哈萨克族食管癌患者组织中细胞外信号调节激酶/丝裂原活化蛋白激酶信号通路的表达

目的 探讨细胞外信号调节激酶(ERK)/丝裂原活化蛋白激酶(MAPK)信号通路在哈萨克族食管鳞癌患者组织中的表达变化及意义.方法 采用Western blot技术检测在血清饥饿条件下、U0126梯度浓度处理下,食管癌细胞系EC9706中磷酸化ERK1(p-ERK1)和磷酸化ERK2(p-ERK2)的表达变化.采用实时荧光定量PCR法检测25例哈萨克族食管癌患者肿瘤组织和癌旁正常组织中总ERK1(t-ERK1)和总ERK2(t-ERK2)mRNA的表达变化.采用Western blot技术检测25例哈萨克族食管癌患者肿瘤组织、癌旁正常组织以及5例哈萨克族非食管癌者正常食管组织中t-ERK1、t-ERK2、p-ERK1和p-ERK2蛋白的表达变化.采用免疫组化染色法,在126例石蜡包埋标本(正常食管黏膜19例,食管原位癌55例,食管浸润癌52例)中验证p-ERK1和p-ERK2蛋白的表达变化.结果 在食管癌EC9706细胞中,ERK/MAPK信号通路呈高度活化状态.血清瞬时刺激10 min后,p-ERK1和p-ERK2的表达达到峰值.EC9706细胞在50 μmol/L的U0126处理下,p-ERK1和p-ERK2的表达几乎完全被抑制.t-ERK1 mRNA在25例哈萨克族食管癌患者肿瘤组织中的表达量为1.92±3.49,明显低于癌旁组织(3.67±7.47,P<0.05);t-ERK2 mRNA在食管癌组织和癌旁组织中的表达量差异无统计学意义(P>0.05).t-ERK1和t-ERK2蛋白在食管癌组织、相应癌旁组织和正常食管黏膜组织中的表达差异无统计学意义(P>0.05);但p-ERK1和p-ERK2蛋白在食管癌组织中的表达量(分别为0.87±0.14和0.79±0.10)均明显低于癌旁组织(分别为1.10±0.13和1.32±0.12,P<0.05)和正常食管黏膜组织(分别为1.50±0.22和1.64±0.18,P<0.05).免疫组化染色验证的结果 显示,p-ERK1和p-ERK2蛋白在浸润性食管癌组织中的阳性表达率均为7.7%(4/52),在癌旁正常食管黏膜组织中的阳性表达率均为31.6%(6/19),在食管原位癌组织中的阳性表达率均为85.5%(47/55),不同组织间的表达差异有统计学意义(P<0.05).结论 在食管癌细胞中,ERK/MAPK信号通路呈活化状态.ERK/MAPK信号通路活化水平的改变可能参与了哈萨克族食管癌患者肿瘤的早期发生.

