Proliferation Of Eca109 Cells Research Articles

RNAi-mediated TCF-3 gene silencing inhibits proliferation of Eca-109 esophageal cancer cells by inducing apoptosis.

Esophageal cancer (EC) remains an important health problem in China. In the present study, through the use of siRNA, specific gene knockdown of transcription factor 3 gene (TCF-3) was achieved in vitro and the effect of TCF-3 gene on human EC Eca-109 cell proliferation and apoptosis. Eca-109 cells were treated using negative control (NC) of siRNA against TCF-3 (siTCF-3) and siTCF-3 group. Colony formation assay was used to detect the colony formation ability in Eca-109 cells. MTT assay was used to measure the cell growth and viability, whereas BrDU assay was used to evaluate cell proliferation, and flow cytometry (FCM) to assess cell apoptosis. Reverse-transcription quantitative PCR (RT-qPCR) was applied to measure TCF-3 gene expression. Protein expressions of TCF-3, apoptosis-related proteins, Bcl-2, Bax, and caspase-3 were determined using Western blotting. Transfection of siTCF-3 successfully down-regulated TCF-3 gene expression. In addition, siTCF-3, reduced Eca-109 cell viability and proliferation, in a time-dependent manner, and inhibited progression of cell cycle from G0/G1 to S-stage. When treated with siTCF-3, the Eca-109 cells exhibited increased apoptosis, with up-regulated cleaved caspase and Bax expressions, whereas Bcl-2 expression was down-regulated. The present study shows that TCF-3 gene silencing inhibits Eca-109 cell growth and proliferation, suppresses cell cycle progression, and promotes apoptosis, which might serve as a new objective for EC treatment.

MiR-214 inhibits the proliferation and invasion of esophageal squamous cell carcinoma cells by targeting CDC25B

BackgroundDysregulation of microRNA(miRNAs) expression was reported in human esophageal squamous cell carcinoma (ESCC). MiR-214 has been found to acts as a tumor suppressor in some tumors including ESCC. The objective of the study was to investigate the functional effect of miR-214 on the regulation of human ESCC progression. MethodsThe expression levels of miR-214 in 57 paired human ESCC tissues and adjacent normal tissues were examined by qRT-PCR. The capacities of cell proliferation and invasion were determined after up-regulation or down-regulation of miR-214 by performing cell viability assay, colony formation assay and transwell assay. Dual luciferase assays, Western blot analysis and qRT-PCR assay were used to demonstrate the association between CDC25B and miR-214. Western blot analysis assessed relative CDC25B protein expression. ResultsWe observed that miR-214 expression exhibited a frequent down-regulation in ESCC tissues and cells, compared to adjacent normal tissues and cells, respectively. Furthermore, up-regulation of miR-214 significantly inhibited cell proliferation and colony formation and cell invasion capacities in Eca9706 and Eca109 cells. However, down-regulation of miR-214 exhibited an opposite effects. Dual luciferase assays showed that CDC25B was identified as a direct target of miR-214. Meanwhile, up-regulation of miR-214 decreased CDC25B expression, whereas, down-regulation of miR-214 increased the CDC25B expression in Eca9706 and Eca109 cells. Moreover, we demonstrated that miR-214 inhibited Eca9706 and Eca109 cells proliferation and invasion through CDC25B. ConclusionOur results indicate that miR-214 function as a tumor suppressor and may be potential therapeutic target for ESCC.

MicroRNA-145 suppresses esophageal squamous cell carcinoma by targeting phospholipase C epsilon 1

