Laryngeal Squamous Cell Carcinoma Hep-2 Research Articles

Investigation of the Functional Role of mir-1825 in Laryngeal Squamous Cell Carcinoma Hep-2 Cell Line

Investigation of the Functional Role of mir-1825 in Laryngeal Squamous Cell Carcinoma Hep-2 Cell Line

Study on the mechanism of inhibiting invasion of human laryngeal squamous cell carcinoma Hep-2 and TU212 cells after the downregulation of miRNA-106b

Objective To investigate the effect of miRNA-106b (miR-106b) on human laryngeal squamous cell carcinoma Hep-2 and TU212 cells and its mechanism. Methods Hep-2 and TU212 cells were divided into miR-106b inhibitory sequence transfected group (the experimental group), miR-106b competitive negative sequence transfected group (the negative control group) and non-intervention group (the blank group). The inhibitory effect of miR-106b inhibitory sequence on the expression of miR-106b was verified by using reverse transcription quantitative polymerase chain reaction (qRT-PCR). Whether phosphatase and tensin homolog (PTEN) was the target gene of miR-106b was analyzed by using bioinformatics and luciferase report vector. PTEN small interfering RNA (siRNA) was used to inhibit the expression of PTEN in Hep-2 and TU212 cells. Transwell method and Western blot were used to detect the change of invasion ability of Hep-2 and TU212 cells after miR-106b silencing or the PTEN intervening, and the expression change of PTEN, epithelial cadherin and vimentin. Results The relative expression levels of miR-106b in Hep-2 and TU212 cells in the experimental group were 0.110 ± 0.037 and 0.074 ± 0.009, respectively, which were lower than those in the negative control group (1.013±0.059 and 1.035±0.062, respectively; all P < 0.05). In Transwell experiments, the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group was less than that in the negative control group [(37.09±4.02) vs. (95.65±4.77), (29.16±2.49) vs. (103.19±6.08), all P < 0.05]. The bioinformatics analysis results showed that 3'-UTR region of PTEN mRNA was complementary to 3'-UTR region of miR-106b. Dual-luciferase reporter system analysis showed that the luciferase reporter activity of wild-type PTEN gene transfected with miR-106b was decreased to (22.84±2.68)%, and that of mutant PTEN gene transfected with miR-106b was almost unchanged [(92.08±3.44)%], and the difference was statistically significant (P < 0.001). The expression level of PTEN protein of Hep-2 and TU212 cells in the experimental group was higher than that in the negative control group. Transwell method showed that the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression was more than that in the experimental group without the inhibition of PTEN expression [(65.08±3.57) vs. (26.72±2.58), (57.38±4.96) vs. (31.81±2.97), all P < 0.05]. Western blot showed that the expression level of epithelial-cadherin was up-regulated and vimentin was down-regulated of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression. Conclusions The human laryngeal squamous cell carcinoma Hep-2 and TU212 cell miR-106b can influence the downstream invasion-related protein of PTEN and change the cell invasion ability through the targeted regulation of PTEN expression. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Neoplasm invasiveness; RNA interference; miRNA-106b; Phosphatase and tensin homolog

AMG-102 inhibits proliferation and induces apoptosis of laryngeal squamous cell carcinoma cells by regulating c-Met/PI3K/Akt pathway

