TE-1 Cells Research Articles

Hsa_circ_0006168 sponges miR-100 and regulates mTOR to promote the proliferation, migration and invasion of esophageal squamous cell carcinoma

Circle RNAs (circRNAs) are the novel noncoding RNAs with the covalent closed-loop structure, which play a crucial role in a variety of pathological processes, including cancer. Nevertheless, the expression profiles and functions of circRNAs in esophageal squamous cell cancer (ESCC) remain largely unknown. In this paper, 10 pairs of ESCC tissues were utilized to screen the circRNA expression profiles by means of microarray assay; further, a novel circular RNA named hsa_circ_0006168 was investigated. Meanwhile, the expression of hsa_circ_0006168 was measured in 52 ESCC tissues and in cell lines. Our results suggested that, hsa_circ_0006168 was remarkably increased not only in ESCC tissues but also in cell lines compared with those in normal cases. Besides, high hsa_circ_0006168 expression was positively connected with lymph node metastasis and TNM stage of ESCC patients. In vitro, the proliferation, invasion and migration capacities of ESCC cells were suppressed through down-regulating hsa_circ_0006168 expression. Besides, RNase R digestion assay confirmed that hsa_circ_0006168 was more stable than its linear CNOT6L mRNA form. Moreover, nuclear and cytoplasmic fraction assay indicated that hsa_circ_0006168 was mainly distributed in the cytoplasm of Kyse450 and TE13 cells. Mechanically, it was discovered in this study that hsa_circ_0006168 might regulate the expression of Mammalian Target of Rapamycin (mTOR) by sponging microRNA-100 (miR-100). Taken together, hsa_circ_0006168 can promote ESCC proliferation, migration and invasion through the competing endogenous RNA (ceRNA) mechanism, which has been first confirmed in our results. In ESCC, hsa_circ_0006168 can serve as a potential diagnostic biomarker and therapeutic target.

Qigesan reduces the motility of esophageal cancer cells via inhibiting Gas6/Axl and NF-κB expression

The present study is mainly to explore the mechanism that how Qigesan (QGS) affects the movement capacity of esophageal cancer (EC) cell. QGS incubates ECA109 and TE1 cell lines and detecting the motility of tumor cells by different experiments. Growth arrest-specific 6 (Gas6) and Anexelekto (Axl) were co-localized, and then detecting Gas6, Axl signaling pathway, and protein expression after QGS intervention. Similarly, Observing the signal localization and protein expression of P-phosphoinositide3-kinases (PI3K), P-AKT protein kinase B (AKT), P-nuclear factor-kappa B (NF-κB), matrix metalloproteinase-2 (MMP2), and matrix metalloproteinase-9 (MMP9). The results showed that the concentration of QGS was less than 200 ug/ml, and the cultured cells did not exceed 24 h, that no obvious cytotoxicity was observed. QGS significantly inhibited the mobility of ECA109 and TE1 cell lines in the concentration-dependent manner. In addition, QGS can regulate the Gas6/Axl pathway, inhibit the formation and localization of the Gas6/Axl complex, and reduce the protein activation of PI3K/AKT, NF-κB, MMP2, and MMP9. Experimental innovation shows that QGS can significantly slow down the mobility of EC cells by regulating the Gas6/Axl complex and downstream signaling pathways, and provides a theoretical basis for the pharmacological effects of QGS in the therapy of EC.

Genome-wide ChIP-seq data with a transcriptome analysis reveals the groups of genes regulated by histone demethylase LSD1 inhibition in esophageal squamous cell carcinoma cells.

Expression of genes is controlled by histone modification, histone acetylation and methylation, but abnormalities of these modifications have been observed in carcinogenesis and cancer development. The effect of the lysine-specific histone demethylase 1 (LSD1) inhibitor, a demethylating enzyme of histones, is thought to be caused by controlling the expression of genes. The aim of the present study is to elucidate the efficacies of the LSD1 inhibitor on the gene expression of esophageal cancer cell lines using chromatin immunoprecipitation (ChIP)-Seq. A comprehensive analysis of gene expression changes in esophageal squamous cell carcinoma (ESCC) cell lines induced by the LSD1 inhibitor NCL1 was clarified via analysis using microarray. In addition, ChIP-seq analysis was conducted using a SimpleChIP plus Enzymatic Chromatin IP kit. NCL1 strongly suppressed the proliferation of T.Tn and TE2 cells, which are ESCC cell lines, and further induced apoptosis. According to the combinatory analysis of ChIP-seq and microarray, 17 genes were upregulated, and 16 genes were downregulated in both cell lines. The comprehensive gene expression study performed in the present study is considered to be useful for analyzing the mechanism of the antitumor effect of the LSD1 inhibitor in patients with ESCC.