Objective To investigate the mechanism of microRNA (miRNA, miR)-145 in modulating the capacity of proliferation and invasion in esophageal squamous cell carcinoma (ESCC). Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-145 and phospholipase C epsilon 1 (PLCE1) mRNA. PLCE1 protein level was evaluated by Western blotting. The potential target gene of miR-145 was predicted by targetscan software. Luciferase reporter gene assay was used to validate the predicted miR-145 binding site in PLCE1 3’untranslated region (3’UTR). Proliferation and invasion capacity were assessed by MTT and wound healing assay separately. Results The relative expression of miR-145 in four types of ESCC cells were 0.63±0.06, 0.72±0.05, 0.55±0.05 and 0.79±0.04 separately, which were obviously lower than in normal esophageal epithilum (P=0.000, P=0.001, P=0.000, P=0.003). While, relative expression of miR-145 in ESCC tissues was 0.68±0.03, which was 31.9% lower than in normal esophageal tissue (P=0.000). An inverse correlation between miR-145 expression and tumor invasion depth and TNM stage were observed (χ2=8.380, P=0.039; χ2=6.810, P=0.033). Luciferase reporter gene assay proved that PLCE1 was a direct target of miR-145. PLCE1 was overexpressed and inversely correlated with miR-145 expression in ESCC (r=-0.828, P=0.002). The relative expression of PLCE1 in four types of ESCC cells were 1.69±0.04, 1.46±0.06, 1.31±0.06 and 1.60±0.08, which were obviously higher than in normal esophageal epithilum (P=0.000, P=0.000, P=0.004, P=0.001). And relative PLCE1 expression was 1.85±0.04 in ESCC tissues, which was 1.85 times higher than in normal esophageal tissue (P=0.000). In addition, overexpression of miR-145 suppressed Eca109 cells proliferation (P=0.006) and decreased invasion capacity by 22.1% (P=0.013). Whereas, overexpression of PLCE1 induced proliferation (P=0.003) and elevated invasion capacity by 24.9% (P=0.029). Enforced expression of miR-145 could partially reversed the promoting effect of PLCE1 (P=0.003). Conclusion MiR-145 suppresses proliferation and invasion of ESCC by targeting PLCE1. Key words: MicroRNA-145; Phospholipase C epsilon 1; Esophageal neoplasms; Proliferation; Invasion

Functional evidence that the self-renewal gene NANOG regulates esophageal squamous cancer development

Cancer cell molecular mimicry of stem cells (SC) follows with enhanced proliferative and renewal capacities. In support, numerous mediators of SC self-renewal have been evinced to exhibit oncogenic potential. More and more researches showed that the embryonic stem cell self-renewal genes express in various cancer cells. In this study, we sought to test the tumorigenic functions of NANOG, particularly, in esophageal cancer (EC). Using quantitative RT-PCR and western blotting, we confirmed that EC cells highly express NANOG mRNA and protein. We then constructed a shRNA-mediated plasmid to knockdown of NANOG mRNA. We observed that NANOG deficiency in Eca109 cells decreased clone formation, cell proliferation, and showed G1 arrest. To further investigate the functions and mechanisms of NANOG in Eca109 cells, we detected the changes of multiple signaling molecules when NANOG deficiency. We foud that NANOG deficiency affected multiple genes, particularly, supressed drug-resistance via down-regulated ABCG2 in Eca109 cells, and caused G1 arrest by down-regulated cyclin D1 (CCND1) expression. The present loss-of-function work, establish the integral role for NANOG in Eca109 cell proliferation, drug resistance, and shed light on its mechanisms of action.

Effect of thrombin on malignant biological behavior of esophageal cancer cell line Eca109

Objective To study the effect of thrombin on proliferation and invasion of esophageal cancer cell line Eca109, and to explore its possible mechanism. Methods The proliferation and invasion of Eca 109 cells treated with thrombin were detected by MTT and Transwell assay, respectively. The activity of matrix metalloproteinase 2 (MMP-2) and MMP-9 in the supernatant of Eca109 cells was detected by gelatin zymography. Reverse transcription polymerase chain reaction (PCR) and immunocytochemistry were used to study the mRNA expression of protease-activated receptor 1 (PAR-1), the important receptor of thrombin, and subcellular localization of PAR-1 protein in Eca109 cells, respectively. Results Thrombin could promote Eca109 cells proliferation in a dose-dependent manner. Cell proliferative rates of 0.5 U/ml and 1.0 U/ml thrombin were 34.38% and 57.19%, respectively (P < 0.05). Compared to that of control group, the number of Eca109 cells incubated with 1.0 U/ml thrombin invading through the basement membrane of Transwell was increased (303.33±6.66 vs. 116.33±11.51, P < 0.05). When treated with various concentrations of thrombin for 24 h, the activities of MMP-2 and MMP-9, especially MMP-9, in the supernatant of Eca109 cells were increased in a dose-dependent manner. Eca109 cells expressed PAR-1 mRNA, and PAR-1 protein was mainly located on the cellular membrane. Conclusion Thrombin increases proliferation and invasion of esophageal cancer Eca109 cells and enhances the activities of MMP-2 and MMP-9 in cells supernatant, which might be induced by activation of PAR-1 located on cellular membrane. Key words: Esophageal neoplasms; Thrombin; Protease-activated receptor 1

