Oral Squamous Cell Carcinoma Tca8113 Research Articles

Study on mechanism of down-regulating ikca1 molecule affecting the increment of oral squamous cell carcinoma

This research aimed to explore the mechanism of mediating the down-regulation of the calcium-activated potassium channel (IKCa1) gene expression in human oral squamous cell carcinoma Tca-8113 cells, thereby affecting cell proliferation and apoptosis. The expression level of IKCa1 in Tca-8113 cell line (oral squamous cell carcinoma) and HOEC cell line (human normal oral epithelial cell) was detected by RT-PCR. Then, after IKCa1 was knocked down in Tca-8113 cell line and HOEC cell line by RNA interference, and then cell proliferation levels were detected by cell counting kit 8 (CCK-8) method. Cell cycle distribution was detected by flow cytometry. Apoptosis was detected by membrane linked protein V-FITC/propidium iodide (PI) double-staining apoptosis detection kit. The protein expression level of IKCa1 was detected by Western Blot method. According to RT-PRC results, IKCa1 was significantly more expressed in Tca-8113 cell line than in HOEC cell line (P< 0.01). In addition, the mRNA expression levels in the normal oral epithelium and oral squamous cell carcinoma showed the same trend. After knocking down IKCa1 in Tca-8113 cell line, the IKCa1siRNA group significantly inhibited cell proliferation compared with the siNC control group. The results of flow cytometry showed that the proportion of apoptotic Tca-8113 cells transfected with IKCa1siRNA was significantly increased. The ratio of early apoptosis and late apoptosis of Tca-8113 cells increased (P< 0.05). To investigate the effect of IKCa1 on apoptosis, we tested the expression levels of apoptosis-related proteins. The results showed that the mRNA level of IKCa1siRNA group was significantly decreased by 44.41% compared with the control group (p< 0.01). Meanwhile, the mRNA level of Bax was significantly increased by 36.0% (p< 0.05). Our results showed that knocking down IKCa1 in Tca-8113 cells could induce cell cycle arrest and apoptosis to produce an anti-proliferation effect, thus inhibiting the expression of IKCa1 has an anti-cancer effect in oral squamous cell carcinoma.

Identification, characterization and microRNA expression profiling of side population cells in human oral squamous cell carcinoma Tca8113 cell lines.

The present study aimed to evaluate the stem cell markers, characteristics and biological functions of cancer stem-like side population (SP) cells in human oral cancer. SP cells were isolated from the human oral squamous cell carcinoma Tca8113 cell line by Hoechst 33342 fluorescence dye and flow cytometry. The colony forming and proliferative capability of SP and non-SP cells were detected using a live-cell analysis system in vitro. The number of cells expressing stem cell markers was compared between SP cells and non-SP cells by flow cytometry. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels of stem cell genes, respectively. Differential expression of microRNAs (miRNAs) in SP and non-SP cells was determined by microarray hybridization and an miRNA regulation network was produced. With regard to the proliferation capability, SP cells reached 60.0% confluence after 40 h of growth compared with 35.1% confluence for non-SP cells (P<0.05). The number of colonies in SP cells was 43.1±9.2 compared with 33.0±8.2 of non-SP cells (P<0.05). The aldehyde dehydrogenase-1 (ALDH1)-positive cell number in the SP cells was increased by 10 times compared with the non-SP cells (P<0.01). The mRNA and protein expression levels of ALDH1, SRY-box 2, POU class 5 homeobox 1 and Nanog homeobox in SP cells were significantly higher compared with non-SP cells (P<0.05). Microarray hybridization demonstrated that 21 miRNAs were upregulated and 13 miRNAs were downregulated in SP cells compared with non-SP cells. SP cells in Tca8113 demonstrated greater capability of proliferation and colony formation compared with non-SP cells in vitro. Stem cell markers were overexpressed in SP cells compared with non-SP cells.

Regulatory effect of MiR103 on proliferation, EMT and invasion of oral squamous carcinoma cell through SALL4.