Abstract：

Objective To investigate the expression variation and significance of ERK1/2 MAPK signaling transduction pathway in the pathogenesis of esophageal squamous cell carcinoma (ESCC) in Kazakh patients. Methods The expression level of p-ERK1/2 after serum starvation and treatment with U0126 inhibitor was detected in esophageal cancer cell line EC9706 by Western blot assay. The mRNA level of total ERK1/2 (t-ERK1/2) and expression level of t-ERK1/2 and p-ERK1/2 proteins of 25 pairs of ESCC and adjacent normal esophageal mucosal tissues of Kazakh patients were examined and identified by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. The expression of p-ERK1/2 protein was verified by immunohistochemistry in 126 paraffin-embeded specimens, including 19 normal esophageal mucosa, 55 esophageal carcinomas in situ and 52 invasive carcinomas. Results ERK1/2 MAPK signaling transduction pathway was in an active status in the EC9706 cells. The expression level of p-ERK1/2 in Ec9706 cells reached a peak at 10 min after transient serum stimulation, and p-ERK1/2 expression was totally restrained after the treatment with 50 μmol/L U0126. In the 25 pairs of ESCC and adjacent normal mucosa, the t-ERK1 mRNA level was 1.92±3.49 in the ESCC tissues and 3.67±7.47 in the adjacent normal mucosa. The t-ERK1 mRNA level in ESCC tissues was significantly lower than that in adjacent normal mucosa (P<0.05), whereas there was no significant difference of t-ERK2 mRNA level between them(P>0.05). The expression levels of p-ERK1 and p-ERK2 proteins were 0.87±0.14 and 0.79±0.10 in the ESCC tissues, and 1.10±0.13 and 1.32±0.12 in the adjacent normal mucosae. p-ERK1/2 protein in the ESCC tissues was significantly lower than that in the adjacent normal tissue (P<0.01). However, there was no significant difference between their t-ERK1/2 protein levels (P>0.05). In the 126 cases of paraffin-embeded specimens, positive expressions of both p-ERK1 and p-ERK2 in esophageal cancer tissues were 7.7% (4/52), significantly lower than those in adjacent normal mucosa (31.6%, 6/19) and carcinoma in situ (85.5%, 47/55, P<0.05). Conclusions ERK1/2 MAPK signaling pathway is in an active status in esophageal cancer and adjacent normal mucosa. Our results imply that the activation of p-ERK1/2 MAPK signaling transduction pathway plays a role in the early pathogenesis of ESCC in Kazakh patients.     

CEA siRNA载体的构建和转染人食管癌EC9706细胞的沉默作用

Objective: To construct the small interfering RNA (siRNA) expression vector of carcino-embryonic antigen (CEA) and inhibit the expression of CEA in EC9706 cells by RNA interference. Methods: Two pairs of oligonucleotide sequences were designed and synthesized according to the encoding sequence of mRNA of CEA. The annealed oligonucleotide frag-ments were cloned into pRNAT-U6.2 expression vector and identified by sequencing. The recombinant plasmid pRNAT-U6.2-CEA was transfected into EC9706 cells. The expression of CEA in the stable transfected cells was assayed by real time PCR and Western blot. Results: DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNAT-U6.2 vector, and CEA expression in the transfected cells was down-regulated significantly by pRNAT-U6.2-CEA at both the mRNA and protein levels. Conclusion: The siRNA expression vector of CEA is successfully constructed and inhibits CEA expression in EC9706 cells. This facilitates further studies of the function of CEA at the molecular level.     

胰岛素样生长因子1受体在食管鳞癌组织中的表达及RNA干扰沉默其表达对EC9706细胞增殖能力的影响

目的 探讨胰岛素样生长因子1受体(IGF-1R)在食管鳞癌组织中的表达及其与患者临床特征之间的关系,以及RNA干扰沉默其表达对人食管癌EC-9706细胞体外增殖能力的影响.方法 采用免疫组化法,检测80例食管鳞癌组织和18例正常食管上皮组织中IGF-1R的表达,通过RNA干扰技术沉默EC9706细胞中IGF-1R的表达,通过绘制生长曲线、四甲基偶氮唑蓝(MTT)实验和平板克隆形成试验,观察IGF-1R对细胞体外增殖能力的影响.结果 食管鳞癌组织中IGF-1R表达的总阳性率为86.3%,强阳性率为51.3%;正常食管上皮组织中IGF-1R表达的总阳性率为61.1%,强阳性率为11.1%.食管鳞癌组织中IGF-1R表达的总阳性率和强阳性率均高于正常食管组织(P＜0.01).有淋巴结转移患者组织中IGF-1R表达的总阳性率和强阳性率均高于无淋巴结转移患者(P＜0.01).IGF-1R的表达随肿瘤组织分化程度的增高而降低,差异均有统计学意义(均P＜0.05).不同年龄组间IGF-1R表达差异无统计学意义(均P＞0.05).Ⅲ～Ⅳ期患者组织中IGF-IR表达的总阳性率和强阳性率均高于Ⅰ～Ⅱ期患者(P＜0.01).稳定干扰后的EC9706细胞IGF-1R蛋白表达下降,生长缓慢,细胞倍增时间较实验对照组和空白对照组延长.培养48 h后,稳定转染细胞抑制率为17.3%,高于实验对照组(2.7%,P＜0.01).稳定转染细胞较实验对照组和空白对照组细胞克隆形成能力减弱(P＜0.05).结论 IGF-1R在食管鳞癌组织中呈高表达,与食管鳞癌的发生、转移、分化程度和临床分期相关;RNA干扰沉默IGF-IR的表达,可以使EC9706细胞的体外增殖能力降低.