Regulatory of miRNA-149-5p for the expression of Aurora-B in esophageal squamous cell carcinoma

Objective To analyze the regulatory of miRNA (miR)-149-5p for the expression of Aurora-B in esophageal squamous cell carcinoma (ESCC). Methods The pathologic histology paraffin blocks of 61 patients with ESCC in Shanxi Provincial People's Hospital from January 2010 to December 2017 were collected. The immunohistochemical staining and tissue section in situ hybridization method were used to observe the expressions of Aurora-B and miR-149-5p in the tumor tissues and adjacent mucosas of the patients, and their relationship with clinicopathological parameters was analyzed. miRNA was predicted by using software TargetScan and miR walk. The relationship between Aurora-B and miR-149-5p were verified by using Western blot in ESCC TE-1 cells. Results The result of immunohistochemical staining showed that in 61 patients, 37 (61%) tumor tissues showed higher expression of Aurora-B compared with adjacent mucosas, the Aurora-B expression in 15 (26%) tumor tissues were in line with benign tissues, the Aurora-B expression in 9 (13%) tumor tissues were inferior to benign tissues. The expression of Aurora-B were not correlated with age (χ 2=0.008, P=0.929), gender (χ 2=0.088, P=0.767), grade of differentiation (χ 2=2.632, P=0.268), but correlated with TNM staging (χ2=15.153, P < 0.01) and lymph node metastasis (χ 2=5.979, P=0.014). The miR-149-5p was predicted to combine with Aurora-B 3'untraslated region (UTR) by using TargetScan and miRwalk software. The result of in situ hybridization showed that the miR-149-5p showed low expression in 22 (36%) tumor tissues. The expression of miR-149-5p was correlated with Aurora-B expression (χ 2=5.622, P=0.018), and not correlated with age (χ 2=2.617, P = 0.106), gender (χ 2 = 1.529, P = 0.216), grade of differentiation (χ 2 = 2.854, P = 0.240), TNM staging (χ 2 = 0.870, P = 0.351) and lymph node metastasis (χ 2 = 0.128, P = 0.351). The Western blot results of TE-1 cells showed that the expression of Aurora-B in simultaneous over-expression of miR-149-5p and Aurora-B group was higher than that in over-expression of miR-149-5p group, and lower than that in over-expression of Aurora-B group. Conclusion The miR-149-5p can be involved in ESCC progression through regulating the expressions of Aurora-B. Key words: Esophageal neoplasms; Carcinoma, squamous cell; Immunohistochemistry; Blotting, Western; Aurora-B; miRNA-149-5p; TE-1 cell line

Tenascin-C is involved in promotion of cancer stemness via the Akt/HIF1ɑ axis in esophageal squamous cell carcinoma

Although tenascin-C (TNC), an extracellular matrix protein, has been shown to be widely expressed in stromal fibroblasts in various cancers, the role of its expression in esophageal squamous cell carcinoma (ESCC) cells remains unclear. Using immunohistochemistry, we investigated the expression of cancer stem-like cell (CSC) markers, epithelial-to-mesenchymal transition (EMT)-related genes, and the Akt/hypoxia-inducible factor-1α (HIF1α) signal pathway in ESCC tissue specimens from 154 patients. We further addressed the effects of TNC on the Akt/HIF1α axis and its putative association with cancer stemness in several ESCC cell lines by immunofluorescence imaging and western blot analysis. Our data suggest that TNC expression was positively correlated with the expression of the CSC marker SOX2 (p = .002), and TNC-expressing cancer cells expressed SOX2 in ESCC tissues. Moreover, TNC expression was strongly associated with EMT-related gene Snail (p = .022) and positively correlated with pAkt-Ser473 (p = .004) and HIF1α (p = .003). Furthermore, TNC-silencing down-regulated the expression of CSC marker SOX2 (p < .001) and EMT-related marker Snail (p < .001). The Akt inhibitor Perifosine inhibited the protein expression of pAkt-Ser473, Akt, HIF1α, and TNC in TE10 (an ESCC cell line) cells. Short-term exposure of TE10 cells to cobalt chloride caused an increase in protein expression of HIF1α, TNC, and SOX2 in a time-dependent manner. Taken together, these results suggest that TNC may enhance the cancer stem-like properties and promote EMT-like changes via the Akt/HIF1α axis.