Effect of Annexin A1 gene on the proliferation and invasion of esophageal squamous cell carcinoma cells and its regulatory mechanisms

The aim of this study was to examine the effect of Annexin A1 (ANXA1) on the proliferation, migration and invasion of esophageal squamous cell carcinoma (ESCC) cells and its possible mechanisms of action. After constructing the ANXA1 overexpression plasmid, we transfected this plasmid and/or microRNA (miRNA)-196a mimic into ESCC cells (Eca109 cell line). Methyl thiazolyl tetrazolium (MTT) assay and Transwell chamber assay were performed to determine cell proliferation, migration and invasion, respectively. Western blot analysis was used to examine the protein expression levels of ANXA1, Snail and E-cadherin. RT-PCR was used to detect the expression of miRNA-196a. Our results revealed that ANXA1 expression was upregulated in the cells transfected with the ANXA1 overexpression plasmid, and cell proliferation, migration and invasion were significantly increased (p=0.004, p<0.001 and p=0.011, respectively). In the cells transfected with the miRNA-196a mimic, miRNA-196a expression was significantly upregulated (p<0.001). However, miRNA-196a expression was downregulated in the cells transfected with the ANXA1 overexpression plasmid. In addition, in the cells transfected with the miRNA-196a mimic, cell proliferation, migration and invasion were significantly decreased (p=0.027, p=0.009 and p=0.021, respectively). In the cells transfected with the ANXA1 overexpression plasmid, the expression of Snail was upregulated and that of E-cadherin was downregulated. However, the opposite was observed in the cells transfected with the miRNA-196a mimic. Our findings thus demonstrate that ANXA1 promotes the proliferation of Eca109 cells, and increases the expression of Snail, whereas it inhibits that of E-cadherin, thus enhancing the migration and invasion of ESCC cells. miRNA-196a negatively regulates the expression of ANXA1, thereby inhibiting the proliferation, invasion and metastasis of ESCC cells.

微小RNA-143抑制食管癌细胞株ECA109的增殖、迁移和侵袭

Objective To investigate the functions of microRNA-143 (miR-143) in esophageal cancer cell line ECA109. Methods ECA109 cells were transfected with negative control (NC), miR-143 mimics or miR-143 inhibitors. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to evaluate the growth of ECA109 cells after transfection. Annexin V-FITC/PI apoptosis test kit was used to detect early apoptosis rate in ECA109 cells. Transwell migration and invasion assays were conducted to compare the migration and invasion capacity of ECA109 among different groups. Real-time PCR and Western blotting were used to analyze the mRNA and protein alteration after transfection. Results Three and four days after transfection, compared with NC (absorbance value: 0.90±0.02 and 1.09±0.07), miR-143 mimics inhibited ECA109 cell proliferation (absorbance value: 0.66±0.05 and 0.80±0.04), while miR-143 inhibitors promoted cell proliferation (absorbance value: 1.13±0.09 and 1.51±0.08), with statistical significances (F=49.16, P=0.000; F=100.34, P=0.000). Early-stage apoptosis rates of ECA109 transfected with NC, miR-143 mimics and miR-143 inhibitors were 3.42%±0.72%, 11.63%±1.15% and 0.94%±0.10%, respectively, with statistical significance (F=151.61, P=0.000). Meanwhile, compared with NC (migration cell number: 336±13, invasion cell number: 147±16), miR-143 mimics inhibited cell migration (148±16) and invasion (75±10), while miR-143 inhibitors promoted cell migration (510±14) and invasion (238±16), with statistical significances (F=470.99, P=0.000; F=90.04, P=0.000). Compared with NC (1.00±0.00), miR-143 mimics down-regulated mRNA (relative expression level 0.22±0.08) and protein expression (relative expression level 0.46±0.08) of K-ras, whereas miR-143 inhibitors up-regulated mRNA (1.55±0.12) and protein expression (1.33±0.05) of K-ras (F=131.36, P=0.000; F=88.17, P=0.000). Conclusion miR-143 functions as a tumor suppressor in esophageal cancer cell line ECA109, probably by down-regulating K-ras expression. Key words: MicroRNAs; Esophageal neoplasms; Genes, tumor suppressor

