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.     

kai1和nm23基因在食管癌中的表达及其临床意义

目的 观察食管癌组织中kai1和nm23基因mRNA的表达及其与临床病理的关系,探讨两种基因在食管癌发生、发展中的作用.方法 应用原位杂交方法检测kai1和nm23基因mRNA在46例食管鳞癌组织和34例手术远端正常食管组织的表达情况.结果 食管癌中kai1 mRNA表达阳性率为91.3%,明显高于癌旁组织的64.7%,差异有统计学意义(χ2=8.65,P=0.003);nm23mRNA表达阳性率为43.5%,明显高于癌旁组织的5.9%,差异有统计学意义(χ2=13.86,P＜0.001);nm23基因表达与肿瘤病理分期和淋巴结转移相关(r=-0.379,P=0.009;χ2=6.47,P=0.011);食管癌组织中kai1和nm23基因mRNA表达呈正相关(r=0.298,P=0.007).结论 nm23基因表达可能与食管癌的发生、发展及病理分期相关,kai1和nm23联合检测可指导食管癌临床治疗.

Abstract：

Objective To investigate the expression of kai1 and nm23 mRNA in esophageal carcinoma, and to explore the clinicopathological significance of kail and nm23 mRNA in the progression of esophageal carcinoma. Methods To detect mRNA expression of kai1 and nm23 in 46 cases of esophageal carcinoma and 34 cases normal esophageal tissues closely adjacent to carcinoma by in situ hybridization.Results The incidence of kai1 (91.3 %＞64.7 %, χ2 =8.65, P =0.003) and nm23 (43.5 %＞5.9 %, χ2=13.86,P ＜0.001) in esophageal carcinoma was significantly higher than in normal esophageal tissues . The expression of nm23 was correlated with the pathological stages (r =-0.379, P =0.009) and lymph node metastasis (χ2 =6.47, P =0.011). The expression of mRNA had positive correlation between kai1and nm23 in esophageal carcinoma (r =0.298, P =0.007). Conclusion The expression of nm23 may play an important role in the occurrence, development and clinicopathological features of esophageal carcinoma. Combined detection of kai1 and nm23 may be helpful in further directing the clinical therapy of esophageal carcinoma.     

人食管癌相关基因4在食管癌细胞系EC9706中表达缺失的机制

目的 探讨人食管癌相关基因4(ECRG4)在食管癌中表达缺失的机制.方法 采用聚 合酶链反应-单链构象多态(PCR-SSCP)和DNA测序的方法检测80对配对食管鳞状细胞癌(ESCC)肿瘤组织和癌旁正常上皮中ECRG4的外显子突变;采用DNA亚硫酸氧盐修饰和序列特异性聚合酶链反应(ssPCR)检测EC9706细胞系ECRG4基因启动子CpG岛甲基化状态;采用逆转录聚合酶链反应(RT-PCR)检测去甲基化药物5-氮杂-2-脱氧胞苷或三氧化二砷(As2O3)处理后ECRG4 mRNA的重新表达.结果 80对ESCC配对标本中,ECRG4的4个外显子编码区均未发现突变.EC9706细胞系ECRG4基因核心启动子区16个CpG岛中,有11个呈高甲基化状态,ECRG4 mRNA不表达.EC9706细胞处理前ECRG4 mRNA不表达,去甲基化药物处理后,ECRG4 mRNA均重新表达.结论 甲基化表观遗传学机制是导致ECRG4基因在食管癌细胞系EC9706中表达缺失的一个机制.

Abstract：

Objective To investigate the mechanism of loss of human esophageal cancer-related gene 4 (ECRG4) expression in esophageal squamous cell carcinoma (ESCC.) Methods PCR-SSCP and DNA sequencing analysis were used to detect the mutation of ECRG4 exons in esophageal cancer and matched adjacent normal tissues of 80 patients. DNA bisulfite-modifying ssPCR sequencing assay was used to examine the methylation status of ECRG4 promoter in human esophageal squamous cell carcinoma EC9706 cells. The re-expression of ECRG4 mRNA was examined by RT-PCR in EC9706 cells, after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. Results No mutation in the four ECRG4 exons was found in all the ESCC and matched normal adjacent tissues. RT-PCR showed that 11 of 16 CpG islands of ECRG4 promoter were hypermethylated, while ECRG4 mRNA expression level was undetectable in the EC9706 cells. The ECRG4 mRNA was re-expressed after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. Conclusion The epigenetic mechanism of methylation is a reason of loss of ECRG4 gene expression in the ESCC cell line EC9706.     