Role of MiR-205/PTEN in cisplatin-resistant esophageal squamous cell carcinoma

Purpose: To evaluate the effect of miRNA-205 on cisplatin-resistant TE13 cell lines, and the interaction of miRNA-205 with downstream signaling pathway.Methods: TE13 cells were treated with cisplatin at a concentration of 1.5 μg/mL every 24 h for 3 months. Cisplatin-resistant cell lines were maintained in the continuous presence of 2 μg/ml cisplatin and supplemented every 72 h. Half-maximal inhibitory concentration (IC50) value was determined by MTT assay, and cell apoptosis was tested by flow cytometry. In addition, luciferase reporter assay was conducted to test the binding of miRNA-205 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) 3’UTR.Results: The cisplatin-resistant (cis-TE13) cell line was well established based on IC50 values (cis-TE13, IC50 = 2.215; TE13, IC50 = 0.304). The levels of miRNA-205, phosphatase, PTEN, and protein kinase B (p-AKT) were abnormally expressed in cis-TE13 cells. Overexpression of miRNA-205 significantly promoted cell viability and decreased cell apoptosis of cis-TE13 cells as indicated by IC50 values (cis-TE13, IC50 = 3.537; TE13, IC50 = 0.580). Moreover, miRNA-205 significantly activated p-AKT expression by inhibiting PTEN expression. In contrast to miRNA-205, PTEN overexpression inhibited cell viability, increased cell apoptosis of cis-TE13 cells (cis-TE13, IC50 = 2.625; TE13, IC50 = 0.246), decreased the expression of p-AKT, and counteracted the regulation of miRNA-205 (control, IC50 = 2.297; miR-205, IC50 = 4.693; miR-205+PTEN, IC50 = 2.011).Conclusion: These findings indicate that miRNA-205 exacerbates esophageal squamous cell via inhibition of PTEN expression, suggesting the potential value of miRNA-205 in thediagnosis or treatment of esophageal squamous cell carcinoma.Keywords: miRNA-205, PTEN, PI3K/AKT signaling pathway, Esophageal squamous cell, Carcinoma, Cisplatin resistance

HOXC6 promotes migration, invasion and proliferation of esophageal squamous cell carcinoma cells via modulating expression of genes involved in malignant phenotypes.

BackgroundHOXC6 is a member of the HOX gene family. The elevated expression of this gene occurs in prostate and breast cancers. However, the role of HOXC6 in esophageal squamous cell carcinoma (ESCC) remains largely uninvestigated.MethodsThe expression of HOXC6 was examined by immunohistochemistry, quantitative real-time PCR and immunoblotting assays. The lentivirus-mediated expression of HOXC6 was verified at mRNA and protein levels. Wound healing and Matrigel assays were performed to assess the effect of HOXC6 on the migration and invasion of cancer cells. The growth curving, CCK8, and colony formation assays were utilized to access the proliferation capacities. RNA-seq was performed to evaluate the downstream targets of HOXC6. Bioinformatic tool was used to analyze the gene expression.ResultsHOXC6 was highly expressed in ESCC tissues. HOXC6 overexpression promoted the migration, invasion, and proliferation of both Eca109 and TE10 cells. There were 2,155 up-regulated and 759 down-regulated genes in Eca109-HOXC6 cells and 95 up-regulated and 47 down-regulated genes in TE10-HOXC6 cells compared with the results of control. Interestingly, there were only 20 common genes, including 17 up-regulated and three down-regulated genes with similar changes upon HOXC6 transfection in both cell lines. HOXC6 activated several crucial genes implicated in the malignant phenotype of cancer cells.DiscussionHOXC6 is highly expressed in ESCC and promotes malignant phenotype of ESCC cells. HOXC6 can be used as a new therapeutic target of ESCC.

Induction of Apoptosis by a Combination of 2-Deoxyglucose and Metformin in Esophageal Squamous Cell Carcinoma by Targeting Cancer Cell Metabolism.

Both mitochondrial dysfunction and aerobic glycolysis are signs of growing aggressive cancer. If altered metabolism of cancer cell is intended, using the glycolysis inhibitor (2-deoxyglucose (2DG)) would be a viable therapeutic method. The AMP-activated protein kinase (AMPK), as a metabolic sensor, could be activated with metformin and it can also launch a p53-dependent metabolic checkpoint and might inhibit cancer cell growth. After treatment with 5 mM metformin and/or 500 µM 2DG, the TE1, TE8, and TE11 cellular viability and apoptosis were assessed by MTT, TUNEL, and ELISA methods. The changes in p53 and Bcl-2 genes expression levels were examined using real-time PCR method. Data were analyzed by Kruskal-Wallis test using the SPSS 17.0 software. Metformin and 2DG, alone and in combination, induced apoptosis in the cell lines. Real-time PCR revealed that metformin induced apoptosis in TE8 and TE11 cells by activating p53, down-regulating Bcl-2 expression. The induced apoptosis by 2DG raised by metformin and the combination modulated the expression of Bcl-2 protein in all cell lines and it was more effective in TE11 cell line. Metformin induced apoptosis in ESCC by down-regulating Bcl-2 expression, and up-regulating p53 and induced apoptosis increased by 2-deoxy-d-glucose. Thus, the combination therapy is an effective therapeutic strategy for esophageal squamous cell carcinoma.