Effect of RAD18-siRNA on proliferation and chemotherapy sensitivity of human esophageal squamous cell carcinoma ECA-109 cells

Objective: To investigate the effect of RAD18-siRNA on cell proliferation and chemotherapy sensitivity of esophageal squamous cell carcinoma (ESCC) ECA-109 cells. Methods: RAD18-siRNA was transfected into human ECA-109 cells by Lipofectamine 3000. Quantitative PCR and Western blot were performed to detect RAD18 and CyclinD1 expression; CCK-8 assay was used to determine cell proliferation and chemotherapy drug sensitivity; flow cytometry was used to determine cell cycle. Correlation between RAD18 and CyclinD1 mRNA expression was analyzed by Pearson's correlation. Results: Compared with non-transfected cells, the expression of RAD18 in RAD18-siRNA group was significantly decreased (P<0.05). The cell proliferation was inhibited (P<0.05) and the cell number of G1 phase was increased, G2/M phase cells decreased (P<0.05) in RAD18-siRNA group. After treatment with different concentrations of cisplatin or 5-FU, the survival rate of the two cell groups was reduced (all P<0.05), and the IC50 of RAD18-siRNA group was significantly lower than that of non-transfected group (P<0.05). The mRNA expression of RAD18 was positively correlated with CyclinD1 expression in ESCC tissues(r=0.478, P<0.01). Conclusion: Down-regulated expression of RAD18 can decrease the cell proliferation and increase chemo-sensitivity of ESCC cells, and CyclinD1 may participate in the process.

Overexpression of Dishevelled-2 contributes to proliferation and migration of human esophageal squamous cell carcinoma.

Dishevelled-2 (Dvl2) was associated with tumor cell proliferation and migration. We aimed to examine the mechanism of Dvl2 in esophageal squamous cell carcinoma (ESCC). Dvl2 was overexpressed in human ESCC tissues and cell lines ECA109 and TE1 cells. CCK-8 and colony formation assay was performed to evaluate the proliferation in ECA109 cells transfected with Dvl2-shRNA. Wound-healing assay and transwell assay were used to examine the activities of migration and invasion in Dvl2-silenced ESCC cells. Knockdown of Dvl2 significantly reduced ECA109 cell proliferation and migration. Moreover, we demonstrated that the proliferation and migration ability of Dvl2 might through the activation of Wnt pathway by targeting the Cyclin D1 and MMP-9. We came to the conclusion that the proliferation and migration effects of Dvl2 might contribute to malignant development of human ESCC.

Downregulation of O-linked N-acetylglucosamine transferase by RNA interference decreases MMP9 expression in human esophageal cancer cells.