Objective: To investigate the effects of c-Met inhibitor AMG-102 on the proliferation and apoptosis of laryngeal squamous carcinoma Hep-2 cells and the underlying mechanism. Methods: Laryngeal squamous carcinoma cell line Hep-2 cells were treated with 2.5, 5 and 10 μmol/L AMG-102, respectively. The proliferation activities of Hep-2 cells were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT). The apoptotic rate of Hep-2 cells was detected by flow cytometry analysis and Hoechst staining. The mRNA expression levels of apoptosis-related genes were detected by real-time quantitative polymerase Chain reaction (RT-qPCR), and the protein expressions of c-Met/PI3K/AKT pathway were detected by western blot. Results: Compared with the control group, the proliferation rates of Hep-2 cells treated with 2.5, 5 and 10 μmol/L AMG-102 for 24 hours were (89.8±1.1)%, (79.8±1.0)% and (69.1±1.2)%, respectively; for 48 hours were (76.8±2.0)%, (60.2±1.1)% and (49.8±1.2)%, respectively; for 72 hours were (50.1±2.0)%, (41.5±1.1)% and (33.6±1.0), respectively, with significant differences (all P<0.05). The apoptotic rates of Hep-2 cells treated with 2.5, 5 and 10 μmol/L AMG-102 for 48 hours were (16.09±1.53)%, (27.51±2.02)% and (36.57±1.42)%, respectively, which were significantly higher than (3.62±0.10) % in the control group (all P<0.05). After treated with 2.5, 5 and 10 μmol/L AMG-102 for 48 hours, the relative expression levels of Bcl-2 mRNA in Hep-2 cells were 0.58±0.13, 0.38±0.12 and 0.20±0.13, respectively; the relative protein expression of p-Met were 80.0±3.8, 50.6±4.2 and 28.5±1.3, respectively; the relative protein expression of p-PI3K were 87.1±0.9, 54.2±1.2 and 21.0±1.2, respectively; the relative protein expression of p-AKT were 98.7±5.6, 56.9±3.2 and 32.2±4.3, respectively; which were significantly lower than those in the control group (all P<0.05). The relative expression levels of Bax mRNA were 1.78±0.13, 2.37±0.14 and 3.05±0.13, respectively, and the relative expression levels of caspase-3 mRNA were 1.98±0.14, 2.47±0.14 and 3.15±0.13, respectively, which were significantly higher than those in the control group (all P<0.05). Conclusion: c-Met inhibitor AMG-102 could inhibit the proliferation and induce apoptosis of laryngeal squamous carcinoma Hep-2 cells by regulating the c-Met/PI3K/Akt pathway.

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells.

Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. MTT assay was used to detect the inhibitory rate of viability. Giemsa's staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. These results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

Descending-SHIP2-mediated radiosensitivity enhancement through PI3K/Akt signaling pathway in laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma (LSCC) is the major type of laryngeal carcinoma. SHIP2 plays a critical role in malignant tumors and is associated with activation of PI3K/Akt signaling pathway. Here, we aimed to explore the impacts of SHIP2 on LSCC Hep-2 cells and the relationship between SHIP2 and radiotherapy. SHIP2 knockdown impairs cell proliferation, invasion, migration and promotes cell apoptosis in this study, suggesting the oncogenic role of SHIP2 in laryngeal cancer. Radiation not only has the similar effect on laryngeal cancer as SHIP2 knockdown, but also causes significant cell cycle G2 arrest, all of which can be significantly enhanced by SHIP2 knockdown. This enhancement effect cause by SHIP2 knockdown derive from the inactivation of PI3K/Akt signaling pathway along with its downstream proteins. Our finding revealed a novel mechanism for sensitivity to radiotherapy caused by SHIP2 knockdown that called descending-SHIP2-mediated radiosensitivity enhancement (DSMRSE).

Oncolytic viro-chemotherapy exhibits antitumor effect in laryngeal squamous cell carcinoma cells and mouse xenografts.