磷脂酰肌醇3-激酶在食管鳞状细胞癌中的表达及其临床意义

目的 探讨磷脂酰肌醇3-激酶(PI3K)在食管鳞状细胞癌(简称食管鳞癌)中的表达及其临床意义.方法 采用免疫组化SP法检测91例食管鳞癌组织和10例正常食管黏膜组织中PI3K蛋白的表达,采用逆转录聚合酶链反应(RT-PCR)检测30例食管鳞癌组织和癌旁正常组织中PI3KmRNA的表达,并分析PI3K表达与患者临床病理指标及预后的关系.结果 PI3K蛋白在食管鳞癌及正常食管黏膜中的阳性表达率分别为86.8%和10.0%,差异有统计学意义(P＜0.001).PI3K蛋白的表达强度与肿瘤分化程度、侵袭深度及临床分期有关(P＜0.05),而与患者性别、年龄及有无淋巴结转移无关(P＞0.05).肿瘤分化越差,PI3K蛋白表达越强(r=0.351,P＜0.05);肿瘤侵袭程度越深,PI3K蛋白表达越强(r=0.210,P＜0.05);肿瘤临床分期越晚,PI3K蛋白表达率越高(r=0.240,P＜0.05).RT-PCR结果显示,PI3K mRNA在食管鳞癌和癌旁正常组织中的相对表达量分别为0.675±0.029和0.349±0.073,差异有统计学意义(P＜0.05).PI3K mRNA的表达与肿瘤分化程度、侵袭深度和临床分期有关(P＜0.05).单因素回归分析显示,PI3K蛋白表达、分化程度、侵袭深度、有无淋巴结转移、临床分期、有无脉管瘤栓、残端是否阳性与预后相关(均P＜0.05).Cox多因素回归模型分析显示,肿瘤临床分期、侵袭深度、残端是否阳性与患者预后相关(均P＜0.05),PI3K蛋白表达与患者预后无关(x2=1.235,P=0.266).结论 PI3K蛋白和mRNA在食管鳞癌组织中的表达水平明显增高,PI3K基因在食管鳞癌发生及发展中起促进作用.PI3K蛋白表达增强提示食管鳞癌患者预后差,可以作为判断食管鳞癌预后的辅助指标.

Abstract：

Objective To explore the expression of PI3K in esophageal squamous cell carcinoma (ESCC) and its clinical significance. Methods The expression of PI3K proteins was detected by immunohistochemical SP staining in 91 ESCC tissues and normal mucosa of 10 cases. RT-PCR was used to detect the expression of PI3K mRNA in 30 ESCC tissues and adjacent normal mucosa. The relation between PI3K expression and the clinicopathological features and prognosis was analyzed. Results The positive rate of expression of PI3K protein in ESCC was 86.8%, significantly higher than that in normal esophageal mucosa (10.0% ,P ＜0. 001 ). There was a positive correlation between P13K expression and the degrees of differentiation, invasion depth, and clinical stage. No positive correlation was found between the expression of PI3K protein and age, gender and lymph node metastasis (P ＞0.05 ). The poorer is the differentiation of ESCC, the stronger is expression of PI3 K protein ( r = 0.351, P ＜ 0.05 ); the deeper is the invasion depth of ESCC, the stronger is expression of PI3 K protein ( r = 0. 210, P ＜ 0.05 ); and the higher is the clinical stage of ESCC, the stronger expression of PI3 K protein ( r = 0. 240, P ＜ 0.05 ). RT-PCR results demonstrated that the level of PI3K mRNA expression in ESCC was 0. 675 + 0. 029, significantly higher than that in the adjacent normal mucosa (0. 349 ±0.073,P ＜0.05). The high expression of PI3K mRNA was correlated with poor differentiation, deep invasion and clinical stage of ESCC ( P ＜ 0.05 ). The result was consistent with SP staining immunohistochemistry. By Kaplan-Meier analyses, following factors were observed to be significantly associated with prognosis: PI3K expression, the degrees of differentiation, invasion depth,lymph node metastasis, clinical stage, tumor embolus and stump invaded ( all P ＜ 0. 05 ). The multivariate analysis showed that clinical stage ( P ＜ 0.05), invasion depth ( P ＜ 0.05 ) and stump invaded ( P ＜ 0.05 )were significantly correlated with the prognosis. The multivariate analysis of Cox model showed that PI3K was not an independent prognostic indicator of prognosis (x2 = 1. 235, P = 0. 266). Conclusions The expression levels of PI3K protein and mRNA in ESCC tissues are significantly increased and PI3K plays a role in the carcinogenesis and development of ESCC. The enhanced PI3K expression may serve as a reference index indicating a poor prognosis in ESCC.     