Upregulated circ RNA hsa_circ_0000337 promotes cell proliferation, migration, and invasion of esophageal squamous cell carcinoma.

BackgroundAs a new class of endogenous ncRNAs, circRNAs have been recently verified to be involved in the carcinogenesis and progression of human cancers. In the current study, we attempted to explore the potential function of a candidate circRNA (hsa_circ_0000337) in esophageal squamous cell carcinoma (ESCC).Patients and methodsThe altered expression of hsa_circ_0000337 was validated in clinical samples from 48 patients with ESCC. The human esophageal carcinoma cell lines KYSE-150 and TE-1, and the normal human esophageal epithelial cell line (HET-1A) were applied for functional analysis of hsa_circ_0000337. Cell proliferation was measured using the Cell Counting Kit-8 assay and the colony formation assay. Cell invasion and migration were detected by Transwell and wound healing assays, respectively. We further performed bioinformatic analysis and luciferase reporter assays to explore the role of hsa_circ_0000337 as a miRNA sponge.Resultshsa_circ_0000337 was significantly upregulated in ESCC tissues compared to adjacent normal-appearing tissues (P<0.0001). In our in vitro experiment, the expression of hsa_circ_0000337 was higher in TE-1 compared to the normal human esophageal epithelial cell line HET-1A (P<0.001), but was not significantly different in KYSE-150 (P>0.05). Knockdown of hsa_circ_0000337 significantly inhibited cell proliferation, migration, and invasion in TE-1 and KYSE-150 cell lines. Bioinformatics predicted and luciferase reporter assay verified that hsa_circ_0000337 could bind to miR-670-5p, a ncRNA involved in carcinogenesis. It is estimated that 21 genes are regulated by miR-670-5p.Conclusionhsa_circ_0000337 was found to be an upregulated circRNA that is related to ESCC and promotes the progression of disease by regulating cell proliferation, migration, and invasion. These findings suggest that this circRNA could be a promising diagnostic biomarker and potential therapeutic target.

MicroRNA-133b suppresses cell proliferation and invasion of esophageal squamous cell carcinoma via downregulating TAGLN2 expression

Objective: To investigate the expression of microRNA-133b (miR-133b) in esophageal squamous cell carcinoma (ESCC), and explore its effect and the underlying molecular mechanisms on cell proliferation and invasion. Methods: Real-time quantitative PCR (qPCR) was used to examine miR-133b expression in 63 ESCC tissues and paired adjacent non-cancerous tissues, several ESCC cells (Eca109, EC9706, EC1, TE1, KYSE70) and normal esophageal epithelial cell Het-1A. MiR-133b mimic, inhibitor and negative control (NC) were transfected into TE1 cells. The effect of miR-133b on cell proliferation and invasion were determined by CCK-8 and Transwell assays, respectively. Subsequently, the target gene of miR-133b was predicted by online tools TargetScan and miRDB, which was verified by dual luciferase reporter assays. Finally, Western blot was utilized to detect the effects of miR-133b overexpression on expression of target gene TAGLN2 as well as EMT-related proteins E-cadherin, N-cadherin, Snail, Slug and Vimentin. Results: Relative levels of miR-133b in ESCC tissues (0.295±0.040) were significantly lower than those in adjacent non-cancerous tissues (1.002±0.011, P<0.001). The expression of miR-133b was tightly associated with clinical staging, lymph node metastasis and prognosis. Moreover, relative levels of miR-133b in ESCC cells Eca109, EC9706, EC1, TE1 and KYSE70 (0.679±0.031, 0.391±0.008, 0.236±0.016, 0.031±0.005 and 0.099±0.020) were evidently lower than that in normal esophageal epithelial cell Het-1A (1.005±0.016, all P<0.001). In TE1 cells, miR-133b mimic significantly increased the level of miR-133b to 6.199±0.627, and suppressed cell proliferation and invasion, whereas miR-133b inhibitor obviously decreased its expression to 0.182±0.023, and promoted cell proliferation and invasion. Most notably, the relative luciferase activities of miR-133b-mimic group (0.320±0.018) in TE1 cells transfected with TAGLN-3'UTR-WT were markedly lower than that in NC group (1.010±0.036, P<0.001), whereas those in TAGLN-3'UTR-MUT transfection cells were 1.019±0.056 and 1.008±0.021, respectively, showing no significantly statistical difference (P>0.05). Furthermore, miR-133b overexpression markedly downregulated TAGLN2, N-cadherin, Snail, Slug and Vimentin levels, and increased E-cadherin expression. Conclusion: MiR-133b plays an important role in the proliferation and invasion of ESCC cells by regulating TAGLN2 expression, and it may be a potential therapeutic target for ESCC patients.