Abstract：

Objective To explore the correlation of IGF-1R expression with clinical features of esophageal squamous cell carcinoma (ESCC) and to investigate the effect of silencing IGF-1R by siRNA on the proliferation of esophageal cancer cell line EC9706 cells. Methods Immunohistochemistry was used to detect the expresion of IGF-1R in 80 specimens of ESCC and 18 specimens of normal esophageal mucosa.IGF-I R siRNA was transfected into esophageal squamous cell carcinoma EC9706 cells, and the effect of RNAi was assessed by Western blot. The proliferation of EC9706 cells was determined by drawing growth curve, MTT assay and plate colony-forming assay. Results The total and strong positive rates of IGF-1R expression were 86.3% and 51.3% in ESCC, and 61.1% and 11.1% in normal esophageal epithelium,respectively. The total and strong positive rates of IGF-1 R expression in patients with lymph node metastasis were 94.4% and 74.1%, significantly higher than 69.2% and 3.9%, respectively, in those without lymph node metastasis (P ＜ 0. 01 ). A significantly higher IGF-1 R expression was associated with lower histological grade ( P ＜ 0.05 ). The total and strong rates of IGF-1 R expression in 39 patients of stages Ⅲ and Ⅳ were 97.4% and 71.8%, significantly higher than the 75.6% and 31.7%, respectively, in 41 cases of stages Ⅰ and Ⅱ (P ＜ 0. 01 ). IGF-1 R RNAi significantly inhibited IGF-1R expression and the growth of EC9706cells. The clone formation rate of RNAi-IGF-1R transfected cells was 19.1%, significantly lower than that of 52. 3% in non-transfected cells and 49.0% in empty vector-transfected EC9706 cells ( P ＜ 0.05 ).Conclusions The overexpression of IGF-1R is colerated with lymph node metastasis, differentiation and clinical stage. Down-regulation of IGF-1R can inhibit the proliferation of esophageal cancer EC9706 cells in vitro.     

食管鳞癌细胞系核苷酸还原酶亚单位M1的表达水平与细胞对吉西他滨敏感性的关系

Objective Ribanucleotide reduetase subunit M1(RRM1)is the intraeellular target of gemeitabine(GEM).The aim of this study is to explore the relationship between the level of RRMI expression and the sensitivity to GEM in the esophageal squamous cell carcinoma cell lines.Methods Four esophageal sqaamous cell carcinoma cell lines(Kyse-150,Kyse-450,9706 and Eca-109)were cultured in vitro.In the same period,RRM1 expression level was measured by RT-PCR and Western blot,and cell sensitivity to GEM was determined by CCK-8 assay.The relation between cell sensitivity and RRM1 expression was further analyzed.Kyse-450 cells were continuously cultured in the medium containing 50 nM GEM.RRM1 expression was measured at different time points to monitor the dynamic changes in the surviving cells.Inhibition of RRM1 expression by RNAi method was applied and the effect on GEMsensitivity was further examined.Results The IC50 of Eca-109,Kyse-150,Kyse-450 and 9706 cells were (0.92±0.17),(0.48±0.11),(0.29±0.06)and(0.02±0.01)mmol/L,respectively.The expressions of RRMI protein and mRNA of Eea-109 cell line were the highest detected by Western blot and RT-PCR,followed by Kyse-150 and Kyse-450,and the lowest one was 9706 cell line.When Kyse-450 cells were continuously treated with 50 nmol/L GEM,the level of RRM1 protein was increasing in the surviving cells.RRM1 siRNA could effeetively knock down the expression of RRM1 and significantly increase the cell sensitivity to GEM(P = 0.035).Conclusion The level of RRM1 expression corelates with the cell sensitivity to gemcitabine.The cells with a lower level of RRM1 expression are more sensitive to gemcitabine.     

