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@#[摘 要] 目的：探讨芝麻酚（SEM）通过腺苷酸活化蛋白激酶（AMPK）/沉默信息调节因子1（SIRT1）/核因子κB（NF-κB）通路影响食管鳞状细胞癌（ESCC）Eca109细胞自噬和凋亡的机制。方法: 用不同浓度的SEM（0、1.562 5、3.125、6.25、12.5、25、50、100、200、400 μmol/L）分别处理Eca109细胞、人食管上皮细胞HEEpiC 48 h，CCK-8法检测细胞增殖率，筛选适宜的SEM浓度用于后续实验。将Eca109细胞分为对照组（CK组，0 µmol/L）、低剂量SEM组（SEM-L组，25 µmol/L）、中剂量SEM组（SEM-M组，50 µmol/L）、高剂量SEM组（SEM-H组，100 µmol/L）、高剂量SEM+Compound C（AMPK抑制剂）组（SEM-H+Compound C组，100 µmol/L+10 µmol/L），所有各组Eca109细胞在对应的药物浓度下处理48 h后，CCK-8法检测Eca109细胞增殖，流式细胞术检测细胞凋亡，透射电镜观察Eca109细胞内自噬小体，WB法检测Eca109细胞中微管相关蛋白1轻链3（LC3）-Ⅱ/LC3-Ⅰ、Beclin-1、B淋巴细胞瘤2（Bcl2）、Bcl2相关X蛋白（BAX）、p-AMPK、SIRT1、p-NF-κB p65的表达。结果: 通过预实验选择SEM实验浓度为25、50、100 μmol/L用于正式研究。在SEM处理下，与CK组比较，SEM-L组、SEM-M组、SEM-H组Eca109细胞的增殖水平（24、48 h）和Bcl2、p-NF-κB p65蛋白表达均显著降低，细胞凋亡率和自噬小体数量、LC3-Ⅱ/LC3-Ⅰ、Beclin-1、BAX、p-AMPK、SIRT1蛋白表达显著升高，且呈剂量依赖性（均P<0.05）；与SEM-H组比较，SEM-H+Compound C组Eca109细胞增殖水平（24、48 h）和Bcl2、p-NF-κB p65蛋白表达均显著升高，细胞凋亡率和自噬小体数量、LC3-Ⅱ/LC3-Ⅰ、Beclin-1、BAX、p-AMPK、SIRT1蛋白表达均显著降低（均P<0.05）。结论:SEM可能通过激活AMPK/SIRT1信号通路而抑制NF-κB活性来促进Eca109细胞自噬与凋亡。
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@#[摘 要] 目的：探讨miR-216b-5p对食管癌Eca109细胞顺铂（DDP）耐药性的影响及其作用机制。方法:采用qPCR法检测miR-216b-5p在食管癌细胞TE-1、KYSE-150、Eca109和耐药细胞Eca109/DDP中的表达水平。利用脂质体转染技术分别将miR-216b-5p mimic及mimic NC、自噬相关蛋白5（ATG5）过表达质粒转染到Eca109/DDP细胞中，用CCK-8、EdU法和FCM分别检测转染后细胞的增殖和凋亡；mRFP-eGFP-LC3双荧光标记实验检测mRFP-eGFP-LC3慢病毒感染后各组细胞自噬发生情况，WB法检测自噬相关蛋白LC3、Beclin 1和P62表达。用荧光素酶报告基因实验验证miR-216b-5p与ATG5的靶向关系，WB法检测ATG5的表达。建立裸鼠Eca109/DDP细胞移植瘤模型，观察miR-216b-5p过表达对移植瘤生长的影响。结果：miR-216b-5p在TE-1、KYSE-150、Eca109和Eca109/DDP细胞中均呈低表达（均P<0.05）。过表达miR-216b-5p可显著抑制Eca109/DDP细胞的增殖并诱导凋亡（均P<0.05），减少细胞中自噬小体数量（P<0.05），下调LC3Ⅱ/LC3Ⅰ比值和Beclin 1蛋白水平、上调P62蛋白水平（均P<0.05）。双荧光素酶报告基因实验证实miR-216b-5p靶向并负调控ATG5的表达（P<0.05），过表达ATG5可使miR-216b-5p mimic对Eca109/DDP细胞增殖、自噬的抑制作用和凋亡的诱导作用明显减弱（均P<0.05），自噬相关蛋白P62表达降低、LC3Ⅱ/LC3Ⅰ比值和Beclin 1表达升高（均P<0.05）。荷瘤实验结果表明，miR-216b-5p过表达可显著抑制裸鼠移植瘤的生长（P<0.05）。结论：miR-216b-5p过表达可逆转食管癌Eca109/DDP细胞对DDP的耐药性，其机制可能与靶向负调控ATG5表达并影响细胞自噬有关。
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Objective: To investigate the effect and its underlying molecular mechanisms of essential oil from Saussurea costus in esophageal cancer cell line Eca109. Methods: The chemical composition of essential oil from Saussurea costus was investigated by gas chromatography-mass spectrometry (GC-MS). The anti-proliferative, anti-migrative, and apoptotic effects of essential oil from Saussurea costus against Eca109 cells were analyzed. Moreover, the expression of proteins associated with cell cycle, metastasis, and apoptosis was determined. Results: GC-MS analysis showed that essential oil from Saussurea costus was predominantly comprised of sesquiterpenes. Saussurea costus essential oil inhibited the viability of Eca109 cells in a dose-and time-dependent manner with IC 50 values of (24.29±1.49), (19.16±2.27) and (6.97±0.86) μg/mL at 12, 24, and 48 h, respectively. The expression levels of target proteins in the cell cycle (phase G 1 /S), including cyclin D1, p21, and p53, were affected by Saussurea costus essential oil. The essential oil also downregulated the expression of metastasis-related proteins MMP-9 and MMP-2. Moreover, it induced apoptosis of Eca109 cells through the mitochondrial pathway, as well as inhibition of STAT3 phosphorylation. Conclusions: The essential oil from Saussurea costus exhibited anti-proliferative, anti-migrative, and apoptotic effects on Eca109 cells, and could be further explored as a potential anti-esophageal cancer agent.