Raltitrexed increases radiation sensitivity of esophageal squamous carcinoma cells

BackgroundRadiation therapy remains an important therapeutic modality, especially for those patients who are not candidates for radical resection. Many strategies have been developed to increase the radiosensitivity of esophageal cancer, with some success.MethodsThis study was conducted to determine whether raltitrexed can enhance radiosensitivity of esophageal squamous cell carcinoma (ESCC). ESCC cell lines 24 h were incubated with raltitrexed or DMSO with or without subsequent irradiation. Cell Counting Kit assay-8 assay and clonogenic survival assay were used to measure the cell proliferation and radiosensitization, respectively. Flow cytometry was utilized to examine cell apoptosis and cell cycle distribution in different groups. Immunofluorescence analysis was performed to detect deoxyribonucleic acid (DNA) double-strand breaks. In addition, the expression levels of proteins that are involved in radiation induced signal transduction including Bax, Cyclin B1, Cdc2/pCdc2, and Cdc25C/pCdc25C were examined by western blot analysis.ResultsThe results indicated that raltitrexed enhanced radiosensitivity of ESCC cells with increased DNA double-strand breaks, the G2/M arrest, and the apoptosis of ESCC cells induced by radiation. The sensitization enhancement ratio of 1.23–2.10 was detected for ESCC cells with raltitrexed treatment in TE-13 cell line. In vitro, raltitrexed also increased the therapeutic effect of radiation in nude mice.ConclusionRaltitrexed increases the radiosensitivity of ESCC. This antimetabolite drug is promising for future clinical trials with concurrent radiation in esophageal cancer.

The anthelmintic drug flubendazole induces cell apoptosis and inhibits NF-κB signaling in esophageal squamous cell carcinoma.

Background and objectivesThe nuclear factor kappa B (NF-κB) signaling is activated in esophageal squamous cell carcinoma (ESCC) and can be used as a potential target for anti-ESCC drug discovery. In this study, we aimed to investigate the function of flubendazole as a novel NF-κB inhibitor in ESCC cells.Materials and methodsCell Counting Kit-8 assay was carried out to assess cell viability of ESCC cells. Flow cytometry and immunoblotting were performed to examine cell apoptosis. Immunoblotting assay was used to analyze the protein expression of NF-κB signaling. Luciferase assay was performed to explore the activation of NF-κB. Plasmids were transfected into ESCC cells using Lipofectamine® 2000.ResultsIn this study, the anthelmintic drug flubendazole was found to inhibit the activation of IκBα kinases (IKKs), block the activation of IκBα, and decrease the phosphorylation of NF-κB p65, which could be a novel NF-κB inhibitor in ESCC cells. We also found that flubendazole inhibited the cell survival of different ESCC cells and induced cell apoptosis in both EC9706 and TE1 cells. Moreover, overexpression of constitutively activated IKKβ markedly decreased the cytotoxic effect of flubendazole on EC9706 and TE1 cells. In addition, flubendazole also showed a synergistic effect on ESCC cells when combined with doxorubicin.ConclusionThe results above demonstrated that flubendazole showed its anti-tumor action by suppressing the NF-κB signaling pathway and suggested that flubendazole might be re-purposed for anti-ESCC therapy in clinic as a single agent or in combination with other anti-tumor drugs.

β-Hydroxy-β-methylbutyrate Suppresses NF-ĸB Activation and IL-6 Production in TE-1 Cancer Cells.