O-linked N-acetylglucosamine transferase (OGT) catalyzes O-linked glycosylation (O-GlcNAcylation). O-GlcNAcylation is a post-translational carbohydrate modification of diverse nuclear and cytosolic proteins by the addition of O-linked β-N-acetylglucosamine. It was recently demonstrated that OGT and the level of O-GlcNAcylation are upregulated in esophageal cancer; however, the physiological consequences of this upregulation remain unknown. The current study reports that OGT knockdown by short hairpin RNA (shRNA) did not affect cell viability; however, cell migration in esophageal cancer Eca-109 cells was significantly reduced. OGT-specific shRNA vectors efficiently decreased the protein and mRNA levels of OGT and the RL2 level (a marker of O-GlcNAcylation levels) in Eca-109 esophageal cancer cells. In addition, colony formation and cell proliferation assays demonstrated that OGT-specific shRNA decreased the proliferation of Eca-109 cells; however, there was no significant statistical difference between OGT-specific shRNA and control shRNA. Notably, transwell assays demonstrated that the migratory ability of Eca-109 cells was significantly suppressed following knockdown of the OGT gene. Correspondingly, western blot analyses demonstrated that OGT knockdown significantly downregulated the expression of matrix metalloproteinase 9 (MMP9) in Eca-109 cells. These results suggest that OGT may promote the migration, invasion and metastasis of esophageal cancer cells by enhancing the stability or expression of MMP9.

FGF2 regulates proliferation, migration, and invasion of ECA109 cells through PI3K/Akt signalling pathway in vitro.

Esophageal cancer is one of the most common malignant cancers that arise from esophagus tissues. Fibroblast growth factor 2 (FGF2) has been implicated in multiple biological functions and was considered as an oncogenic factor in tumorigenesis. However, the effects of FGF2 in esophageal carcinoma are yet to be fully elucidated. To better understand the function of FGF2 in esophageal cancer, we used the esophageal cancer cell line ECA109 as a cell model and downregulated FGF2 expression using RNAi; the results showed that insufficient expression of FGF2 inhibited cells proliferation, migration, and invasion of ECA109 cells. Meanwhile, the proliferation, migration, and invasion abilities were stimulated after treatment of exogenous FGF2. In addition, a PI3K/Akt signalling pathway inhibitor (LY294002) alleviated the tumorigenic effects of FGF2. These findings implied that the oncogenic effects of FGF2 was mediated, at least in part, through the PI3K/Akt signalling pathway and FGF2 may be a potential therapeutic target to constrain the tumorigenesis of esophageal cancer.

Tonglian Decoction () arrests the cell cycle in S-phase by targeting the nuclear factor-kappa B signal pathway in esophageal carcinoma Eca109 cells.

To investigate the anti-tumor activity and molecular mechanism of Tonglian Decoction (, TLD) on esophageal carcinoma Eca109 cells. Eca109 cells were treated with TLD and its separated formulae, including the clearing-heat and detoxification formula (Q), activating-blood and promoting-qi formula (H) and nourishing-yin and blood formula (Z). Cell proliferation was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, cell morphology was observed using a microscope, the cell cycle was measured using flow cytometry and the activity of the nuclear factor-kappa B (NF-κB) signal pathway was detected by Western blot. The half maximal inhibitory concentrations of TLD, Q and H were 386, 771 and 729 mg/L, respectively. TLD, Q and H significantly inhibited cell proliferation, with 69.43%, 60.84% and 61.90% of treated cells in the G phase of the cell cycle. The percentage of cells in S phase increased significantly after treatment with TLD, Q, and H compared with the control group (P<0.05), and TLD showed the strongest effect. Z had no influence on the cell cycle compared with the control group (P>0.05). Western blot detection indicated slight differences in the inhibition of the NF-κB pathway by the different formulae. TLD formula strongly inhibited IKKβ, NF-κB, interleukin-6 and tumor necrosis factor-α expression compared with the control group. TLD inhibited Eca109 cell proliferation by arresting cells in S phase. The possible mechanism might be related to inhibiting the NF-κB transduction cascade. The combination of the herbs found in the three separate formulae, H, Q and Z, work synergistically in TLD to produce the inhibitory effects of TLD treatment on Eca109 proliferation.

Secreted recombinant human IL-24 protein inhibits the proliferation of esophageal squamous cell carcinoma Eca-109 cells in vitro and in vivo.