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@# [摘 要] 目的： 探讨长链非编码RNA（lncRNA）LINC01140在食管鳞状细胞癌（esophageal squamous cell carcinoma，ESCC）组织及细胞中的表达及其对Eca109细胞增殖与侵袭的影响及其分子机制。方法：选取2012年3月至2015年5月河北医科大学第四医院收治的133例ESCC患者的临床资料和GEPIA数据库中收集的182例ESCC组织及286例食管正常黏膜组织的LINC01140表达数据，以及ESCC细胞系Kyse150、Eca109和TE13。用qPCR法检测癌组织和细胞中LINC01140的表达水平，分析其表达水平与患者临床病理特征及预后的关系。分别将pcDNA3.1-LINC01140、阴性对照（pcDNA3.1-NC）或miR-452-5p mimic及阴性对照（miR-NC）转染到Eca109细胞，MTS、Transwell实验分别检测细胞的增殖与侵袭能力。用双荧光报告基因实验及TOP/FOP报告基因系统检测LINC01140与miR-452-5p的靶向结合作用及LINC01140对Wnt/β-catenin通路活化水平的影响。结果：LINC01140在ESCC组织和细胞中表达均显著下调（均P<0.01），LINC01140低表达与ESCC患者年龄、淋巴结转移、TNM分期及OS密切相关（均P<0.05）。LINC01140过表达明显抑制Eca109细胞的增殖及侵袭能力（均P<0.01）。机制研究表明，LINC01140可能通过竞争结合miR-452-5p影响Wnt/β-catenin信号通路的活化水平继而调控Eca109细胞的恶性生物学行为。结论：LINC01140通过靶向miR-452-5p/Wnt/β-catenin轴促进ESCC细胞的增殖与侵袭能力，其有望成为ESCC患者靶向治疗的潜在靶点及预后评估的标志物。
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investigate the clinical significance and expression of microRNA(miR)-561 in esophageal squamous cell carcinoma. Methods From January 2015 to February 2016, 96 specimens of adjacent tissues and cancer tissues from patients with esophageal squamous cell carcinoma who underwent surgery in Dongfeng Hospital Affiliated of Hubei University of Medicine were selected to detect the expression level of miR-561. Cell culture experiments were used to detect the expression level of miR-561 in Het-1a, Kyse150 and Eca109 cell lines. The correlation between clinical features and the expression level of miR-561 was counted. The expression level of miR-561 in patients with esophageal squamous cell carcinoma was analyzed. The relationship between the level of miR-561 and prognosis was investigated. Results Relative expression of miR-561 in esophageal squamous cell carcinoma cell lines Eca109 and Kyse150 (1.61 ± 0.30, 1.21 ± 0.28) was significantly lower than that in Het-1a (2.56 ± 0.51), and the difference was statistically significant (P < 0.05). The relative expression of miR-561 in esophageal squamous cell carcinoma tissues was significantly lower than that in adjacent tissues (0.80 ± 0.17 vs. 1.51 ± 0.42), and the difference was statistically significant (P<0.05). The expression of miR-561 in esophageal squamous cell carcinoma was correlated with alcohol drinking history, lymph node metastasis, differentiation degree and tumor stage (P<0.05). Cox multivariate regression analysis showed that the independent risk factors affecting the prognosis of patients were the expression of miR-561, TNM stage and lymph node metastasis (P<0.05). The survival rate of patients with esophageal squamous cell carcinoma whose expression of miR-561 did not decrease at follow-up was significantly higher than that of patients with decreased expression of miR-561 (P<0.05). Conclusions In patients with esophageal squamous cell carcinoma, the expression of miR-561 is low, and the development, occurrence and prognosis of esophageal squamous cell carcinoma are closely related with it.