Stress reactions, especially those related to surgery, cause poor convalescence of cancer patients. β-Hydroxyβ-methylbutyrate (HMB) is known to regulate excessive inflammation in the body. The objective of this work was to investigate the capacity of HMB to suppress activation of nuclear factor-kappa B (NF-ĸB) and production of interleukin-6 (IL-6) in a human esophageal squamous cell carcinoma cell line (TE-1). Cell proliferation was measured using the water-soluble tetrazolium-1 method, while tumor necrosis factor alpha (TNFα)-induced IL-6 production was measured using an enzyme-linked immunosorbent assay (ELISA) assay. Nuclear translocation of NF-ĸB was detected by immunofluorescence staining. HMB did not affect cell proliferation. However, HMB suppressed the TNFα-induced increase in IL-6 production in TE-1 cells by inhibiting NF-ĸB activation. HMB did not influence TE-1 cell proliferation, but inhibited activation of NF-ĸB and IL-6 production. This result may be useful for improving excessive stress reactions during and after surgery.

Cyclin-Dependent Kinase Inhibitor 3 Promoted Cell Proliferation by Driving Cell Cycle from G1 to S Phase in Esophageal Squamous Cell Carcinoma.

Background and aims. Cyclin-dependent kinase inhibitor 3 (CDKN3) has been found playing a varying role in carcinogenesis, but its biological function in esophageal squamous cell carcinoma (ESCC) is unclear. The aim of this study was to demonstrate the role of CDKN3 in ESCC.Materials and Methods: Real-time PCR and Western blot was performed in 15 pairs of ESCC tissues and adjacent normal esophageal tissues. Then cell proliferation ability, cloning ability, cell cycle status and migration and invasion ability were explored in CDKN3 overexpressed TE1 cell line and CDKN3 siRNA transfected TE1 and KYSE70 cell lines. Finally, cell cycle related proteins CyclinD1, CDK4, pAKT, P53, P21, and P27 were tested by Western blot.Results: mRNA level was higher in 11 ESCC tissues compared to adjacent normal tissues, and an increased protein expression was further detected in 8 of those 11 ESCC tissues. Functional assays showed that CDKN3 overexpression promoted ESCC cell proliferation, colony formation, migration and invasion, and facilitated G1/S transition. Opposite results were also got after transfected with CDKN3 siRNA. Cell cycle associated protein pAKT, CyclinD1, CDK4 and P27 were upregulated and P53, P21 and were downregulated under CDKN3 overexpression. All the protein levels were found changed in the opposite direction when CDKN3 expression was disturbed by siRNA.Conclusions: Our study suggested that CDKN3 acted as an oncogene in human ESCC and may accelerate the G1/S transition by affecting CyclinD-CDK4 complex via regulating pAKT-p53-p21 axis and p27 independent of AKT.

Radiosensitization of esophageal carcinoma cells by knockdown of HMGB1 expression.

Radiotherapy (RT) is a traditional and important treatment for carcinoma of the esophagus along with surgery and chemotherapy. High mobility group box 1 (HMGB1) plays a crucial part in inhibiting the apoptosis of cancer cells after irradiation treatment. The present study, was designed to analyze the function of HMGB1 in esophageal cancer progression and elucidate the effects of HMGB1 on the radiosensitivity of human esophageal cancer cell lines. In the present study, an immunohistochemical evaluation of HMGB1 was performed on 77 biopsies, and the results revealed that HMGB1 overexpression was positively correlated with gross tumor volume (GTV), tumor‑node‑metastasis (TNM) stage, T classification, distant metastasis, and relapse and negatively correlated with patient survival rates, suggesting that HMGB1 acts as a key factor in the development of esophageal cancer. An shRNA targeting HMGB1 was designed for the knockdown of HMGB1 in ECA109 and TE13 cells, and the transfection efficiency of the shRNA was assessed using quantitative real‑time reverse transcription polymerase chain reaction and western blot analysis. CCK‑8 and clonogenic assays were used to analyze the effect of HMGB1 on the proliferation and radiosensitivity, respectively, of esophageal cancer cells in vitro. The influence of HMGB1 on radiation‑induced changes in the migration, invasion, and cell cycle as well as apoptosis of tumor cells was examined by wound‑healing and Transwell assays and flow cytometry, respectively. In addition, xenograft tumor models were constructed to observe the effect of HMGB1 on tumor growth in vivo. The results of the study in vitro revealed that the proliferation of the HMGB1‑shRNA group decreased after irradiation, and the radiation treatment reduced the tumor volume of the xenograft model which was more marked in HMGB1‑shRNA group. Moreover, HMGB1 was involved in the phosphorylation of H2AX after irradiation, and HMGB1 knockdown blocked the cell cycle in the G0/G1 phase and increased apoptosis. HMGB1 deficiency was also correlated with the upregulation of p16, Bax and caspase‑9 and the downregulation of MMP‑2, MMP‑9, cyclin D1, CDK4, γH2AX and Bcl‑2. These data indicated that the overexpression of HMGB1 prior to treatment was correlated with poor clinical outcome in esophageal carcinoma and that knockdown HMGB1 expression in human esophageal cancer cell lines increased their radiosensitivity by allowing the induction of apoptosis and G0/G1 arrest after exposure to radiation.

