative in the stratum corneum of normal epithelia. Compared with normal epithelia, was significantly decreased in dysplastic and cancerous oral epithelia the staining. The positive rates of Cx43 were respectively 100.00% (10/10), 85.71% (12/14), 66.67% (8/12), 40.00% (4/10), and 33.33% (4/12) in tongue carcinogenesis (in normal, mild, moderate and severe dysplasia, and squamous cell carcinoma tissues). The differences were statistically significant (P<0.05).

CONCLUSIONExpression level of Cx43 protein was dramatically decreased with the development of rat tongue carcinoma induced by 4NQO, suggesting that abnormal expression of Cx43 protein is involved in oral mucosa carcinogenesis. Decreased Cx43 expression is an early sign of oral mucosa carcinogenesis.

4-Nitroquinoline-1-oxide ; toxicity ; Animals ; Carcinoma, Squamous Cell ; chemically induced ; chemistry ; etiology ; Connexin 43 ; analysis ; genetics ; physiology ; Male ; Rats ; Rats, Sprague-Dawley ; Tongue Neoplasms ; chemically induced ; chemistry ; etiology