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Objective@#To investigate the clinical significance and expression of microRNA(miR)-561 in esophageal squamous cell carcinoma.@*Methods@#From January 2015 to February 2016, 96 specimens of adjacent tissues and cancer tissues from patients with esophageal squamous cell carcinoma who underwent surgery in Dongfeng Hospital Affiliated of Hubei University of Medicine were selected to detect the expression level of miR-561. Cell culture experiments were used to detect the expression level of miR-561 in Het-1a, Kyse150 and Eca109 cell lines. The correlation between clinical features and the expression level of miR-561 was counted. The expression level of miR-561 in patients with esophageal squamous cell carcinoma was analyzed. The relationship between the level of miR-561 and prognosis was investigated.@*Results@#Relative expression of miR-561 in esophageal squamous cell carcinoma cell lines Eca109 and Kyse150 (1.61 ± 0.30, 1.21 ± 0.28) was significantly lower than that in Het-1a (2.56 ± 0.51), and the difference was statistically significant (P < 0.05). The relative expression of miR-561 in esophageal squamous cell carcinoma tissues was significantly lower than that in adjacent tissues (0.80 ± 0.17 vs. 1.51 ± 0.42), and the difference was statistically significant (P < 0.05). The expression of miR-561 in esophageal squamous cell carcinoma was correlated with alcohol drinking history, lymph node metastasis, differentiation degree and tumor stage (P < 0.05). Cox multivariate regression analysis showed that the independent risk factors affecting the prognosis of patients were the expression of miR-561, TNM stage and lymph node metastasis (P < 0.05). The survival rate of patients with esophageal squamous cell carcinoma whose expression of miR-561 did not decrease at follow-up was significantly higher than that of patients with decreased expression of miR-561 (P < 0.05).@*Conclusions@#In patients with esophageal squamous cell carcinoma, the expression of miR-561 is low, and the development, occurrence and prognosis of esophageal squamous cell carcinoma are closely related with it.
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@#Objective: To investigate the expression of lncRNA LINC00886 in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines, and its effects on proliferation, migration and invasion of Eca109 cells. Methods: The cancer tissues and corresponding para-cancerous tissues of 69 ESCC patients were collected in the biological specimen bank of the Fourth Hospital of Hebei Medical University from June 2014 to December 2016; the ESCC cell lines Eca109, TE13, TE1, Kyse150, Yes-2 and Kyse170 were also collected. LINC00886 gene expression in ESCC tissues and cell lines was detected by qPCR. Eca109 cells were transfected with pIRES2-LINC00886 and pIRES2-NC, respectively, and the overexpression efficiency of LINC00886 gene in Eca109 cells was detected by qPCR; MTS, clone formation assay, wound-healing assay and Transwell invasion assay were respectively used to detect the effect of LINC00886 over-expression on proliferation, migration and invasion ability of Eca109 cells. Results: The expression of LINC00886 gene in ESCC tissues was significantly lower than that in para-cancerous tissues (P<0.01), and its expression level was associated with tumor TNM stage and lymph node metastasis (both P<0.05). The expression level of LINC00886 gene in ESCC cell lines was also lower than that of the control group (all P<0.01). Compared with control group, the expression level of LINC00886 gene was significantly higher in Eca109 cells transfected with pIRES2-LINC00886 (both P<0.05). Compared with the control group, LINC00886 overexpression significantly inhibited the proliferation, migration and invasion abilities of Eca109 cells (all P<0.01). Conclusion: The decreased expression of LINC00886 gene may be related to the occurrence and development of ESCC. Over-expression of LINC00886 gene inhibits the proliferation, migration and invasion abilities of ESCC cells.