Background: Oncolytic virus can specifically replicate in and then lyse tumor cells, but seldom in normal cells. Further studies have shown the significant therapeutic effect of oncolytic virotherapy combining with other strategies, such as chemo-, radio-, and immunotherapy et al. In this study, we investigated the combinational effect of oncolytic virus ZD55-TRAIL and chemotherapy drug doxorubicin (DOX) on human laryngeal squamous cell carcinoma (LSCC).Methods: The effect of ZD55-TRAIL combined with DOX on cell growth was assessed in LSCC Hep2 cells and normal cells by MTT assay. Hochest 33342 staining was performed to observe cell morphological changes. Western blot was used to detect the expression of apoptotic activation proteins. The in vivo antitumor efficacy of combination treatment was estimated in laryngeal cancer xenograft models.Results: The combination of ZD55-TRAIL and DOX exhibited enhanced inhibitory effects on laryngocarcinoma cell growth, and had few side effects to normal cells in vitro. Chemotherapy drug increased the inducement of tumor cell apoptosis mediated by oncolytic virus. In vivo experiment confirmed that the combination treatment significantly inhibited Hep2 laryngocarcinoma xenografts growth in mice.Conclusion: The oncolytic viro-chemotherapy is a potent therapeutic approach for in vitro cytotoxicity evaluation of Hep2 cells and xenograft growth in vivo.

Long chain non-coding RNA MALAT-1 gene knockdown inhibits growth and migration and promotes apoptosis of human laryngeal squamous cell carcinoma Hep-2 cells in vitro

To investigate the effect of knocking down long chain non-coding RNA MALAT-1 gene on the biologicalbehaviors of human laryngeal squamous cell carcinoma Hep-2 cells. With immortalized nasopharyngeal epithelial(NPE) cell line NP-69 as the reference, MALAT1 expression in FaDu, Hep-2 and nasopharyngeal carcinoma CNE-2Z cells weredetected using real-time PCR. Hep-2 cells were transfected with shmalat1 lentivirus and the expression of MALAT1 wasdetected. MTT assay, flow cytometry, Transwell assay and M Atrigel invasiveness test were used to evaluate the effect ofMALAT-1 knockdown on the proliferation, cell cycle, cell apoptosis, migration, and invasiveness of Hep-2 cells. Compared with NP-69 cells, Hep-2 cells, FaDu cells, and CNE-2Z cells all showed significantly increased MALAT-1expression. In Hep-2 cells, knockdown of MALAT-1 significantly inhibited the cell proliferation, increased the cell percentagein S phase (P < 0.01), decreased the cell percentage in G2/M phase (P < 0.01), and attenuated the migration and invasiveness of thecells. MALAT-1 is over-expressed in laryngeal squamous cell carcinoma, and knocking down MALAT-1 gene cansignificantly suppress the proliferation, invasion and migration and promotes apoptosis of the cancer cells.

MiR-154 inhibits the growth of laryngeal squamous cell carcinoma by targeting GALNT7.

MicroRNAs are critical regulators of the development and progression of laryngeal squamous cell carcinoma (LSCC). However, the role of microRNA-154 (miR-154) in the development and progression of LSCC has not been clarified. We found that down-regulated miR-154 expression in LSCC tissues was associated with poorer prognosis in LSCC patients. MiR-154 over-expression inhibited the proliferation, clonogenicity, and migration of LSCC cells and induced cell cycle arrest, which were reversed by miR-154 inhibition. MiR-154 targeted GALNT7 expression by reducing GALNT7-regulated luciferase activity in LSCC cells while up-regulating GALNT7 mRNA transcription in LSCC tissues and cells. GALNT7 silencing significantly attenuated the proliferation, clonogenicity, and migration of LSCC cells and induced cell cycle arrest. Finally, intravenous treatment with lentivirus for miR-154, but not scrambled control miRNA, significantly restrained the growth of implanted LSCC Hep-2 tumors and decreased the tumor mass by reducing GALNT7 expression in mice. Therefore, miR-154 may serve as a novel prognostic marker and therapeutic target for LSCC.

Knockdown of Snail inhibits epithelial-mesenchymal transition of human laryngeal squamous cell carcinoma Hep-2 cells through the vitamin D receptor signaling pathway.

