Proliferation Of Laryngeal Carcinoma Cells Research Articles

Effects of Epidermal Growth Factor Receptor Signal Pathway on Proliferation, Invasion and E-Cadherin/Vimentin Protein Expression in Laryngeal Carcinoma Hep-2 Cells

Epidermal growth factor receptor (EGFR) is one transmembrane receptor with a high expression in more than 90% of head-neck squamous carcinoma cells. This study investigated the role of EGFR signal pathway on proliferation, invasion and expression of related proteins E-cadherin/Vimentin expression in laryngeal carcinoma cells. Laryngeal carcinoma Hep-2 cells were treated with EGFR agonist EGF or inhibitor Gefitinib followed by measuring cell cycle, proliferation index (PI) and apoptosis by flow cytometry. Transwell assay was employed for cell invasion and migration. Western blotting was further employed to test E-cadherin and Vimentin level. Compared to blank control group, EGF-treated cells had significantly lower S percentage of cell cycle at 6 h, 12 h and 24 h, plus higher PI. With prolonged incubation time, S ratio and PI were further significantly elevated, with more potent cell invasion and migration abilities, lower E-cadherin and Vimentin protein levels (p < 0.05). Gefitnib significantly elevated S ratio at 6 h, 12 h and 24 h cell cycle, reduced PI, invasion or migration ability, as upregulated E-cadherin and downregulated Vimentin protein (p < 0.05). Suppressing EGFR signal pathway could inhibit proliferation of laryngeal carcinoma cells, decrease cell invasion or migration, upregulate E-cadherin and downregulate Vimentin.

PRMT5/Wnt4 axis promotes lymph-node metastasis and proliferation of laryngeal carcinoma

Metastasis is the main cause of laryngeal cancer-related death; its molecular mechanism remains unknown. Here we identify protein arginine methyltransferase 5 (PRMT5) as a new metastasis-promoting factor in laryngeal carcinoma, and explore its underlying mechanism of action in regulating laryngeal cancer progression. We illustrated that PRMT5 expression was positively correlated with tumor stages, lymphatic metastasis, and unfavorable outcome. Functional assays revealed that PRMT5 promoted laryngeal carcinoma cell proliferation, migration, and invasive capacity in vitro, as well as lymph-node metastasis in vivo. The ectopic expression of PRMT5 induced EMT with downregulation of E-cadherin and upregulation of N-cadherin, snail, and MMP9. Mechanistic results revealed that the metastatic effects could be attributed to PRMT5-mediated activation of Wnt signaling, and Wnt4 is an important driver of Wnt/β-catenin signaling pathway. Wnt4 silencing could reverse PRMT5-induced cell proliferation, migration, and invasion capacities. Furthermore, inhibition of the Wnt/β-catenin signaling pathway abolished the effect of PRMT5-induced proliferation, whereas activation of the pathway enhanced the effect of PRMT5 overexpression on cell proliferation. These results demonstrated that the oncogenic role of PRMT5 could be attributed to PRMT5/Wnt4 axis-mediated activation of the Wnt/β-catenin signaling pathway. PRMT5 may serve as a novel prognostic marker and a therapeutic target for lymphatic metastasis of laryngeal carcinoma.

LncRNA WTAPP1 promotes proliferation of laryngeal carcinoma cells through regulating microRNA-592.

The aim of this work was to investigate the mechanism by which long non-coding RNA (lncRNA) WTAPP1 promotes the malignant progression of laryngeal cancer. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) examined the expression of lncRNA WTAPP1 in 49 pairs of tumor tissue specimens and paracancerous normal ones collected from laryngeal cancer patients. Subsequently, in the laryngeal squamous cell carcinoma cell lines AMC-HN-8 and Hep-2, WTAPP1 overexpression and knockdown vectors were constructed using lentivirus, and cell counting kit-8 (CCK-8), cell colony formation and 5-ethynyl-2'-deoxyuridine (EdU) assays were carried out to analyze the impact of lncRNA WTAPP1 on the function of laryngeal cancer cells. Finally, Luciferase reporting assay and recovery experiments were carried out to further explore whether lncRNA WTAPP1 has an impact on the malignant progression of laryngeal cancer via modulating microRNA-592. QRT-PCR results revealed a significantly higher expression of lncRNA WTAPP1 in tumor tissues of patients with laryngeal cancer than that in adjacent normal ones. Compared with patients with low expression of WTAPP1, those with higher expression had a more advanced pathological stage. Meanwhile, the proliferation ability of cells in sh-WTAPP1 group was remarkably attenuated when compared with that in sh-NC group. In addition, microRNA-592 and WTAPP1 mRNA levels were found negatively correlated in laryngeal carcinoma tissue specimens. Luciferase reporter gene assay indicated that WTAPP1 can be targeted by microRNA-592 through certain binding sites. Moreover, we demonstrated through some recovery experiments that WTAPP1 can indeed serve as an oncogene accelerating the malignant progression of laryngeal cancer through the modulation of microRNA-592. LncRNA WTAPP was markedly highly expressed both in laryngeal carcinoma tissues and cell lines, which was also found to be closely relevant to the pathological stage of laryngeal cancer patients. Additionally, lncRNA WTAPP1 is able to enhance the proliferation capacity of laryngeal carcinoma cells via regulating microRNA-592.