Long non-coding RNA XIST promotes the development of esophageal cancer by sponging miR-494 to regulate CDK6 expression

ObjectiveEsophageal cancer is one of the deadliest cancers worldwide occurring at upper gastrointestinal tract. This study aimed to explore the possible role of long non-coding RNA X Inactive Specific Transcript (XIST) in the development of esophageal cancer. Material and methodsThe lncRNA XIST expressions both in esophageal cancer tissues and in cells were investigated. The TE-1 and SKGT-4 cells were transfected with LV-sh-XIST and LV-scramble for the further detection of the effects of XIST expression on cell biological processes, including proliferation, apoptosis and the expression of apoptosis-related proteins, migration, invasion and the expression of epithelial-mesenchymal transition markers. Additionally, the regulatory relationships between lncRNA XIST and miR-494, between miR-494 and CDK6, between miR-494/CDK6 and JAK2/STAT3 pathway were investigated. ResultsLncRNA XIST was overespressed in esophageal cancer tissues and cells. Suppression of XIST significantly inhibited the proliferation, migration and invasion, but induced apoptosis of two kinds of cells, TE-1 and SKGT-4. Moreover, miR-494 was down-regulated in esophageal cancer tissues and cells. XIST could sponge miR-494 and inhibition of miR-494 reversed the effects of XIST suppression on the malignant behaviors of TE-1 cells. Also, CDK6 was a target of miR-494 and CDK6 knockdown reversed the effects of miR-494 inhibition on the malignant behaviors of TE-1 cells. Besides, the expression of p-JAK2 and p-STAT3 was increased after miR-494 inhibition, which was reversed after inhibition of miR-494 and CDK6 at the same time. ConclusionsThe data presented in this study revealed that XIST abnormal expression may play an oncogenic role in the development of esophageal cancer via miR-494/CDK6 axis through JAK2/STAT3 signal pathway. This study may provide theoretical basis for the molecular mechanism investigation of esophageal cancer.

Casticin inhibits esophageal cancer cell proliferation and promotes apoptosis by regulating mitochondrial apoptotic and JNK signaling pathways.

Casticin, a flavonoid isolated from Vitex species, has been found to have anti-tumor property in multiple human cancers. The present study aimed to investigate the effect of casticin on the proliferation and apoptosis of esophageal cancer (EC) cells, and further illustrate the underlying mechanisms. In in vitro studies, human EC cell lines TE-1 and ECA-109 were treated with various concentrations of casticin (low-, middle-, and high-dose groups). The results showed that casticin dose-dependently inhibited the proliferation and clonogenicity of EC cells and induced cell cycle arrest in sub-G1 and G2 phases. Furthermore, casticin markedly enhanced EC cell apoptosis as detected by flow cytometry and Hoechst 33342 staining. The level of anti-apoptotic Bcl-2 protein was decreased, while the levels of pro-apoptotic Bax, cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP were conversely increased in casticin-treated TE-1 and ECA-109 cells. Moreover, casticin decreased the mitochondrial membrane potential and increased the release of mitochondrial cytochrome C into cytoplasm. In addition, the JNK signaling pathway was involved in casticin-medicated anti-proliferation and pro-apoptosis. Cells pretreated with SP600125, a JNK pathway inhibitor, partially abolished the effect of casticin. Finally, the anti-tumor property of casticin was confirmed in in vivo xenograft models. Overall, we provided both in vitro and in vivo evidences that casticin inhibited the proliferation and induced apoptosis of EC cells, and the anti-tumor action of casticin was mediated, in part, by the mitochondrial-dependent apoptosis and the activation of JNK signaling pathway.

MiR-130b promotes the progression of oesophageal squamous cell carcinoma by targeting SASH1.