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@#Objective: To investigate the expression of lncRNA01296 in esophageal cancer (EC) tissues and its effect on the proliferation and migration of EC TE-2 cells. Methods:Atotal of 36 pairs of esophageal cancer tissues and corresponding para-cancerous tissues were collected from EC patients admitted to the Department of Thoracic Surgery,Affiliated Hospital of Chengde Medical College from January 2017 to September 2018. The human normal esophageal epithelial (HEEC) cells and human esophageal cancer cell lines ECA109, TE-1 and TE-2 were cultured. qPCR was used to detect the mRNAexpressions of lincRNA01296, SNRPA(small nuclear ribonucleoproteinA) and NGF (nerve growth factor) in EC tissues and cells. Recombinant lentiviral interference vectoror control vector were used to transfect EC cell lines, as sh-lncRNA01296#1,#2 and Mock groups. WB was used to detect the protein expressions of SNRPAand NGF in transfected cells. MTS assay was used to detect cell proliferation, and Transwell assays were used to detect cell invasion and migration of TE-2 cells after transfection. Results: The mRNAexpressions of lncRNA01296, SNRPAand NGF were significantly increased in esophageal cancer tissues and cell lines (all P<0.01), and these expressions in poorly differentiated TE-2 cells were higher than those in highly differentiated ECA109 and TE-1 cells (all P<0.05). The mRNAexpressions of lncRNA01296 and NGF in sh-lncRNA01296#1 and sh-lncRNA01296#2 groups were significantly lower than those in Mock group (all P<0.01), while the mRNAexpression of SNRPAshowed no statistical difference among three groups (P>0.05). The protein expressions of lncRNA01296 and NGF in sh-lncRNA01296#1 and sh-lncRNA01296#2 groups were significantly lower than those in Mock group (all P<0.01). The relative proliferation ability of cells in sh-lncRNA01296#1 and shlncRNA01296#2 groups was significantly lower than that of Mock group at 48 and 72 h after transfection (P<0.05 or P<0.01). The number of invasive cells was (72.0±6.3), (36.6±4.3) and (33.9±3.7) in Mock, sh-lncRNA01296#1 and sh-lncRNA01296#2 groups, respectively; and the number of migrated cells was (85.2±9.9), (47.5±8.1) and (43.8±6.5), respectively, indicating that the numbers of invasive and migrated cells in sh-lncRNA01296#1 and sh-lncRNA01296#2 groups were significantly less than those in Mock group(all P<0.01). Conclusion: lncRNA01296 can up-regulate SNRPAexpression to promote NGF-mediated proliferation and metastasis of EC cells, which may provide new target for the diagnosis and treatment of esophagealcancer.
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@#Objective: To detect the expression of non-receptor protein tyrosine phosphatase 6 (PTPN6) in different esophageal squamous cell carcinoma (ESCC) cell lines, and to investigate its effect on proliferation, migration and invasion ability of Eca109 and Yes-2 cell lines. Methods: qPCR was applied to detect the mRNA expression of PTPN6 in different ESCC cell lines (TE1, Eca109, Kyse150, Kyse170 and Yes-2). pcDNA3.1-PTPN6 plasmid was transiently transfected into Eca109 and Yes-2 cells respectively. The expression of PTPN6 was detected by real-time PCR and Wb. The effects of PTPN6 over-expression on the biological behaviors of ESCC cells were detected by MTS, colony formation assay, wound healing assay and Transwell assay, respectively. Results: The mRNA expression of PTPN6 was remarkably reduced in ESCC cell lines (TE1, Eca109, Kyse150, Kyse170 and Yes-2) compared to normal esophageal epithelial cells (HEEpiC) (P<0.05). Compared to the mock cells, significant up-regulation of PTPN6 was detected in pcDNA3.1-PTPN6 transfected Eca109 and Yes-2 cells (all P<0.05 or P<0.01); PTPN6 over-expression led to a significant inhibition in migration and invasion ability of Eca109 and Yes-2 cells (all P<0.05). Conclusions: Over-expression of PTPN6 may inhibit the proliferation, migration and invasion of ESCC cells, which might be an important factor influencing the biological characteristics of ESCC cells.