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.     

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.     

抑制酰基-辅酶A去饱和酶1表达对食管鳞癌细胞增殖和凋亡的影响及其分子机制探讨

目的:检测酰基-辅酶A去饱和酶1(stearoyl-CoA desaturase-1,SCD-1)在食管鳞癌组织和细胞中的表达,分析其表达下调对食管鳞癌EC1细胞增殖和凋亡的影响,并探讨其相关的分子机制.方法:采用免疫组织化学和原位杂交法检测60例食管鳞癌组织及其相应癌旁正常食管黏膜组织中SCD-1 mRNA和蛋白的表达,实时荧光定量PCR和蛋白质印迹法检测正常食管组织(作为阴性对照)和食管鳞癌细胞株中SCD-1 mRNA和蛋白的表达.不同浓度SCD-1小干扰RNA (small interfering RNA,siRNA)转染食管鳞癌EC1细胞后,采用实时荧光定量PCR和蛋白质印迹法检测SCD-1 mRNA和蛋白的表达,应用CCK-8法检测转染前后EC1细胞增殖的变化,FCM检测转染前后EC1细胞凋亡的改变,蛋白质印迹法检测总Akt、p-Akt、bcl-2和bax蛋白的表达.结果:食管鳞癌组织中SCD-1 mRNA和蛋白表达的阳性率显著高于正常食管黏膜组织(P＜0.05),其表达上调与肿瘤组织分级、TNM分期和淋巴结转移密切相关(P＜0.05).此外,与正常食管组织相比,食管鳞癌EC9706、Eca109和EC1细胞中SCD-1 mRNA和蛋白表达均显著上调(P＜0.05),其中EC1细胞中SCD-1的表达水平最高.50 nmol/L SCD-1 siRNA能显著下调EC1细胞中SCD-1 mRNA和蛋白的表达.SCD-1表达下调可显著抑制EC1细胞的增殖,诱导细胞凋亡;同时,显著上调bax蛋白的表达,并下调p-Akt和bcl-2蛋白的表达,但不改变总Akt的表达水平.结论:SCD-1可能在食管鳞癌的发生和发展中具有重要作用,抑制SCD-1的表达有望成为食管鳞癌重要的分子治疗策略之一.     

组蛋白去乙酰化酶在食管鳞癌中的表达及其表达下调对EC9706细胞生物特性的影响

目的:探讨组蛋白去乙酰化酶(histone deacetylase2,HDAC2)在食管鳞癌组织中的表达,并研究其表达下调对食管鳞癌EC9706细胞增殖、细胞周期和细胞凋亡的影响,以及分析其相关的分子机制。方法:采用免疫组织化学法检测食管鳞癌组织中HDAC2蛋白的表达。将特异性针对HDAC2基因的小分子干扰RNA(small interfering RNA,siRNA)和对照siRNA分别转染食管鳞癌EC9706细胞,实验分3组:未处理组、对照siRNA组和HDAC2siRNA组。蛋白质印迹法检测各组EC9706细胞中HDAC2蛋白的表达。CCK-8计数法检测转染前后细胞的增殖情况。FCM法检测细胞周期和细胞凋亡的变化。蛋白质印迹法检测与细胞增殖、细胞周期和细胞凋亡相关蛋白的表达变化。结果:HDAC2蛋白在食管鳞癌组织中表达的阳性率为79.71%,显著高于癌旁不典型增生组织的51.11%和正常食管黏膜组织的23.19%,3者之间差异具有统计学意义(χ2=44.121,P=0.000);此外,HDAC2蛋白表达与患者的年龄和性别无关(P均>0.05),但是与组织学分级、浸润深度、TNM分期和淋巴结转移均显著相关(P均<0.05)。HDAC2siRNA能有效下调食管鳞癌EC9706细胞中HDAC2蛋白的表达,明显抑制食管鳞癌EC9706细胞的增殖,促使细胞周期静止在G0/G1期,并诱导细胞凋亡。蛋白质印迹法检测结果显示,HDAC2表达下调能明显提高p21和凋亡相关蛋白bax的表达量,同时降低细胞周期蛋白cyclin D1和bcl-2的表达量。结论:HDAC2可能在食管鳞癌的发生、发展中具有重要作用,其表达下调介导的食管鳞癌细胞增殖抑制、细胞周期静止以及细胞凋亡可能与p21、bax的表达升高和cyclin D1、bcl-2表达降低密切相关。     