As2O3诱导Raji细胞凋亡的基因分析

Objective The effect of arsenic trioxide on apoptosis gene expression of Raji cell was explored when Raji cells were incubated with 0.5μmol/L of arsenic trioxide for 6h。Methods Cell culture,extraction and isolation of mRNA,preparation of probes labeled with fluorescence,hybridization technique of DNA chip(each chip containing 200 apoptosis genes,Chinese Shanghai Biostar,In.)were used.Results Arsenic trioxide induced significant changes in 10%(20/200 genes)of the apoptosis genes:18 genes were downregulated,only two upregulated.In particular,inhibitors of apoptosis protein,such as X-linked inhibitor of apoptosis protein,were significantly downregulated.P53 and the other apoptosis genes were also downregulatec.Of the upregulated genes,high expression of heat-shock protein could promote apoptosis of Raji cells.Conclusion The inhibitors of apoptosis protein play an important role in the process of arsenic trioxide-induced apoptosis of Raji cells.     

Expression of ECRG4 is an independent prognostic factor for poor survival in patients with esophageal squamous cell carcinoma

In this study, we examined the expression of esophageal cancer-related gene 4 (ECRG4) mRNA and evaluated its clinical significance in esophageal squamous cell carcinoma (ESCC). ECRG4 mRNA expression was quantified by real-time RT-PCR in 63 ESCC and corresponding normal esophageal mucosal samples. ECRG4 mRNA expression levels were significantly lower in ESCC tissues compared with corresponding normal esophageal mucosa (P<0.0001), in patients with locally invasive T2-4 tumors compared with less invasive T1 tumors (P=0.0229) and in stage 4 tumors compared with stage 0-3 tumors (P=0.0120). Furthermore, low ECRG4 mRNA expression levels were associated with significantly shorter survival after surgery compared with high ECRG4 mRNA expression levels (P=0.0150) in ESCC patients. On the basis of multivariate analysis, we conclude that ECRG4 mRNA expression level could be a candidate for an independent prognostic factor for ESCC patients.     

CpG island methylation status of miRNAs in esophageal squamous cell carcinoma

Previous studies on esophageal squamous cell carcinoma (ESCC) indicated that it contains much dysregulation of microRNAs (miRNAs). DNA hypermethylation in the miRNA 5' regulatory region is a mechanism that can account for the downregulation of miRNA in tumors (Esteller, N Engl J Med 2008;358:1148-59). Among those dysregulated miRNAs, miR-203, miR-34b/c, miR-424 and miR-129-2 are embedded in CpG islands, as is the promoter of miR-34a. We investigated their methylation status in ESCC by bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP). The methylation frequency of miR-203 and miR-424 is the same in carcinoma and in the corresponding non-tumor tissues. The methylation ratio of miR-34a, miR-34b/c and miR-129-2 is 66.7% (36/54), 40.7% (22/54) and 96.3% (52/54), respectively in ESCC, which are significantly higher than that in the corresponding non-tumor tissues(p < 0.01). Quantitative RT-PCR analysis in clinical samples suggested that CpG island methylation is significantly correlated with their low expression in ESCC, 5-aza-2'-deoxycytidine (DAC) treatment partly recovered their expression in EC9706 cell line. We conclude that CpG island methylation of miR-34a, miR-34b/c and miR-129-2 are frequent events and important mechanism for their low expression in ESCC. DNA methylation changes have been reported to occur early in carcinogenesis and are potentially good early indicators of carcinoma (Laird, Nat Rev Cancer 2003;3:253-66). The high methylation ratio of miR-129-2 indicated its potential as a methylation biomarker in early diagnosis of ESCC.     