Interleukin-24 (IL-24) displays cancer-specific apoptosis-inducing properties in a broad spectrum of human tumors without harmful effects on normal cells. The human IL-24 protein is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and a potent antiangiogenic molecule. However, the function of secreted recombinant human IL-24 (srhIL-24) protein in esophageal squamous cell carcinoma (ESCC) cells has not been studied. In the present study, we prepared a stable site-specific-integrated cell line, Flp-InTMCHO/IL-24 (FCHO/IL-24), which secreted rhIL-24 at a higher level than three random-integrated cell lines. In vitro, we identified that the purified srhIL-24 inhibited proliferation and induced the apoptosis of ESCC Eca-109 cells and activated STAT3, which was related with the IL-20 receptors. In vivo, the tumorigenicity of Eca-109 cells was significantly inhibited by s.c. injection of FCHO/IL-24 cells. Decreased tumor microvessel density and an increased number of TUNEL-positive tumor cells were associated with tumor growth inhibition, indicating the presence of antiangiogenic activity and induction of apoptotic activity. In summary, the present study demonstrated that srhIL-24 induced growth inhibition and apoptosis in ESCC Eca-109 cells in vitro and in vivo, which may be mediated by the receptor pathway.

MiR141 expression is downregulated and negatively correlated with STAT5 expression in esophageal squamous cell carcinoma.

The aim of the present study was to investigate the association between microRNA-141 (miR141) and signal transducer and activator of transcription 5 (STAT5) expression levels in human esophageal squamous cell carcinoma (ESCC) and to investigate the effects of miR141 on ESCC cells. A total of 45 consecutive patients with ESCC were enrolled in the study. The expression of miR141 in ESCC tissue samples was detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The expression of STAT5 in the ESCC tissues was detected using immunohistochemical staining and western blotting. In addition, Eca109 cells were transfected with miR141 mimic, and the levels of STAT5 were detected using western blotting. The effects of miR141 on the proliferation, invasion and migration of the cells were also detected using MTT, scratch and Transwell invasion assays, respectively. The miR141 expression level in the ESCC tissue samples was significantly decreased compared with that in the adjacent normal tissues (P<0.05). The expression of miR141 in the tissues from patients with lymph node metastasis was significantly decreased compared with that in the tissues of patients without such metastasis (P<0.05). The expression levels of STAT were significantly increased in the ESCC tissues compared with those in the adjacent normal tissues (P<0.05). Furthermore, the levels of STAT5 were significantly increased in the tissues from patients with lymph node metastasis compared with those without such metastasis (P<0.05); however, no statistically significant differences in miR141 expression were observed according to gender, age, tumor size, lesion location, differentiation and invasion (P>0.05). The results suggest that the miR141 mimic significantly inhibited the proliferation, migration and invasion of Eca109 cells in vitro. miR141 and STAT5 expression levels exhibited a negative association in the ESCC tissues, and were both closely associated with the progression of ESCC. Therefore, it appears that miR141 plays an important role in the development, invasion and metastasis of ESCC by regulating the expression of STAT5.

Gene silencing of galectin-3 changes the biological behavior of Eca109 human esophageal cancer cells.

Galectin-3 is a multifunctional β-galactoside-binding lectin that is involved in multiple biological functions which are upregulated in malignancies, including cell growth, adhesion, proliferation, progression and metastasis, as well as apoptosis. A previous study has confirmed the roles of galecin-3 overexpression in the biological behavior of Eca109 human esophageal cancer (EC) cells. In the present study, small interfering (si)RNA-mediated galectin-3 silencing was performed to analyze the effects of decreased galectin-3 expression on the biological behavior of EC cells. Western blot and quantitative polymerase chain reaction analyses were utilized to confirm galectin-3 knockdown at the protein and mRNA level (P<0.05 vs. siRNA-control and untransfected groups). Cell proliferation was assessed using the Cell Counting Kit-8 assay. At 72 and 96 h after transfection, the proliferation of Eca109 cells in the siRNA-Gal-3 group was decreased compared with that in the siRNA-Control and untransfected groups (P<0.001 and P=0.004, respectively). Furthermore, Transwell assays demonstrated that inhibition of galecin-3 significantly reduced the migration and invasion of Eca109 cells compared with that in the other groups (P<0.05). Finally, apoptosis of Eca109 cells was detected using Annexin V/7-amino-actinomycin double-staining and flow cytometric analysis. Galectin-3 knockdown significantly enhanced the apoptotic rate of Eca109 cells compared with that in the siRNA-control and untreated groups (P=0.031 and P=0.047, respectively). In conclusion, following successful knockdown of galecin-3 expression in Eca109 cells, the cell proliferation, migration and invasion were reduced, while the apoptosis was enhanced, which indicates that galectin silencing may represent a therapeutic strategy for EC.