Oral squamous cell carcinoma (OSCC) is a common tumor of head and neck cancer. MiR-103 is involved in several tumors. However, the role and mechanism of miR103 in OSCC remain unclear. Oral cancer Tca8113 cells were cultured in vitro and randomly divided into control group, miR-103 mimics group, and miR-103 inhibitor group, followed by analysis of miR-103 expression by Real Time-PCR, SALL4 expression by Real Time-PCR and Western blot, cell survival by MTT assay, and cell invasion by transwell chamber assay on tumor. Real Time-PCR was performed to measure MMP-9 and MMP-2 expression. Western blot was conducted to detect E-cadherin and Vimentin expression. The Dual-Luciferase reporter system validated the relationship between miR103 and SALL4. Transfection of miR-103 mimics into Tca8113 cells significantly upregulated miR-103 expression, decreased SALL4 mRNA and protein expression, inhibited proliferation and invasion of Tca8113 cell, downregulated MMP-9 and MMP-2 mRNA expression, increased E-cadherin, and decreased Vimentin protein expression (p<0.05). However, miR-103 inhibitor transfection down-regulated miR-103 expression, promoted proliferation and invasion of Tca8113 cells, increased MMP-9 and MMP-2 mRNA expression, decreased E-cadherin expression, and elevated Vimentin expression. Compared with the control group, the differences were statistically significant (p<0.05). The Dual-Luciferase reports confirmed a targeted relationship between miR103 and SALL4. The overexpression of miR-103 inhibits MMP-9 and MMP-2 expression by negatively regulating SALL4, inhibiting proliferation and invasion of oral squamous cell carcinoma Tca8113 cells.

Role and mechanism of Galectin-3 gene in proliferation, invasion, and apoptosis of oral squamous cell carcinoma

The effect and mechanism of Galectin-3 gene expression on proliferation, invasion, and apoptosis of oral squamous cell carcinoma (OSCC) were investigated. Reverse transcription-polymerase chain reaction (RT- PCR) and Western blotting were used to detect the mRNA and protein of Galectin-3 gene in OSCC. OSCC Tca8113 was divided into control, negative control, and Galectin-3 transfection groups. Western blotting was used to detect the expression of Galectin-3, matrix metalloproteinase (MMP)-2, MMP-9, Cleaved Caspase-3, β-catenin, and Cyclin D1 protein after transfection for 48h in each group. Cell proliferation was detected by CCK8. Cell invasion ability was detected by using a Transwell chamber. Cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Galectin-3 gene in OSCC were significantly higher than those in adjacent tissues (P<0.01). Galectin-3 protein expression in Tca8113 cells significantly decreased after RNA interference. Cell survival rate and invasion as well as MMP-2, MMP-9, β-catenin, and Cyclin D1 protein expression were significantly lower than the blank group. Apoptosis rate and Cleaved Caspase-3 protein expression were significantly higher than the control group (P<0.01). Inhibition of Galectin-3 gene expression in OSCC can significantly reduce the proliferation and invasion of cancer cells and induce apoptosis. The mechanism is related to downregulation of the Wnt/β-catenin signaling pathway.

Identification of differentially expressed genes in oral squamous cell carcinoma TCA8113 cells.

Previous studies have demonstrated that cancer cells with increased levels of aldehyde dehydrogenase ‘bright’ activity (ALDHbr) exhibit stem cell properties compared with cells exhibiting decreased ALDH activity (ALDHlow). To screen possible biomarkers of cancer stem cells in tongue squamous cell carcinoma, ALDHbr and ALDHlow cells were isolated from the tongue squamous cell carcinoma TCA8113 cell line, and suppression subtractive hybridization was performed to identify differentially expressed genes in the two subpopulations. A total of 240 positive clones were randomly selected for sequencing and were functionally characterized using bioinformatical tools. The results of the present study identified the differential expression of 104 clones, 62 of which corresponded to known genes and 42 of which corresponded to unknown genes. Cluster analysis revealed that the known genes were involved in the regulation of the cell cycle and cell differentiation. In addition, analysis of 10 signaling pathways revealed that genes were markedly altered in the ALDHbr cell subpopulation. Additional study is required to identify the function that these genes serve in the biomolecular regulatory mechanisms of cancer stem cells and to assist in explaining the biological behavior of oral squamous cell carcinoma.