MiR‐130b and SAM and SH3 domain containing 1 (SASH1) play an important role in many types of human cancers. The aim of our research was to study their interactions in the process of the proliferation and aggressiveness of oesophageal squamous cell carcinoma (ESCC) cells. Microarray analysis was done to screen the differentially expressed genes in the ESCC tissues. miR‐130b and SASH1 mRNA levels in the ESCC tissues and cells were detected by qRT‐PCR. Dual luciferase reporter system was used to verify the target relationship between miR‐130b and SASH1. The effects of miR‐130b on SASH1 expression were explored by western blot in KYSE30 and TE1 cell lines. CCK‐8 assay, flow cytometry, Transwell, and wound healing assays were conducted to explore the effects of miR‐130b and SASH1 in vitro. In addition, in vivo experiments were conducted to study the roles of miR‐130b and SASH1. miR‐130b was highly expressed, while SASH1 was the opposite in both the ESCC tissues and cells. The expression of SASH1 was inhibited by the direct binding of miR‐130b. The inhibition of miR‐130b reduced the proliferation and aggressiveness of ESCC cells, while it also induced apoptosis and cell cycle arrest in the ESCC cells by suppressing SASH1. The in vivo assay suggested that the overexpression of miR‐130b promoted the growth of ESCC tumours. MiR‐130b was up‐regulated in the ESCC tumour tissues and cells, acting as a tumour promoter. A stimulating effect was demonstrated on ESCC cell growth and aggressiveness by suppressing SASH1, which is an anti‐oncogene.

Low-density lipoprotein promotes lymphatic metastasis of esophageal squamous cell carcinoma and is an adverse prognostic factor.

The purpose of the current study was to investigate the prognostic role of preoperative serum lipid levels in patients with esophageal squamous cell carcinoma (ESCC) and to preliminarily explore the mechanism of serum lipids in this disease. Preoperative lipids, including total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and triglyceride levels, were assessed in 242 patients with ESCC. To eliminate the influence of nutritional status, all patients had previously undergone esophagectomy. Univariate and multivariate Cox regression analyses were performed to identify predictors of overall survival (OS). Associations between significant lipid targets and clinical features were then analyzed and the results were validated using TE-1 and ECa109 esophageal cancer cell lines. The cell proliferation was evaluated with a Cell Counting Kit-8 (CCK8) assay and the cell cycle was assessed with propidium iodide staining and flow cytometry. Univariate analysis revealed that HDL (P=0.048), LDL (P=0.020), Pathological T-staging status (pT status) (P=0.001), Pathological N-staging status (pN status) (P=0.001) and histological differentiation (P=0.002) were significantly associated with OS. Based on multivariate analysis, LDL [hazard ratio (HR)=2.164, P=0.005], pT status (HR=1.714, P=0.001), pN status (HR=1.966, P=0.001) and histological differentiation (HR=4.083, P=0.002) were risk factors in patients with ESCC. A high LDL level (>3.12 mmol/l) was associated with sex (P=0.001), tumor location (P=0.004) and a higher susceptibility to lymphatic metastasis (P=0.007). A CCK8 assay demonstrated that LDL promoted TE-1 and ECa109 cell proliferation, and flow cytometry analysis revealed that treatment with LDL at an appropriate concentration resulted in an accumulation of cells in G2 phase and decreased the number of cells in G1 phase. In summary, the current study identified that preoperative LDL serum level serves an important role in predicting ESCC outcome as LDL promotes lymphatic metastasis. Furthermore, a preliminary mechanism for this association has been validated in vitro.

Steroidal dimer by001 inhibits proliferation and migration of esophageal cancer cells via multiple mechanisms.

To investigate the potential inhibitory effects of structurally novel steroidal dimer by001 in esophageal cancer in vitro. The cytotoxicity of by001 on esophageal, gastric, neuroblastoma and prostate cancer cells was examined MTT assay and colony formation assay. By001 induced apoptosis and production of intracellular reactive oxygen species on esophageal cancer cells Ec109, TE-1 and human normal gastric epithelial cells GES-1 was detected by flow cytometry. The effect of by001 on mitochondrial membrane potential was detected by fluorescence microscope through JC-1 staining. The level of intracellular reactive oxygen species was measured by fluorescence microscope and flow cytometry via DCFH-DA staining. The effect of by001 on members of Bcl-2 family, Fas, LC3, PARP and caspases was determined by Western blot. The effect of by001 on migration was measured by transwell assay. By001 effectively inhibited proliferation of esophageal, gastric, neuroblastoma and prostate cancer cells in a time- and concentration-dependent manner in vitro. By001 reduced the number and the size of colonies at low micromolar concentrations, elevated cellular ROS levels and caused mitochondrial dysfunction in esophageal cancer cells. Molecular mechanistic studies showed that by001 triggered apoptosis through regulating members of Bcl-2 family and Fas. These findings suggested that by001 may inhibited proliferation of esophageal cancer cells through mitochondria and death receptor-mediated apoptotic pathways, autophagy induction, as well as suppressed migration of esophageal cancer cells.