lncRNA NORAD促进食管鳞状细胞癌EC9706 细胞的增殖和迁移

目的：探讨长链非编码RNA（long non-coding RNA,lncRNA）DNA损伤激活的非编码RNA（non-coding RNA-activated by DNA damage,NORAD）对食管鳞状细胞癌（esophageal squamous cell carcinoma,ESCC）细胞株EC9706 增殖和迁移能力的影响及其机制。方法：采用RT-PCR 法检测不同ESCC细胞（EC9706、TE1、YES-2、KYSE150）中NORAD mRNA表达水平,通过RNA干扰技术将NORAD的小干扰RNA（siRNA）转染到EC9706 细胞（si-NORAD组）以建立NORAD低表达细胞,另设置空白对照组（Ctrl 组,不转染任何序列）及阴性对照组（NC组,转染siRNA 阴性对照序列）,qPCR验证其转染效果。用MTT、平板克隆形成和划痕愈合实验检测敲低NORAD前后EC9706 细胞增殖和迁移能力的变化,Western blotting 检测敲低NORAD前后EC9706 细胞中上皮钙黏蛋白（E-cadherin）、神经钙黏蛋白（N-cadherin）和锌指转录因子Snail 的表达变化。结果：在4 种ESCC 细胞中NORAD mRNA 均呈高表达状态,同时与TE1、YES-2、KYSE150 细胞相比,EC9706 细胞中NORAD mRNA 呈显著高表达（P<0.01）。与Ctrl 组和NC 组比较,转染NORAD-siRNA 后,si-NORAD 组EC9706 细胞中NORAD 表达水平显著降低（均P<0.01）,EC9706 细胞的增殖和迁移能力显著降低（均P<0.05）;敲低NORAD 表达后,EC9706 细胞中E-cadherin 表达升高而N-cadherin 和Snail 表达降低（均P<0.05）。结论：NORAD 在EC9706 细胞中呈高表达状态,敲低NORAD 表达可通过上调E-cadherin、下调N-cadherin 和Snail表达而抑制EC9706 细胞的增殖和迁移能力。     

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。     

EphB1通过PI3K/AKT信号通路促进食管鳞状细胞癌EC-9706细胞增殖、迁移、侵袭并抑制其凋亡

目的:研究促红细胞生成素产肝细胞受体B1(erythropoietin-producing human hepatocellular receptor B1,EphB1)对食管鳞状细胞癌EC-9706细胞增殖、迁移、侵袭及凋亡的影响,并探索其可能的作用机制。方法:qRT-PCR法分析40对食管鳞状细胞癌手术患者的癌组织及邻近正常组织中EphB1表达情况,并分析其临床特征。在EC-9706细胞中分别转染si-EphB1 RNA和oe-EphB1 RNA及其阴性对照。通过qRT-PCR和蛋白免疫印迹实验检测转染效率;CCK-8实验分析EphB1对细胞活力的影响;Hoechst 33258染色和流式细胞学实验检测细胞凋亡;划痕愈合实验及Transwell侵袭实验明确细胞迁移及侵袭能力;蛋白免疫印迹实验检测EphB1蛋白、增殖相关蛋白［增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）］、凋亡相关蛋白［Bad、Bcl-2、Caspase 3 及剪切型-Caspase 3（cleaved-Caspase 3）］、侵袭转移相关蛋白［基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2）、Snail、波形蛋白(Vimentin)、N-cadherin、E-cadherin］以及PI3K/AKT信号通路相关蛋白（PI3K、AKT、p-AKT）的表达水平。结果:EphB1在食管鳞状细胞癌组织及细胞系中高表达(P均＜0.05),EphB1的表达水平与食管鳞状细胞癌患者的TNM分期、有无淋巴结转移和远处转移具有显著相关性(P均＜0.05)。与对照组相比,通过细胞转染技术沉默EphB1和过表达EphB1使EC-9706细胞的增殖、迁移、侵袭能力明显降低或增强,并诱导或抑制细胞凋亡(P均＜0.05)。在蛋白水平上,si-EphB1和oe-EphB1处理EC-9706细胞48 h后,增殖相关蛋白PCNA蛋白表达水平分别显著降低或增加（P均＜0.05）,并分别增加或降低促凋亡相关蛋白Bad、cleaved-Caspase 3的表达水平（P均＜0.05）,下调或上调抗凋亡蛋白Bcl-2、Caspase 3的蛋白表达（P均＜0.05）,抑制或促进侵袭转移相关蛋白MMP-9、MMP-2、Snail、Vimentin、N-cadherin的蛋白活性,但却上调或下调 E-cadherin的表达水平（P均＜0.05）。同时,Western blotting实验结果还证实EphB1可诱导食管鳞状细胞癌EC-9706细胞中通路蛋白PI3K、p-AKT的活性增强（P均＜0.05）。结论:EphB1可能通过调控PI3K/AKT信号通路促进食管鳞状细胞癌EC-9706细胞的增殖、迁移及侵袭,并抑制其凋亡。     