SRY-box 17基因在食管鳞状细胞癌中异常甲基化及表达的研究

目的:检测食管磷癌(esophageal squamous cell cancer,ESCC)细胞株及组织标本中Wnt通路相关因子SRY-box 17基因的甲基化状态及表达情况,探讨其与食管鳞癌发生的相关性.方法:分别采用甲基化特异性-PCR(methylation specific-PCR,MSP)和RT-PCR的方法检测食管癌细胞株TE1、TE13及109例食管鳞癌及相应癌旁非肿瘤组织中SRY-box 17基因的甲基化状态及mRNA表达情况,并分析其与Wnit通路中心因子β-catenin蛋白表达的关系.结果:在食管癌细胞株TE1和TE13中,SRY-box 17基因mRNA均呈阴性或弱阳性表达,用甲基化抑制剂5-氮-2’-脱氧胞苷(5-aza-2’-deoxycytidine,5-Aza-dC)处理后,其mRNA全部恢复阳性表达;MSP检测结果显示,在食管癌细胞株中SRY-box 17基因均呈高甲基化状态;在食管癌组织标本中,SRY-box17基因的甲基率为89.0％ (97/109),明显高于癌旁组织的53.2％ (58/109)(P＜0.01);癌组织中SRY-box 17基因的甲基化率在Ⅲ和Ⅳ期肿瘤患者中明显高于Ⅰ和Ⅱ期患者(P＜0.05),而该基因的甲基化率与肿瘤患者的组织学分级无相关性;在癌组织中该SRY-box17mRNA的阳性表达率为28.4％(31/109),明显低于癌旁组织(P＜0.01).其mRNA表达的缺失率及通路中心因子β-catenin蛋白的异质表达率均与该基因的甲基化状态有相关性(P＜0.05).结论:食管鳞癌组织及细胞株中SRY-box 17基因均呈高甲基化状态,该基因的高甲基化可能是引起mRNA表达下调的重要机制之一,并可能通过Wnt/β-catenin信号转导通路的激活在食管癌的发生、发展中具有重要作用;对该基因的甲基化检测可能对食管癌的预后判断有一定的临床指导意义.     

siRNA靶向沉默MALAT-1基因对人食管癌EC9706细胞体外侵袭迁移的影响

目的:研究长链非编码RNA MALAT-1对食管癌EC9706细胞系迁移、侵袭能力的影响。方法:化学合成针对MALAT-1的siRNA,脂质体法转染EC9706细胞,转染48h后RT-PCR和Western blot检测各细胞MALAT-1在mRNA和蛋白质水平的表达。Transwell实验检测迁移、侵袭能力改变。结果:小干扰RNA下调MALAT-1表达后,RT-PCR和Western blot证实食管癌EC9706细胞中MALAT-1的表达在mRNA水平和蛋白质水平均明显下调,Transwell实验证实食管癌EC9706细胞迁移、侵袭能力下降。结论:抑制MALAT-1表达能够使食管鳞癌细胞的侵袭转移能力明显降低。     

Promoter methylation of heat shock protein B2 in human esophageal squamous cell carcinoma

Hypermethylation of gene promoters and the corresponding loss of gene expression are recognized as a hallmark of human cancer, and DNA methylation has emerged as a promising biomarker for the detection of human esophageal squamous cell carcinoma (ESCC). To identify novel genes methylated in ESCC, we screened 35 candidate genes identified from an oligonucleotide microarray. Among them, the heat shock protein B2 (HSPB2) was methylated in 95.7% (67/70) of primary ESCCs, whereas no methylation was found in normal esophageal tissues from ESCC patients (0%, 0/20). RT-PCR analysis revealed that HSPB2 expression was silenced or weakly expressed in most ESCC cell lines, and re-activated by the demethylating agent 5-aza-2'-deoxy-cytidine. These results indicate that promoter methylation of HSPB2 is one of the causal factors for loss or down-regulation of HSPB2 expression. mRNA expression of HSPB2 in ESCC tissues was significantly down-regulated compared to normal tissues. Our data suggest that promoter methylation of HSPB2 deserves further attention as a novel molecular biomarker in human ESCC.     