It has been well documented that Snail plays a decisive role in various tumors. However, the direct effect of Snail on laryngeal squamous cell carcinoma (LSCC) has not been elaborated. In this study, we firstly detected the expression of Snail in 14 samples of patients with LSCC and found that its content was high in cancer tissues compared with adjacent tissues. Then we established LSCC Hep-2 cells with Snail silencing and validated the knockdown efficiency by Western blotting and real-time PCR. Results showed that silencing of Snail significantly inhibited the ability of adhesion, migration, and invasion of Hep-2 cells. Further study revealed that knockdown of Snail suppressed the epithelial-mesenchymal transition (EMT) process of Hep-2 cells, as evidenced by downregulation of matrix metallopeptidase (MMP)-2, MMP-9, integrin subunit beta 1 (ITGβ1), β-catenin, vimentin, N-cadherin, and fibronectin and upregulation of vitamin D receptor (VDR) and E-cadherin. Additionally, transfection with the small interfering RNA of VDR reversed the effect induced by Snail silencing in Hep-2 cells. Taken together, these results demonstrate that knockdown of Snail can inhibit the EMT process of LSCC cells through the VDR signaling pathway in vitro.

MicroRNA-204-5p inhibits invasion and metastasis of laryngeal squamous cell carcinoma by suppressing forkhead box C1.

Background and aim: Understanding the molecular biological mechanisms underlying laryngeal squamous cell carcinoma (LSCC) invasion and metastasis is crucial for diagnosis, treatment, and prognosis. We aimed to examine the expression of the tumor suppressor microRNA-204-5p (miR-204-5p) and its target gene, forkhead box C1 (FOXC1), in human LSCC and explore their roles in the malignant behaviors of LSCC Hep-2 and TU-177 cells.Methods: The regulatory effects of miR-204-5p on the 3′ untranslated region of FOXC1 predicted by bioinformatics were tested by dual-luciferase reporter assay. Quantitative RT-PCR was used to detect mRNA expression in 43 fresh samples of LSCC and corresponding adjacent normal mucosa (ANM). FOXC1 protein expression was examined by immunohistochemistry. miR-204-5p mimics and FOXC1 siRNA were transfected into LSCC cell lines Hep-2 and TU-177 to observe malignant behavior. miR-204-5p mimics were injected into Hep-2 or TU-177 xenograft tumors in nude mice to examine tumor growth. Results: The miR-204-5p mRNA level was lower in all 43 LSCC samples than in the ANM samples, but the FOXC1 level was higher in the LSCC samples than in the ANM samples. The miR-204-5p level was lower for stage III and IV cancer and lymph node N+ status samples than for stage I and II cancer and N0 status samples. FOXC1 mRNA and protein levels were higher for N+ than for N0 LSCC. The miR-204-5p mRNA levels were lower in Hep-2 and TU-177 cells than in ANM tissues, but FOXC1 mRNA levels were higher in Hep-2 and TU-177 cells than in ANM tissues. Dual-luciferase reporter assays demonstrated the targeted regulatory effects of miR-204-5p on the FOXC1 3′ UTR. Cell proliferation and colony formation was facilitated with miR-204-5p mimics and FOXC1 siRNA, with weaker cell migration and invasion than the controls. Moreover, miR-204-5p overexpression or FOXC1 knockdown inhibited the EMT process in LSCC cells. In vivo experiments demonstrated that injection of miR-204-5p into Hep-2 and TU-177 xenograft tumors in nude mice significantly inhibited tumor growth.Conclusions: miR-204-5p is involved in the invasion and metastasis of LSCC. It has a targeted regulatory effect on FOXC1 expression; malignant LSCC behaviors, including cell proliferation, invasion, and metastasis, are suppressed, and tumor growth in vivo is inhibited. This suggests that miR-204-5p may be a target for molecular therapy of LSCC in the future.

Ascorbic Acid Induces Necrosis in Human Laryngeal Squamous Cell Carcinoma via ROS, PKC, and Calcium Signaling.

Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.