RNA interference for epidermal growth factor receptor enhances the radiosensitivity of esophageal squamous cell carcinoma cell line Eca109.

The present study investigated the effects of small interfering RNAs (siRNAs) specific to the epidermal growth factor receptor (EGFR) gene, on the radiosensitivity of esophageal squamous cell carcinoma cells. EGFR gene siRNAs (EGFR-siRNA) were introduced into esophageal cancer Eca109 cells using Lipofectamine® 2000. The EGFR messenger (m)RNA expression levels, EGFR protein expression and cell growth were assessed using reverse transcription-polymerase chain reaction analysis, western blot analysis and a Cell Counting Kit-8 (CCK-8), respectively. In addition, colony assays were used to determine the inhibitory effects of X-ray radiation on EGFR-silenced cells. EGFR mRNA and protein levels were reduced in the Eca109 cells transfected with EGFR-siRNA. The relative EGFR mRNA expression levels were reduced to 26.74, 9.52 and 4.61% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These mRNA levels were significantly reduced compared with the those of the control group (42.44%; P<0.0001). Transfection with siRNA3 resulted in the greatest reduction in EGFR mRNA expression, with an inhibition rate of 85%. The relative EGFR protein expression levels were reduced to 24.05, 34.91 and 34.14% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These protein levels were significantly reduced compared with those of the control group (78.57%; P<0.0001). Transfection with siRNA1 resulted in the greatest reduction in EGFR protein expression, with an inhibition rate of 72.84%. This reduction in EGFR expression inhibited the proliferation of Eca109 cells, which was identified using the CCK-8 assay. The proliferation inhibition ratio was 28.2%. The cells treated with irradiation in addition to EGFR-siRNA, demonstrated reduced radiobiological parameters (D0, Dq and SF2) compared with those of cells treated with irradiation only, with a sensitization enhancing ratio of 1.5. In conclusion, suppression of EGFR expression may enhance the radiosensitivity of esophageal cancer Eca109 cells and therefore may represent a promising approach for future clinical practice.

β-Elemene inhibits the proliferation of esophageal squamous cell carcinoma by regulating long noncoding RNA-mediated inhibition of hTERT expression.

The study aimed to clarify the relationship between β-elemene, a long noncoding RNA (lncRNA), and human telomerase reverse transcriptase (hTERT) in esophageal carcinoma ECA-109 cells. The proliferation of ECA-109 cells was measured using a CCK-8 kit and flow cytometry. PCR microarray and real-time RT-PCR were designed to determine lncRNA expression in ECA-109 cells before and after treatment with β-elemene. Western blot was used to detect the hTERT level after the differentially expressed lncRNAs in ECA-109 cells were interfered with small interfering RNA (siRNA). On treatment with β-elemene, the proliferation of ECA-109 cells was notably inhibited, and about 85% of the lncRNAs showed higher expression levels in ECA-109 cells than in those untreated cells, from which, CDKN2B-AS1 was screened out. A specific siRNA (si-CDKN2B-AS1) that targets the β-elemene-mediated lncRNA CDKN2B-AS1 was designed, synthesized, and applied to treat ECA-109 cells. Its interference efficiency reached as high as 89.6%. When ECA-109 cells were transfected with the siRNA, the hTERT level was increased by 84.7%. The CCK-8 assay showed that the proliferation of ECA-109 cells treated with β-elemene was significantly promoted after siRNA transfection (P<0.01). It was also shown by flow cytometry that, compared with the scramble-treated group (negative control), the proliferation index value of ECA-109 cells in the si-CDKN2B-AS1 treatment group was notably increased (25.7 vs. 51.7%) and the TERT protein level was increased by 67.25% after the cells were treated with si-CDKN2B-AS1. The chemotherapeutic drug β-elemene suppressed the proliferation of esophageal carcinoma ECA-109 cells by regulating the inhibition of hTERT expression by lncRNA CDKN2B-AS1.