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@#Objective: To investigate the effects of ezrin enhancer knockout on ezrin gene expression, cell proliferation and migration of human esophageal carcinoma Eca-109 cells. @*@#Methods: The CRISPR/Cas9 recombinant plasmids targeting upstream/downstream of human ezrin enhancer were co-transfected into human esophageal carcinoma Eca-109 cells, and the cell line Eca-C2 with ezrin enhancer knockout was screened by purinomycin. Then the expression levels of ezrin mRNAand protein in Eca-C2 cells were detected by Real-time quantitative PCR (qPCR) and Western blotting, respectively; The expression levels of MAPK-pathway-related proteins were detected by protein array technology; and the effects of ezrin enhancer knockout on the proliferation and migration of Eca-C2 cells were analyzed by WST-1 method and wound-healing assay, respectively. @*@# Results:The human esophageal carcinoma cell line Eca-C2 with stable ezrin enhancer knockout was established successfully. Compared with control cells, the mRNA and protein expressions of ezrin in Eca-C2 cells were significantly reduced (all P<0.05).Among the 17 detected MAPK pathway related proteins in Eca-C2 cells, 9 proteins (AKT, CREB, GSK3b, MKK6, mTOR, P38, P53, P70S6K and RSK1) were down-regulated, and the cell proliferation and migration were significantly inhibited (all P<0.05).@*@# Conclusion: ezrin enhancer knockout can significantly inhibit the cell proliferation and migration of human esophageal carcinoma Eca-109 cells.
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Objective: To investigate effects of cinnamaldehyde (CA) on the proliferation and apoptosis of oesophageal squamous cell carcinoma Eca109 cells and to explore its mechanism. Methods: The effects of different concentrations (1.88, 3.75, 7.50, 15.00, 30.00 μg/mL) of CA on the proliferation of Eca109 cells were detected by MTS assay; Morphological changes were observed by phase contrast microscope; Flow cytometry was used to detect cell cycle distribution and apoptosis rate of Eca109 at different concentrations of CA; The expression of apoptosis-related protein Caspase-3/Caspase-9, pro-apoptosis protein Bax and anti-apoptosis protein Bcl-2 and Mcl-1 in ECA109 cells treated with different concentrations of CA for 24 h was detected by Western blotting. Results: After treated by CA with a concentration of 15.00, 30.00 μg/mL for 24 h, compared with the control group, the cell proliferation of Eca109 cells was significantly inhibited [(42.91 ± 2.15)%, (36.04 ± 2.97)% vs (100.00 ± 0.00)%, P < 0.05]; After treated by CA, Eca109 cells showed obvious apoptotic morphological changes; After treated with different concentrations of CA for 24 h, the proportion of cells in G0/G1 phase was decreased significantly (P < 0.05), while the proportion of cells in G2/M phase was increased significantly (P < 0.05); After treated by CA for 24 h, the apoptosis rate of Eca109 cells was increased significantly [(4.3 ± 0.11)%, (4.8 ± 0.07)%, (9.1 ± 0.13)% vs (1.0 ± 0.03)%, P < 0.05]; Compared with the control group, the fragment of protein Caspase-3 and Caspase-9 was significantly cleaved in Eca109 cells treated with different concentrations of CA for 24 h (P < 0.05); The expression levels of anti-apoptotic protein Bcl-2 and Mcl-1 were significantly decreased (P < 0.05), while the pro-apoptotic protein Bax expression was significantly up-regulated (P < 0.05). Conclusion: Cinnamaldehyde can inhibit the proliferation of oesophageal squamous cell carcinoma Eca109 cells and promote its apoptosis. The mechanism may be associated with the up-regulation of Caspase-3, Caspase-9, pro-apoptotic protein Bax and down-regulation of anti-apoptotic protein Bcl-2 and Mcl-1.