Effect of DJ-1 silencing by RNA interference on growth of xenografted human laryngeal squamous cell carcinoma Hep-2 cells in nude mice

Objective: To investigate the effect of silencing DJ-1 on xenografted human laryngeal squamous cell carcinoma (LSCC) Hep-2 cells in nude mice. Methods: Xenograft model of human LSCC was established by subcutaneous transplantation of Hep-2 cells in 24 nude mice. The LSCC-bearing nude mice were randomly divided into 3 groups (n=8 in each):DJ-1 siRNA low dose group and DJ-1 siRNA high dose group were injected in tumors with 20 μg of DJ-1 siRNA or 40 μg of DJ-1 siRNA in 50 μL, respectively; control group was injected with 5% glucose solution in 50 μL, twice a week for 3 weeks. The weight and size of tumors were measured before injection. The animals were sacrificed 48 h after the final treatment, and the tumors were harvested and weighed. The apoptosis and proliferation of tumor cells were determined; the expressions of Caspase-3 and Ki-67 in tumor specimens were detected with immunohistochemistry. The expression of DJ-1, PTEN, survivin mRNA and protein in tumor tissues were detected by RT-PCR and Western blotting, respectively. Results: Tumor weight in low dose group[(0.66±0.15)g] and high dose group[(0.48±0.11)g] were significantly lower than that in control group[(0.83±0.16)g, all P<0.05]. The inhibition rates of low dose group and high dose group were (20.48±0.18)% and (42.16±0.13)%, respectively. Immunohistochemistry showed that the expression of Caspase-3 was increased and Ki-67 was reduced in tumor specimens, compared with the control group (all P<0.05). RT-PCR and Western blot results showed that in low dose group and high dose group the mRNA and protein expression of DJ-1 and survivin significantly decreased (all P<0.05), while PTEN mRNA and protein content increased (all P<0.05). Conclusion: High dose DJ-1 siRNA can inhibit the tumor growth in human LSCC xenograft nude mouse model, which indicates that down-regulating DJ-1 and survivin, and up-regulating PTEN expression may lead to blockage of PI3K-PKB/Akt signaling pathway and promoting tumor cell apoptosis.

Effect and mechanism of miR-34a on proliferation, apoptosis and invasion of laryngeal carcinoma cells.

To discuss the effect and mechanism of miR-34a on the proliferation, apoptosis and invasion of laryngeal carcinoma cells. The laryngeal squamous carcinoma Hep2 cells were transiently transfected with miR-34a mimics and miR-34a NC. The MTT, colony-forming assay, Hoechst staining and AnnexinV-PI double staining flow cytometry were employed to detect the effect of miR-34a on the viability and apoptosis of laryngeal squamous carcinoma Hep2 cells; Transwell assay to defect the effect of miR-34a on the migration and invasion of laryngeal squamous carcinoma Hep2 cells; western blot and RT-PCR assay to defect the effect of miR-34a mimics on the expression of survivin and Ki-67 mRNA in laryngeal squamous carcinoma Hep2 cells. Compared with miR-34a NC group, the cell viability in miR-34 mimics group was significantly decreased (P<0.01), the cell apoptosis rate was significantly increased (P<0.01), the abilities of cell migration and invasion were significantly reduced (P<0.01) and the expression of survivin and Ki-67 mRNA was significantly decreased (P<0.01). The increased expression of miR-34a can induce the apoptosis of Hep2 laryngeal carcinoma cells and inhibit the cell proliferation and invasion, which is related to the down-regulated expression of survivin and Ki-67.

The anti-tumour activity of rLj-RGD4, an RGD toxin protein from Lampetra japonica, on human laryngeal squamous carcinoma Hep-2 cells in nude mice.