Notch1基因在食管鳞癌细胞株EC9706中的表达及其对细胞凋亡的影响

背景与目的:早期的研究显示,Notch1信号途径与肿瘤的发生密切相关,在细胞的生长、增殖、分化和凋亡中起着十分重要的作用.本研究旨在研究Notch1基因在食管鳞癌EC9706细胞中的表达及其对EC9706细胞凋亡的影响.方法:通过免疫细胞化学方法检测Notch1基因在EC9706细胞中的表达.采用RT-PCR技术扩增Notch1基因,构建真核表达载体pcDNA3.1-Notch1(命名为pcNICD),转染EC9706细胞,利用RT-PCR及Western blot检测稳定转染pcNICD 载体、转染pcDNA3.1空载体及未处理的EC9706细胞中Notch1的表达,另通过流式细胞仪检测未处理、转染pcDNA3.1和转染pcNICD的EC9706细胞的凋亡.结果:EC9706细胞中发现Notch1基因的表达.与未处理的和转染pcDNA3.1的EC9706细胞相比,转染pcNICD的EC9706细胞中Notch1基因的mRNA和蛋白表达水平均明显增加,大约增加3倍(P＜0.05);但未处理的和转染pcDNA3.1的EC9706细胞中Notch1的表达差异无统计学意义(P＞0.05).流式细胞检测结果显示,在未处理的和转染pcDNA3.1的EC9706细胞中,细胞凋亡率差异无统计学意义(P＞0.05);但与与未处理的和转染pcDNA3.1的EC9706细胞相比.转染pcNICD的EC9706细胞转染后24 h、48 h和72 h时细胞凋亡率明显增加(P＜0.01).结论:Notch信号途径的激活引起食管鳞癌EC9706 细胞的凋亡.提示Notch1基因有可能成为治疗食管鳞癌的新靶点.     

Expression of esophageal cancer related gene 4 (ECRG4), a novel tumor suppressor gene,in esophageal cancer and its inhibitory effect on the tumor growth in vitro and in vivo

The ECRG4 gene was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no. AF325503). We revealed the expression of ECRG4 protein was downregulated in 68.5% (89/130) ESCC samples using tissue microarray. The low ECRG4 protein expression was significantly associated with regional lymph node metastasis, primary tumor size, and tumor stage in ESCC (p < 0.05). ECRG4 mRNA expression was downregulated in ESCC due to the hypermethylation in the gene promoter. The treatment with 5‐aza‐2′‐deoxycytidine, which is a DNA methyltransferase inhibitor restored ECRG4 mRNA expression in ESCC cells. The result indicated that promoter hypermethylation may be 1 main mechanism leading to the silencing of ECRG4. The high expression of ECRG4 in patients with ESCC was associated with longer survival compared with those with low ECRG4 expression by Kaplan‐Meier survival analysis (p < 0.05). ECRG4 protein was an independent prognostic factor for ESCC by multivariable Cox proportional hazards regression analysis (p < 0.05). The restoration of ECRG4 expression in ESCC cells inhibited cell proliferation, colony formation, anchorage‐independent growth, cell cycle progression and tumor growth in vivo (p < 0.05). The transfection of ECRG4 gene in ESCC cells inhibited the expression of NF‐κB and nuclear translocation, in addition to the expression of COX‐2, a NF‐κB target gene, was attenuated. Taken together, ECRG4 is a novel candidate tumor suppressor gene in ESCC, and ECRG4 protein is a candidate prognostic marker for ESCC. © 2009 UICC