A novel pyrazolone-based derivative induces apoptosis in human esophageal cells via reactive oxygen species (ROS) generation and caspase-dependent mitochondria-mediated pathway

Pyrazolone complexes have strong bio-activity but the anti-tumor mechanism of pyrazolone-based metal complexes is not fully understood. In this study, the inhibitory effect and possible mechanism of a novel pyrazolone-based derivative compound (Cd–PMPP-SAL) on human esophageal cancer cells were investigated. We found that Cd–PMPP-SAL inhibited the proliferation of Eca-109 cells in a dose-dependent manner and induced the apoptosis in the cells. Interestingly, Cd–PMPP-SAL promoted the production of ROS, loss of mitochondrial membrane potential, PARP cleavage and activation of caspase-3/9. These results suggest Cd–PMPP-SAL-induced apoptosis might be mediated by the increased production of ROS and caspase-dependent mitochondria-mediated pathway. These results suggest that Cd–PMPP-SAL is a potential candidate for the treatment of esophageal cancer.

Construction of shRNA-Bmi-1 plasmid and its effect on proliferation of ECA109 cells.

To explore the role of Bmi-1 gene in the proliferation of squamous carcinoma cells and whether the silencing Bmi-1 can inhibit the growth of squamous cell carcinomas cells. The expressions of Bmi-1 in primary cultured Fibroblasts, karatinocyte cell line Hacat,squamous carcinoma cell line A431, and ECA109 were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Recombinant plasmid inserted with Bmi-1 gene short hairpin RNA (shRNA) expression vector PGPU6/GFP/Neo-shBmi-1 was constructed and transfected into ECA109 cells with control set. After transfection for 48 and 72 hours,the mRNA and protein levels of Bmi-1 were examined with RT-PCR and Western blot analysis, respectively. The proliferation of the ECA109 cells was evaluated by MTT method and flow cytometry. Bmi-1 was highly expressed in A431 and ECA109 cells than in Fibroblast cells and Hacat cells. The mRNA and protein expressions of Bmi-1 were significantly silenced in ECA109 cells after recombinant expression vector PGPU6/GFP/Neo-shBmi-1 transfection (P<0.05). Compared with the control groups,the proliferation of ECA109 transfected with PGPU6/GFP/Neo-shBmi-1 was significantly inhibited (P<0.05), and cells in G1 phase increased while in S phase decreased (P<0.05). Bmi-1 is involved in the proliferation of squamous carcinoma cells. After the silencing of Bmi-1 expression,the proliferation ECA109 cells is suppressed due to the altered cell cycle.

Silencing stathmin-modulating efficiency of chemotherapy for esophageal squamous cell cancer with paclitaxel.

Paclitaxel (PTX) is broadly considered the drug of choice for treating human esophageal squamous cell cancer (ESCC). However, PTX resistance often ultimately leads to treatment failure. stathmin, or Op18, is a ubiquitously expressed 19-kDa cytosolic phosphoprotein that can integrate various cellular regulatory signals. stathmin overexpression could lead to resistance to chemotherapeutic agents. In this study we investigated the effect of stathmin gene silencing, using small interfering RNA (stathmin siRNA), on the efficacy of PTX in ESCC. Transfection of stathmin siRNA could significantly inhibit stathmin mRNA and protein levels in ESCC cell lines EC9706 and Eca-109. The silencing of stathmin combined with PTX significantly inhibited the proliferation of EC9706 and Eca-109 cells, with a significantly higher proportion of cells at G2/M phase and this antiproliferative effect was accompanied by an increase in apoptosis rates and morphology changes of EC9706 and Eca-109. Thus, combined chemotherapeutic agent PTX and stathmin siRNA could potentially enhance the therapeutic outcomes of PTX in treating ESCC.