A biflavonoid-rich extract from Selaginella moellendorffii Hieron. induces apoptosis via STAT3 and Akt/NF-κB signalling pathways in laryngeal carcinoma.

Selaginella moellendorffii Hieron. (SM), a perennial evergreen plant, has been used in the treatment of acute infectious hepatitis, thoracic and hypochondriac lumbar contusions, systemic oedema and thrombocytopaenia. However, the role of a biflavonoid‐rich extract from SM (SM‐BFRE) in anti‐larynx cancer has rarely been reported. In this study, the in vitro and in vivo anti‐laryngeal cancer activity and potential mechanisms of SM‐BFRE were investigated. An off‐line semipreparative liquid chromatography‐nuclear magnetic resonance protocol was carried out to determine six biflavonoids from SM‐BFRE. In vitro, MTT assay revealed that SM‐BFRE inhibited the proliferation of laryngeal carcinoma cells. A wound healing assay indicated that SM‐BFRE suppressed the migration of laryngeal cancer cells. Hoechst 33 258 and Annexin V‐FITC/PI double staining assays were performed and verified that SM‐BFRE induced apoptosis in laryngeal carcinoma cells. The Hep‐2 bearing nude mouse model confirmed that SM‐BFRE also exhibited anticancer effect in vivo. In addition, Western blot analysis demonstrated that SM‐BFRE exerted its anti‐laryngeal cancer effect by activating the mitochondrial apoptotic pathway and inhibiting STAT3 and Akt/NF‐κB signalling pathways. All results suggested that SM‐BFRE could be considered as a potential chemotherapeutic drug for laryngeal cancer.

Molecular mechanism of targeted inhibition of HMGA2 via miRNAlet-7a in proliferation and metastasis of laryngeal squamous cell carcinoma.

MiRNAlet-7a is associated with the tumorigenesis of laryngeal squamous cell carcinoma (LSCC). Our study was designed to infer whether let-7a targets high-mobility AT-hook 2 (HMGA2) and suppresses laryngeal carcinoma cell proliferation, invasion, and migration. The expression levels of let-7a and HMGA2 were measured in 30 LSCC clinical specimens by qRT-PCR and their correlation was analyzed. Cell model and mice xenograft model with or without let-7a overexpression were constructed to evaluate the effects of let-7a on LSCC. Moreover, luciferase assay was performed to reveal the interaction between let-7a and HMGA2, which was further verified in xenograft. Let-7a was significantly down-regulated and HMGA2 was up-regulated in LSCC tissues compared with normal tissues (P<0.05), both of which were significantly correlated with TNM stage and lymph node metastases of LSCC patients (P<0.05). We also observed a negative correlation between let-7a and HMGA2 expression in LSCC samples (r = −0.642, P<0.05). In vitro and in vivo experiments demonstrated that let-7a overexpression could inhibit cell proliferation and tumor growth of LSCC and simultaneously down-regulate the expression of HMGA2. Moreover, the regulation of HMGA2 by let-7a was also proved by luciferase assay. Our results revealed that let-7a promotes development and progression of LSCC through inhibiting the expression of HMGA2. Therefore, let-7a may thus be a potential diagnostic biomarker and therapeutic target for treating LSCC.

MiR-632 Promotes Laryngeal Carcinoma Cell Proliferation, Migration, and Invasion Through Negative Regulation of GSK3β.