The objective of this study is to investigate the antiproliferative activity and mechanism of integrin-binding rLj-RGD4 in a Hep-2 human laryngeal carcinoma-bearing nude mouse model. Human laryngeal squamous carcinoma cells (Hep-2) were inoculated subcutaneously into the axilla of nude mice to generate a Hep-2 human laryngeal carcinoma-bearing nude mouse model. When the Hep-2 xenograft model was successfully established, the animals were randomly separated into five groups. Three groups were treated with different dosages of rLj-RGD4. Cisplatin was administered to the positive control group, and normal saline (NaCl) was administered to the negative control group for 3 weeks. The body weights and the survival of the nude mice were evaluated, and the volumes and weights of the solid tumours were measured. The mechanism underlying rLj-RGD4 inhibition of tumour growth in transplanted Hep-2 human laryngeal carcinoma-bearing nude mice was evaluated by haematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), measurement of intratumoural microvessel density (MVD), Western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The tumour volumes and weights of the treatment groups were reduced compared with the model group, and survival times were improved by rLj-RGD4 treatment in Hep-2 human laryngeal carcinoma-bearing nude mice. The number of apoptotic Hep-2 human cells and intratumoural MVD significantly decreased after the administration of rLj-RGD4. In the xenograft tissue of animals treated with rLj-RGD4, FAK, PI3K, and Akt expression was unaltered, whereas P-FAK, P-PI3K, Bcl-2, P-Akt, and VEGF levels were down-regulated. In addition, activated caspase-3, activated caspase-9, and Bax levels were up-regulated. rLj-RGD4 exhibits potent invivo activity and inhibits the growth of transplanted Hep-2 human laryngeal carcinoma cells in a nude mouse model. Thus, these results indicate that the recombinant RGD toxin protein rLj-RGD4 may serve as a potent clinical therapy for human laryngeal squamous carcinoma.

The role of miR-145 in stem cell characteristics of human laryngeal squamous cell carcinoma Hep-2 cells.

The cancer stem-like cells (CSLCs) are tumorigenic cells promoting initiation, progression, and spread of the tumor. Accumulating evidences suggested the presence of CSLCs in distinct tumors including laryngeal squamous cell carcinoma (LSCC). MicroRNAs have been proposed as significant regulators of carcinogenesis, and several of them have been demonstrated to have direct roles in survival of CSLCs. In this study, we aimed to explore the role of miR-145, which is downregulated in LSCC, on cancer stem cell potency of laryngeal cancer cells. We initially showed the downregulation of miR-145 expression in tumor tissue samples and in CD133-enriched CSLCs. Quantitative reverse-transcription PCR (qRT-PCR) analysis of miR-145-transfected Hep-2 cells demonstrated the inhibitory role of miR-145 on stem cell markers like SOX2, OCT4, KLF4, and ABCG2. We, then, investigated the stem cell features of miR-145-overexpressing Hep-2 cells by sphere formation assay, single-cell cloning assay, and aldehyde dehydrogenase (ALDH) assay, which all demonstrated the inhibition of stem cell potency upon miR-145 overexpression. Further qRT-PCR analysis demonstrated altered expression of epithelial to mesenchymal transition markers in miR-145-overexpressing Hep-2 cells. In conclusion, we demonstrated the regulatory role of miR-145 in stem cell characteristics of Hep-2 cells. Based on these results, we propose that miR-145 might carry crucial roles in LSCC tumorigenesis, prognosis, metastasis, chemoresistance, and recurrence through regulating stem cell properties of tumor cells.

MicroRNA-101 is a potential prognostic indicator of laryngeal squamous cell carcinoma and modulates CDK8.