Abstract 3006: Arsenic trioxide inhibits overexpression of NAMPT in oral cancer

Abstract Purpose: Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme for a rate-limiting step in the nicotinamide adenine dinucleotide (NAD) salvage pathway. Tumor cells are reliant on the NAMPT salvage pathway for NAD regeneration, thus making this enzyme an attractive therapeutic target for cancer treatment. Overexpression of NAMPT has been observed across a broad range of solid tumors including ovarian, breast, and prostate cancers. However, it is unknown regarding the expression and function of NAMPT in oral cancer. Arsenic trioxide (ATO) has been approved for the treatment of acute promyelocytic leukemia, and accumulating evidence indicate that it is a promising therapeutic drug for certain solid malignant tumors. Up to now, the effect of ATO on NAMPT in oral cancer remains unclear. In the present study, we investigated the expression of NAMPT in different differentiated degree oral cancer and examined the effects of ATO on NAMPT in oral cancer cell line. Materials and methods: Tissue microarray (TMA) was performed by immunohistochemistry to examine the NAMPT expression in well differentiated (n = 39), moderately differentiated (n = 6), and poorly differentiated (n = 5) tissue samples collected from surgical specimens of the oral cancer patients. Human oral squamous cell carcinoma Tca-8113 cells were treated with ATO for 48 hrs. PrestoBlue activity assay was used to detect cell activity and NAMPT mRNA expression was measured by RT-PCR. Results: In paracancer tissue, NAMPT was mainly distributed in cytoplasma and lightly located in nuclear. Intensely increased staining of NAMPT was found in all differentiated grade oral cancer by immunohistochemistry. Moreover, while increased NAMPT are mainly located in cytoplasma in well-differentiated cancer, the marked increase of NAMPT occurred both in cytoplasma and nuclear in moderate- and poor-differentiated cancer. TMA results showed that comparing with normal oral tissue, the protein expression of NAMPT increased by 2.20, 2.66, and 3.03 fold in well-, moderate-, and poor- differentiated oral cancer, respectively. ATO (1, 2.5, 5, and 10 μmol/L) pretreatment of Tca8113 cells significantly reduced the cell activity in an ATO concentration-dependent manner. More importantly, NAMPT mRNA expression was markedly decreased 8.60%, 26.92%, 71.01%, and 78.26%, respectively, compared with control cells. Conclusion: Our novel findings suggest that increased NAMPT expression were highly associated with cellular differentiation in oral cancer, indicating that NAMPT may participate in the development of oral cancer. ATO can inhibit cell activity by depressing NAMPT transcription in oral cancer. These findings may have implications for exploring the NAMPT pathway for oral cancer treatment. Citation Format: Jin Zhi Wang, Yan Liu, Xin Yue Wang, Li Chi Han, Fu Yin Zhang, Ke Liu, Bin Xiang. Arsenic trioxide inhibits overexpression of NAMPT in oral cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3006.

MiRNA-99b-3p functions as a potential tumor suppressor by targeting glycogen synthase kinase-3β in oral squamous cell carcinoma Tca-8113 cells.

Dysregulation of microRNAs (miRNAs) has been associated with carcinogenesis in oral squamous cell carcinoma (OSCC). In the present study, we investigated the expression and function of miR-99b-3p in human OSCC. We found that the expression levels of miR-99b-3p decreased in 21 clinical OSCC samples (84%). Furthermore, ectopic expression of miR-99b-3p inhibited OSCC cell proliferation by downregulating glycogen synthase kinase-3β (GSK3β), an miR-99b-3p' target gene, at the mRNA and protein levels, both in vitro and in vivo. Moreover, the silencing of GSK3β recapitulated the cellular and molecular effects in a similar manner to the overexpression of miR-99b-3p, which included inhibition of OSCC cell proliferation and suppression of p65 (RelA) and G1 regulators (cyclin D1, CDK4 and CDK6) in vitro. Our data suggest that miR-99b-3p functions as a tumor suppressor in OSCC via GSK3β downregulation.