Laryngeal cancer, one of the most common head and neck malignancies, is an aggressive neoplasm. Increasing evidence has demonstrated that microRNAs (miRNAs) exert important roles in oncogenesis and progression of diverse types of human cancers. miR-632, a tumor-related miRNA, has been reported to be dysregulated and implicated in human malignancies; however, its biological role in laryngeal carcinoma remains to be elucidated. The present study aimed at exploring the role of miR-632 in laryngeal cancer and clarifying the potential molecular mechanisms involved. In the current study, miR-632 was found to be significantly upregulated both in laryngeal cancer tissues and laryngeal cancer cell lines. Functional studies demonstrated that miR-632 accelerated cell proliferation and colony formation, facilitated cell migration and invasion, and enhanced the expression of cell proliferation-associated proteins, cyclin D1 and c-myc. Notably, miR-632 could directly bind to the 3′-untranslated region (3′-UTR) of glycogen synthase kinase 3β (GSK3β) to suppress its expression in laryngeal cancer cells. Mechanical studies revealed that miR-632 promoted laryngeal cancer cell proliferation, migration, and invasion through negative modulation of GSK3β. Pearson’s correlation analysis revealed that miR-632 expression was inversely correlated with GSK3β mRNA expression in laryngeal cancer tissues. Taken together, our findings suggest that miR-632 functions as an oncogene in laryngeal cancer and may be used as a novel therapeutic target for laryngeal cancer.

MicroRNA‐379 inhibits laryngeal carcinoma cell proliferation and invasion through directly targeting TCF‐4

The expression pattern, functions, and detailed regulatory mechanisms of miR-379 in laryngeal carcinoma remain unknown. In the present study, we aimed to uncover novel potential miR-379 targets and shed light on its roles in laryngeal carcinoma. The expression level of miR-379 was measured based on the data obtained from the TCGA database and the cell lines. After miR-379 mimic transfection, cell proliferation, invasion and migration assay, and wound-healing assay in HEp-2 cell line were implemented to evaluate the effects of miR-379 on laryngeal carcinoma in vitro. The target genes for miR-379 in silico were predicted and validated using luciferase reporter assay. The miR-379 expression level was reduced in laryngeal carcinoma tissues and cell lines. Moreover, miR-379 overexpression inhibited the cell proliferation, migration, and invasion of laryngeal carcinoma cells. TCF-4 was detected as a direct target of miR-379 in laryngeal carcinoma. Furthermore, restored TCF-4 expression rescued the inhibitory roles of miR-379 overexpression on cell proliferation, migration, and invasion.Our study for the first time demonstrated that miR-379/TCF-4 might involve in the progression of laryngeal carcinoma, and miR-379 appears to serve as a novel tumor suppressor in laryngeal carcinoma.

Down-regulation of SOX18 inhibits laryngeal carcinoma cell proliferation, migration, and invasion through JAK2/STAT3 signaling.

Laryngeal carcinoma is one of the most common malignant tumors of the head, neck, and respiratory tract. The aim of the present study is to explore the biological function of SRY-related HMG-box 18 (SOX18) in laryngeal carcinoma cells and study the molecular mechanism involved. Initial findings indicate that the expression of SOX18 was increased in laryngeal carcinoma cell lines and tissues. The effect of SOX18 on laryngeal carcinoma cell proliferation, cell cycle, apoptosis, invasion, and migration was also identified. The results indicated that down-regulation of SOX18 significantly inhibited cell proliferation, migration, and invasion, and induced cell-cycle arrest in G0/G1 phase and apoptosis of laryngeal carcinoma cells. However, overexpression of SOX18 promoted cell proliferation, invasion, and migration, and inhibited cell apoptosis. The expression of cyclin D1, active-caspase-3, N-cadherin, MTA1, MMP-2, and MMP-7 was also regulated by the overexpression of siSOX18 or SOX18. In addition, it was found that SOX18 could also accelerate the phosphorylation of JAK2/STAT3 signaling in laryngeal carcinoma cells. Furthermore, our study indicated that SOX18 could stimulate cell proliferation, migration, and invasion of laryngeal carcinoma cells via regulation of JAK2/STAT3 signaling, which could provide a new strategy for laryngeal carcinoma diagnosis and molecular therapies.

Fasudil inhibits proliferation and migration of Hep-2 laryngeal carcinoma cells.

BackgroundRho-kinase signal pathway is a new target for cancer therapy. Fasudil, a selective Rho-kinase inhibitor, is found to exert antitumor effects on several types of cancer, but whether fasudil has antitumor effects on laryngeal carcinoma is still unknown. The aim of this study was to determine the effects of fasudil on laryngeal carcinoma and explore the underlying molecular mechanisms in this process.MethodsAfter treatment with fasudil, changes in biological behaviors, including the growth, proliferation, clone formation, apoptosis, and migration of human laryngeal carcinoma cells (Hep-2 cells) were observed. The influences on apoptotic protease activity factor-1 (APAF-1)-mediated apoptosis pathway and the activities of matrix metalloproteinases (MMP-2 and MMP-9) were measured by Western blotting and gelatin zymography assay.ResultsHalf-maximal inhibitory concentration of fasudil to Hep-2 cells was ~3.40×103 µM (95% CI: 2.53–4.66×103 µM). Moreover, fasudil treatment significantly decreased the ability of growth, proliferation, clone formation, and migration of Hep-2 cells, while remarkably increased the apoptosis rate. Furthermore, the expressions of APAF-1, caspase-9, and caspase-3 significantly increased in fasudil treatment group. Meanwhile, fasudil led to a remarkable decrease in the expressions and activities of MMP-2 and MMP-9.ConclusionOur findings first demonstrate that fasudil not only inhibits the proliferation of laryngeal carcinoma cells through activating APAF-1-mediated apoptosis pathway, but also prevents migration by inhibiting the activities of MMP-2 and MMP-9. Therefore, fasudil is an attractive antitumor drug candidate for the treatment of laryngeal carcinoma.