BackgroundVarious microRNAs (miRNAs) negatively modulate genes that are involved in cellular proliferation, differentiation, invasion, and apoptosis. In many types of cancer, the expression profiles of these miRNAs are altered. Recently, miR-101 was identified as a tumour suppressor and was found to be expressed at low levels in various types of tumours, including prostate, breast, endometrium, and bladder cancers. However, the function(s) of miR-101 in laryngeal carcinoma remain unknown.MethodsThe expression levels of miR-101 in laryngeal squamous cell carcinoma (LSCC) tissues and cells were detected by qPCR. Cell proliferation, migration, cell cycle, and apoptosis assay were applied to assess the function(s) of miR-101 in vitro. Nude mice subcutaneous tumour model was used to perform in vivo study. Moreover, we identified Cyclin-dependent kinase 8 (CDK8) as the target of miR-101 by a luciferase assay. The possible downstream effectors of CDK8 were investigated in Wnt/β-catenin signaling pathway. Changes of CDK8, β-catenin, and cyclin D1 protein levels were analyzed by western blotting and immunohistochemical staining. The prognostic effect of miR-101 was evaluated using the Kaplan–Meier method.ResultsExpression of miR-101 was down-regulated in the LSCC tissues compared with the adjacent normal tissues. Furthermore, downregulation of miR-101 correlated with T3–4 tumour grade, lymph node metastasis, and an advanced clinical stage in the LSCC patients examined (P < 0.05). The low level of miR-101 expression was associated with poor prognosis (P < 0.05). CDK8 was identified as the target gene of miR-101 by luciferase reporter assay. Moreover, we showed that up-regulation of miR-101 expression suppressed humen LSCC Hep-2 cells proliferation and migration, and induced cell-cycle arrest. Increased expression of miR-101 induced cells apoptosis both in vitro and in vivo. Correspondingly, exogenous expression of miR-101 significantly reduced the growth of tumour in a LSCC xenograft model. Furthermore, the miR-101 level was inversely correlated with levels of CDK8, β-catenin, and cyclin D1 in western blotting assay and immunohistochemical staining assay.ConclusionsThese results indicate that miR-101 is a potent tumour repressor that directly represses CDK8 expression. Thus, detection and targeting of miR-101 may represent a novel diagnostic and therapeutic strategy for LSCC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0626-6) contains supplementary material, which is available to authorized users.

Regulatory effects of WRAP53 on radiosensitivity of laryngeal squamous cell carcinoma cells.

Telomere length is closely associated with cellular radiosensitivity and WRAP53 is required for telomere addition by telomerase. In this research we assessed radiosensitivity of laryngeal squamous cell carcinoma Hep-2 cell lines after WRAP53 inhibition, and analyzed the molecular mechanisms. phWRAP53-siRNA and pNeg-siRNA were constructed and transfected into Hep-2 cells with lipofectamine. Expression of WRAP53 was analyzed by RT-PCR and Western-blottin, radiosensitivity of Hep-2 cells was assessed colony formation assay, and the relative length of telomeres was measured by QPCR. The data revealed that the plasmid of phWRAP53-siRNA was constructed successfully, and the mRNA and protein levels of WRAP53 were both obviously reduced in the Hep-2 cell line transfected with phWRAP53-siRNA. After Hep-2 cells were irradiated with X-rays, the D0 and SF2 were 2.481 and 0.472, respectively, in the phWRAP53-siRNA group, much lower than in the control group (D0 and SF2 of 3.213 and 0.592) (P<0.01). The relative telomere length in the phWRAP53-siRNA group was 0.185±0.01, much lower than in the untreated group (0.523±0.06) and the control group (0.435±0.01). Decreasing the expression of WRAP53 using RNA interference technique can enhance the radiosensitivity of Hep-2 cell lines by influencing the telomere length. WRAP53 is expected to be a new target to regulate the radiosensitization of tumor cells.

Effects of curcuma extract-elemene on proliferation, invasion and tumor-associated calcium signal transducer-2 gene expression of various cancer cells