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Objective: To investigate the inhibitory effect of nitidine chloride (NC) on human esophageal carcinoma cell line Eca109 and the molecular mechanism of its induction. Methods: CCK-8 method was used to detect the inhibition rate of human esophageal cancer Eca109 cells with different concentrations of NC and different intervention time. According to the result of CCK-8 method, the experiment was divided into four groups, and the concentrations of NC in each group were 0, 5, 10, and 15 μmol/L, respectively, and the drug action time was 48 h. The apoptotic rate was detected by flow cytometry with Annexin V-FITC/PI double staining. The mRNA expressions of Caspase-3, Caspase-9, and Noxa were detected by qRT-PCR. The expressions of cleaved Caspase-3 and Bcl-2, p53, and Noxa protein levels were detected by Western blotting. Results: NC had inhibitory effect on Eca109 cells in a certain range of time and dose-dependent manner. Flow cytometry showed that NC at 5 μmol/L mainly induced early apoptosis (P < 0.01); NCs at 10 and 15 μmol/L mainly induced late apoptosis (P < 0.01). qRT-PCR results showed that the mRNA expression of Caspase-3, Caspase-9 and Noxa was increased with the increase of NC concentration, of which 10 and 15 μmol/L group increased significantly. The results of Western blotting showed that the protein levels of cleaved Caspase-3, p53, and Noxa were both increased with the increase of NC concentration (P < 0.01), but the increase of Noxa was not significant in 5 μmol/L group (P < 0.01). The expression of Bcl-2 protein was decreased with the increase of NC concentration, and which was significantly higher in 10 and 15 μmol/L groups (P < 0.05, P < 0.01). Conclusion: The inhibitory effect of NC on human esophageal carcinoma Eca109 cells is mainly through apoptosis. The apoptosis of NC induced of Eca109 cells is associated with increased expression of p53 and Noxa, downregulation of Bcl-2, and activation of Caspase-3.
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Objective To discuss the effect of α-asarone on the expression level of Cyt-c,Smac,Caspase3 mRNA and protein in human esophageal carcinoma Eca-109 cell mitochondria. Methods The Eca-109 cells were cultured in vitro,and divided into the negative control group and the α-asarone treatment groups(final concentration:25,50,100 μg·mL-1).After 48 h,the morphological changes of Eca-109 cells were observed by fluorescence inversion microscope.The total RNA of cells were extracted by TRIzol method,the expressions of Cyt-c、Smac and Caspase3 were measured by RT-PCR and Western blotting. Results After Eca-109 cells were treated with different concentrations of α-asarone for 48 h,and obvious changes in the morphology were observed,the expressions of Cyt-c,Smac and Caspase3 genes and protein were increased significantly compared to the negative control group( P<0.05). Conclusion α-asarone can induce the human Eca-109 cells apoptosis by regulating expressions of mitochondrial apoptosis pathway correlation genes such as Cyt-c,Smac and Caspase3.
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@# Objective: To investigate the expression of long non-coding RNA NUP50-AS1 (lncRNA NUP50-AS1) in esophageal squamous cell carcinomas (ESCC) tissues and cell lines, and to explore its effect on proliferation, migration and invasion of human esophageal cancer Eca109 cells. Methods: 49 pairs of ESCC tissues and corresponding para-cancerous tissues obtained from the Biological Specimen Base of the Fourth Hospital of Hebei Medical University during Jan. 2015 to Jan. 2016 were used in this study. qRT-PCR method was applied to detect the expression of NUP50-AS1 in collected tissues samples and five esophageal cancer cell lines (TE1, TE13, Eca109, Kyse150 and Kyse170). ShRNAs were transiently transfected into Eca109 cells to interfere the expression of NUP50AS1 gene, and finally, sh2-NUP50-AS1 was used for the following experiments. The effect of NUP50-AS1 gene knockdown on the proliferation of Eca109 cells was detected by MTS and colony formation assay; the effect of NUP50-AS1 gene knockdown on the migration of Eca109 cells was detected by scratch test, and the effect on cell invasion was detected by Transwell assay. Results: The expression of NUP50-AS1 in ESCC was correlated with the lymphnode metastasis and TNM stage (all P<0.01). The expression of NUP50AS1 in ESCC tissues was significantly higher than that in corresponding normal tissues (2.003±0.870 vs 1.000±0.000, P<0.05). The expression of NUP50-AS1 in five esophageal cancer cell lines was significantly up-regulated (P<0.05), and it had the highest expression in Eca109 cell line. After transfection, sh2-NUP50-AS1 had the highest transfection efficiency, and knocking down NUP50-AS1 gene significantly inhibited the proliferation, invasion and migration of the Eca109 cells. Conclusion: The expression of lncRNA NUP50AS1 in ESCC tissues was significantly higher than that in the para-cancerous tissues, and correlated with the TNM stage and lymphnode metastasis. The down-regulation of NUP50-AS1 inhibited the proliferation, invasion and migration of esophageal cancer cells. The high expression of NUP50-AS1 gene may be closely related to the occurrence and development of ESCC.