Gelsolin regulates proliferation, apoptosis, migration and invasion in human oral carcinoma cells

Gelsolin (GSN) is one of the most abundant actin-binding proteins, and is involved in several pathological processes, including Alzheimer's disease, cardiac injury and cancer. The aim of the present study was to assess the effect of GSN on the growth and motility of oral squamous cell carcinoma Tca8113 cells. The overexpression vector pcDNA3.1-GSN was transfected into Tca8113 cells and the stable GSN overexpression cell line was identified based on G418 antibiotic selection. The effect of GSN overexpression on the proliferation, apoptosis, migration and invasion of Tca8113 cells was examined using a cell counting kit-8 assay, flow cytometry and Transwell assays. The results revealed that GSN overexpression significantly promoted the cell proliferation and apoptosis of Tca8113 cells. In addition, Transwell assays demonstrated that the migration and invasion abilities of Tca8113 cells were enhanced by GSN overexpression. Therefore, the upregulation of GSN promotes cell growth and motility, indicating that it may perform a vital function in the progression of human oral cancers.

Application of quantum dots as vectors in targeted survivin gene siRNA delivery

Gene silencing using short interfering RNA (siRNA) is becoming an attractive approach for probing gene function in mammalian cells. This study evaluated the specificity and efficiency of quantum dots (QDs) as non-viral gene vectors for delivery of survivin siRNA and downregulation of survivin gene expression in oral squamous cell carcinoma Tca8113 cells. Water-dispersible cationically-modified QDs were electrostatically attached to anionic siRNA molecules and complexed with siRNA for downregulating expression of the survivin gene. Cellular uptake and allocation of QD–siRNA complexes in Tca8113 cells were monitored using confocal laser scanning microscopy. Real-time polymerase chain reaction (PCR) was used to quantify survivin messenger RNA (mRNA) levels. CdSe QDs were observed with high intensity fluorescence under confocal laser scanning microscopy. Tca8113 cells were successfully transfected by QDs with survivin siRNA, and the red fluorescence from CdSe QDs and green fluorescein amidite fluorescence from siRNA could both be easily observed after 6 hours of incubation. The release of siRNA into the cytoplasm was verified through real-time PCR quantification that showed reduced survivin mRNA levels. In this study, survivin siRNA successfully complexed with water-soluble CdSe QDs and exhibited excellent fluorescent properties and downregulated the expression of the survivin gene in oral squamous cell carcinoma Tca8113 cells. QDs are a novel non-viral gene delivery vector.

Effects of RhoA siRNA on the proliferation, adhesion, migration and invasion of tongue squamous cell carcinoma Tca8113 cells in vitro

to investigate the effect of RhoA on the metastasis of tongue squamous cell carcinoma Tca8113 cells in vitro. a group of RhoA specific small interfering RNAs (siRNA) was constructed and confined by sequencing analysis. The siRNA of RhoA gene was transfected into human tongue squamous cell carcinoma Tca8113 cells line by Lipofectamine(TM) 2000. The protein transient expression of RhoA in the transfectants was examined 48 h after transfection. The cell line with stable expression of siRNA of RhoA was obtained by blasticidin screening and colony culture. Cell growth rate was determined with a cell counting. Millicell chambermodel and wound healing assay were used to examine the abilities of migration and invasion, respectively in vitro. RhoA was overexpressed in tongue squamous cell carcinoma Tca8113 cell line. Silencing of endogenous RhoA gene expression in Tca8113 cells resulted in inhibition of the proliferation, adhesion, chemotaxis and invasion of Tca8113 cells in vitro. RhoA siRNA inhibits the proliferation, adhesion, chemotaxis and invasion of oral squamous cell carcinoma Tca8113 cell lines. siRNA of RhoA is a potential factor controlling the proliferation and metastasis of Tca8113 cells. RhoA may play an important role in metastasis of oral squamous cell carcinoma.