EZH2 promotes cell proliferation by regulating the expression of RUNX3 in laryngeal carcinoma.

Enhancer of zeste homolog 2 (EZH2) is a highly conserved histone methyltransferase, which is overexpressed in different types of cancers such as breast and prostate cancer. It is reported that EZH2 can directly down-regulate RUNX3 by increasing histone H3 methylation. However, the role of EZH2 in the development and progression of laryngeal carcinoma has not yet been investigated, and the relationship between EZH2 and RUNX3 in laryngeal carcinoma is rarely reported. The current study aims to determine the role of EZH2 in the progression of laryngeal carcinoma, and investigate the interaction between EZH2 and the tumor suppressor RUNX3. Our study found that EZH2 is overexpressed in laryngeal carcinoma patients, and silencing EZH2 by EZH2 siRNA significantly inhibited the proliferation of laryngeal carcinoma cells. Besides, we also found that RUNX3 is repressed in laryngeal carcinoma patients. Moreover, RUNX3 as a downstream target protein of EZH2 is up-regulated by EZH2 siRNA accompanied by a decrease in the trimethylation modification pattern of H3K27. RUNX3 siRNA inhibits the decreased proliferation induced by EZH2 siRNA. Furthermore, β-catenin protein expression is down-regulated by EZH2 siRNA and up-regulated by RUNX3 siRNA, and RUNX3 siRNA inhibits the down-regulation effect of EZH2 siRNA on β-catenin protein expression. Additionally, the Wnt/β-catenin activator BIO reverses the inhibitory effect of EZH2 siRNA on Hep-2 cell proliferation. Taken together, our results suggest that EZH2 regulates cell proliferation potentially by targeting RUNX3 through the Wnt/β-catenin signaling pathway in laryngeal carcinoma.

Zerumbone, a Sesquiterpene, Controls Proliferation and Induces Cell Cycle Arrest in Human Laryngeal Carcinoma Cell Line Hep-2

ABSTRACTZerumbone (ZER), a sesquiterpene found in Zingiber zerumbet Smith, has been shown to possess antiproliferative, anticancer, antioxidant, and anti-inflammatory activity against various types of human carcinoma. The molecular mechanism by which ZER mediates its activity against many cancer types is revealed by many studies. Upregulation of proapoptotic molecules and suppression of antiapoptotic gene expression are few of the mechanisms by which ZER mediates its effect. The present study is focused on investigating the effect of ZER on proliferation of laryngeal carcinoma cells (Hep-2). MTT assay results showed that ZER (0.01–100 μM) induced death of Hep-2 cells in a concentration-dependent manner; significant suppression of proliferation of Hep-2 cells was seen with a IC50 value of 15 µM. ZER at a concentration of 15 and 30 μM for 48 h showed early signs of apoptosis as evidenced by confocal microscopy imaging. Flow cytometry studies showed that ZER induced cell cycle arrest. ZER arrested Hep-2 proliferation at S and G2/M phases of cell cycle. In conclusion, these results indicate that ZER has antiproliferative effect and arrests cell cycle in Hep-2 cells in vitro. This could be a potential anticancer drug against laryngeal carcinoma.

MicroRNA-203 leads to G1 phase cell cycle arrest in laryngeal carcinoma cells by directly targeting survivin

Previous studies have shown that miR-203 acts as a tumor-suppressive microRNA in various cancers, but its roles in laryngeal carcinoma are still contradicted. Here, we found that miR-203 inhibited the growth of laryngeal cancer cells and survivin was a direct target of miR-203. Moreover, silencing of survivin recapitulated the effect of miR-203 on cell cycle progression, whereas overexpression of survivin reversed this effect. Additionally, qRT-PCR showed the reciprocal relationship between miR-203 and survivin in laryngeal cancer tissues. These findings indicate that miR-203 inhibits the proliferation of laryngeal carcinoma cells by directly targeting survivin, suggesting its application in anti-cancer therapeutics.