Objective To study the effects and mechanism of curcuma extract-elemene on proliferation,invasion and tumor-associated calcium signal transducer-2 (TROP-2) gene expression of various cancer cells.Methods Human esophageal carcinoma TE-1 and Ca-ES-17 cells,laryngeal squamous cell carcinoma Hep-2 cells,lung cancer NCI-H1975 cells,colorectal cancer SW-480 cells,and prostate cancer PC-3 cells were prepared.After all group cancers were treated with different doses of elemene,and proliferation of cancer cells was studied by methyl thiazol tetrazolium (MTT) assay,and invasion ability was determined by Boyden chamber assay.TROP-2 mRNA level was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).Results Elemene has an inhibitory effect on all group cancer cells in a dose-dependent manner.The MTT assay showed that the inhibition rate of proliferation was 43.919,189.036,48.690,75.538,35.145,and 31.063 mg/L in TE-1,Ca-ES-17,Hep-2,NCI-H1975,SW-480,and PC-3 cells,respectively.The Boyden chamber test showed that the inhibition rate of invasion was 25.6％,18.9％,23.8％,22.2％,26.5％,and 27.8％ in TE-1,Ca-ES-17,Hep-2,NCI-H1975,SW-480,and PC-3,respectively.The FQ-PCR assay showed that the inhibition rate of TROP-2 mRNA was 35.8％,25.6％,32.5％,25.6％,29.8％,and 33.6％ in TE-1,Ca-ES-17,Hep-2,NCI-H1975,SW-480,and PC-3,respectively.Conclusion Elemene could reduce cell proliferation and invasion of various cancer cells through down-regulation of TROP-2 gene. Key words: Curcuma; Elemene; Cancer cells ; Proliferation; Invasion; Trophoblast cell-surface antigens-2

Proteasome inhibitor bortezomi-induced the apoptosis of laryngeal squamous cell carcinoma Hep-2 cell line via disrupting redox equilibrium

Bortezomib, a proteasome inhibitor, has been therapeutic effects in some solid tumors. In the present work, the effects of bortezomib on laryngeal squamous cell carcinoma Hep-2 cell line was investigated and the role of redox equilibrium was explored. The results showed that bortezomib decreased the cell viability and increased the apoptosis in Hep-2 cells. In addition, bortezomib overproduced reactive oxygen species (ROS) and induced the loss of mitochondrial membrane potential (ΔΨm). And the activity of caspase-3 increased. N-acetyl-L-cysteine (L-NAC), a ROS scavenger, alleviated oxidative stress and inhibited the apoptosis induced by bortezomib. However, buthionine sulfoximine (BSO), an inhibitor of GSH synthetase, aggravated bortezomib-induced oxidative stress and apoptosis. So bortezomi-induced the apoptosis of Hep-2 cells via disrupting redox equilibrium.

Down-regulation of histone deacetylase 2 induces cell apoptosis and inhibits cell proliferation and migration of laryngeal squamous cell carcinoma cells

To investigate the effects of histone deacetylase 2 (HDAC2) expression on cell proliferation, apoptosis and migration of laryngeal squamous cell carcinoma (LSCC) Hep-2 cells. HDAC2 siRNA and control siRNA were transfected into LSCC Hep-2 cells by lipofectamine 2000, and cells were divided into three experimental groups: untreated group, control siRNA group and HDAC2 siRNA transfection group. Western blotting was utilized to detect the expression of HDAC2 protein in Hep-2 cells. Cell proliferation and apoptosis were investigated by CCK-8 kit and flow cytometry, respectively. Boyden chamber was used to study cell migration. Expressions of cell apoptosis and cell migration related proteins were detected by Western blotting. HDAC2 siRNA significantly down-regulated the expression of HDAC2 protein in LSCC Hep-2 cells. Down-regulation of HDAC2 expression coincided with an inhibition of cell proliferation and migration along with an induced cell apoptosis of Hep-2 cells. Moreover, down-regulation of HDAC2 expression significantly increased the expressions of caspase-3 and caspase-9 proteins but decreased the expressions of matrix metalloproteinases (MMP)-2 and MMP-9 proteins. HDAC2 may play a pivotal role in the initiation and development of LSCC. Down-regulation of HDAC2 expression mediates cell apoptosis. Cell migration inhibition may be tightly associated with overexpression of caspase-3 and caspase-9 along with down-regulation of MMP-2 and MMP-9 expressions.