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Objective To investigate the radiosensitization effect of stattic on the xenograft of esophageal squamous cell carcinoma ( ESCC ) ECA109 cells in nude mouse and explore the underlying mechanisms. Methods Male nude mice inoculated subcutaneously with ECA109 cells were used to examine the radiosensitization effect of stattic in vivo. When average volume of tumors was about 150 mm3 , mice were randomly divided into 4 groups:control group, 25 mg/kg stattic alone group, ionizing radiation (IR) (6 Gy) alone group, and 25 mg/kg stattic plus IR group. During the term of treatment, the volume of tumors was measured every 2 days. On 25 days after treatment, the mice were killed and the expressions of pSTAT3, STAT3, HIF-1α and VEGF in ECA109 xenografts were assessed by Western blot and immunohistochemistry analysis. Results The volume of tumor in nude mice was (705. 1 ± 75. 5) mm3 in stattic plus IR group, which was significantly smaller than that in IR group (113. 5 ± 101. 4) mm3 and stattic alone group(1696.5 ±100.6)mm3(t=4.35, 14.14,P<0.0). The inhibition rate was (66.1 ± 3. 2)％ in stattic plus IR group. The expression levels of pSTAT3, HIF-1α and VEGF were obviously decreased in the stattic plus IR group compared with other groups (t=17. 07, 5. 05, 3. 54, P<0. 05). Conclusions Stattic play a radiosensitization role in the xenograft of esophageal carcinoma in nude mice probably by inhibiting the signaling pathways of pSTAT3, HIF-1α and VEGF.
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Objective To investigate the effects of inhibition of MDC1 protein expression on xenografted tumors in nude mice,and to observe the histopathological and cellular changes in nude mice.Methods Three pairs of effective and control short hairpin RNA targeting MDC1 mRNA were designed and cloned into the pSIH1-H1-copGFP vector.Real-time PCR and Western blot were used to determine the mRNA and protein expression of MDC1.After selection by copGFP reporter gene,cells were divided into negative transfection group (ECA109-N) and MDC1 transfection group (ECA109-M).The transfected cells were injected into nude mice.The mice were divided into ECA109 group,ECA109-N group,and ECA109-M group.Each group was divided into irradiation subgroup and non-irradiation subgroup.The changes in tumor size after irradiation were evaluated in each group.Western blot was used to measure the expression of CHK1,CHK2,and CHK2T68 in xenografted tumors.Flow cytometry was used to analyze the cell cycle distribution and apoptosis of tumor cells in nude mice.The variance analysis was used to compare the mean of multiple groups,and the SNK-q test was used in the two two groups.Results The pMDC1-shRNA plasmid was successfully constructed and used to transfect ECA109 cells.ECA109-M cells were obtained by stable transfection with the recombinant plasmid.All inoculated nude mice survived with visible xenografted tumors at the underside of the paw in about one week.There was no swelling and wound in inoculation sites.There was no significant difference in tumor size between different groups (P＞0.05).The tumor growth in the ECA109 group and the ECA109-N group significantly slowed down after irradiation with a dose of 15 Gy (P＜0.05).Compared with the other two groups,the ECA109-M group had a significant smaller tumor size,significantly slower relative tumor growth,and significantly higher growth inhibition (all P＜0.05).The q value of the ECA109-M group was 1.36.In the ECA109-M group,there were no significant changes in the protein expression of CHK1 and CHK2 after irradiation (P＞ 0.05);however,the phosphorylation of CHK2T68 protein was significantly reduced after irradiation (P＜0.05).There were no significant differences in cell cycle distribution or the proportion of apoptotic cells in tumor tissue between the three groups (P＞0.05).Conclusions Inhibition of MDC1 protein expression by RNA interference can effectively inhibit the growth of xenografted tumors after irradiation in the nude mice by increasing their radiosensitivity.
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Objective Studies show that miRNA plays an important role in regulating the development, metastasis, inva-sion, and angiogenesis of carcinoma.This study aimed to investigate the expression of miR-155-5p in esophageal squamous cell carci-noma ( ESCC) and its role in the occurrence and progression of ESCC. Methods We collected 20 surgical specimens of ESCC and another 20 from the adjacent normal tissue at least 5 cm from the lesion and determined the expression of miR-155-5p in the tissues by qPCR.We transfected the miR-155-5p mimic, inhibitor and negative control plasmid into the Eca109 cells, measured the proliferation and apoptosis of the cells by CCK8 assay and flow cytometry, and examined their migration using the scratch test. Results The ex-pression of miR-155-5p was significantly up-regulated in the ESCC tissue as compared with that in the normal tissue (529.42% vs 100%, P<0.05).The scratch test showed a markedly increased migration of the cancer cells when miR-155-5p was overexpressed. No remarkable differences were observed in the apoptosis rates of the cells transfected with the miR-155-5p mimic ([5.43 ± 3.09]%), inhibitor ([5.28 ±1.98]%), and negative control plasmid ([5.67 ±1.99]%) (P<0.05). Conclusion The ex-pression of miR-155-5p is significantly up-regulated in the ESCC tissue, which may act as a potential maker for the diagnosis of ESCC. The up-regulated expression of miR-155-5p does not significantly in-fluence the proliferation and apoptosis but enhances the migration of Eca109 cells, which may be related to early metastasis of ESCC.
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<p><b>OBJECTIVE</b>To investigate the anti-tumor activity and molecular mechanism of Tonglian Decoction (, TLD) on esophageal carcinoma Eca109 cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Subject(s)
Humans , Blotting, Western , Cell Count , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Cell Shape , Drugs, Chinese Herbal , Pharmacology , Esophageal Neoplasms , Metabolism , Pathology , Flow Cytometry , Inhibitory Concentration 50 , NF-kappa B , Metabolism , S Phase , Signal TransductionABSTRACT
Objective:To investigate the effects of epidermal growth factor (EGF)on cell cycle and cell cycle-related regulatory factors of human esophageal squamous cell carcinoma (ESCC) cell line Eca109.Methods: Serum starved Eca109 cells were treated with 20 ng/ml recombinant human EGF(rhEGF)for 24 h.The cell cycle phase distribution was detected by flow cytometry.The mRNA and protein expression levels of p21CIP1/WAF1(p21) and p27KIP1(p27) were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot,respectively.Results: The proportions of G1 phase cells in EGF group and control group were ( 54.90 ±0.82 )% and ( 65.94 ±0.74 )%.The mRNA and protein expression levels of p 21 in EGF group was significantly higher ,and p27 was significantly lower than that in control group ( P<0.01 ) .Conclusion: EGF facilitates G1-S phase transition,and promotes the proliferation of Eca 109 cells,which may be associated with the up-regulation of p21 and down-regulation of p27.
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Objective To investigate the expression of Nek8 in esophageal squamous cell carcinoma(ESCC) tissues and cell line , and to evaluate its correlation with the clinicopathological features of ESCC and the survival rate of ESCC patients after operation . Methods The expression of Nek8 mRNA in human ESCC Eca109 cell line and two pairs of ESCC tissues and adjacent normal e‐sophageal mucosal epithelium were detected by semi‐quantitative reverse transcription polymerase chain reaction (RT‐PCR) .Immu‐nohistochemistry and tissue microarray technique were used to examine the expression of Nek 8 protein in ESCC tissues and tumor‐adjacent tissues .The correlation between Nek8 expression and clinicopathological features of ESCC and survival rate of ESCC pa‐tients was then analyzed .Results The expression of Nek8 mRNA was positive in Eca109 cells and two cases of ESCC tissue ,and it was negative in paired normal esophageal mucosal epithelium specimens .In tissue microarray ,the expression of Nek8 protein in ES‐CC tissues ,which was mainly in the cytoplasm ,was significantly higher than that in tumor‐adjacent tissues(P= 2 .16E‐13) .The high expression of Nek8 was associated with tumor size (P=0 .008) ,but not with sex ,age ,histological grade ,infiltration degree , lymph node metastasis ,and the survival rate(P>0 .05) .Conclusion The expression of Nek8 is up‐regulated in ESCC tissues and cell line ,and may be involved in tumorigenesis and development of ESCC .Nek8 could act as a potential biomarker for ESCC diag‐nose and target for therapy .