Nuclear expression of Twist promotes lymphatic metastasis in esophageal squamous cell carcinoma

Twist-1 protein (also called Twist) has been suggested to be involved in tumor epithelial-mesenchymal transition (EMT) related progression, however, the mechanism by which twist promotes lymph node metastasis is not fully understood. In the present study, we found that nuclear twist expression is clearly correlated with lymph node (LN) metastasis as determined by immunohistochemistry (IHC). A highly invasive EC109 cell subline, EC109-P, was established by repeated in vitro transwell isolations for the cell model. Immunofluorescence (IF) assay demonstrated that nuclear twist expression was markedly higher in the highly invasive EC109-P cell line when compared with EC109 and EC9706 cells. Based on our cell model, the function and mechanism by which twist regulates LN metastasis in ESCC was investigated. The results showed that the overexpression of Twist could significantly increase the invasion and VEGF-C expression of EC9706 cells, whereas the knockdown of twist expression results in the opposite effects. This finding was further strengthened by the results of the analysis of co-expression of twist and VEGF-C by IHC in ESCC clinical samples. In summary, our study indicates that nuclear twist plays an important role in ESCC lymphatic metastasis by increasing the expression of VEGF-C. The combination of twist and VEGF-C detection could be a reliable prediction of LN metastasis in ESCC.     

小干扰RNA沉默Bcl-2表达增强食管癌细胞放射敏感性研究

目的 探讨Bcl-2基因的特异性小干扰RNA (siRNA)对食管癌细胞(EC9706细胞)Bcl-2蛋白表达和凋亡的影响及放射增敏作用。方法 化学合成Bcl-2基因的siRNA,通过脂质体转染法转入EC9706细胞。流式细胞仪检测转染前后Bcl-2蛋白表达及细胞凋亡,成克隆分析siRNA联合X线照射对EC9706细胞的放射敏感性影响。结果 Bcl-2 siRNA1、Bcl-2 siRNA2、Bcl-2 siRNA3转染后Bcl-2蛋白在EC9706细胞中的阳性表达率分别为25.13％、38.87％、30.55％,较对照组的84.28％明显下降(t =4.01、3.04、3.64,P均＜0.05)。Bcl-2 siRNA1转染组凋亡率为33.86％,明显高于空白对照组的5.51％(t=6.55,P＜0.01)和阴性siRNA1转染组的5.59％(t=6.54,P＜0.01);Bcl-2 siRNA1联合X线照射的细胞凋亡率为56.76％,明显高于单纯照射组的24.51％(t=3.59,P＜0.05)。成克隆分析的Bcl-2 siRNA1转染加照射组D0、Dq、SF2值均低于单纯照射组,放射增敏比为1.33(D0值之比)。结论 Bcl-2基因的特异性siRNA可明显增强EC9706细胞的放射敏感性,具有良好临床应用前景。     

下调HDAC6表达对食管鳞癌细胞周期、细胞迁移的影响及其分子机制探讨

目的:探讨组蛋白去乙酰化酶6（HDAC6）表达的下调对食管鳞癌细胞周期、细胞迁移的影响及其分子机制。方法:将食管鳞癌EC9706细胞分为三组,即未处理组、对照siRNA组和HDAC6 siRNA组,后两组分别转染对照siRNA和HDAC6 siRNA。采用半定量反转录-聚合酶链反应、蛋白印迹方法检测各组细胞中细胞增殖和周期相关因子p21 mRNA和蛋白以及细胞迁移相关因子E-cadherin mRNA和蛋白的表达水平的变化。结果:HDAC6 siRNA组中p21及E-cadherin mRNA和蛋白的表达水平分别是0.440±0.120、0.840±0.070和0.580±0.090、0.450±0.080,且均明显高于未处理组（0.165±0.090、0.090±0.020;0.088±0.009、0.054±0.011）和对照siRNA组（0.163±0.021、0.070±0.040;0.820±0.070、0.066±0.007）中p21及E-cadherin mRNA和蛋白的表达水平,其表达差异均具有统计学意义（P均<0.05）,而未处理组和对照siRNA组之间p21及E-cadherin mRNA和蛋白的表达无差异（P＞0.05）。结论:HDAC6表达下调对食管鳞癌细胞周期的影响可能与p21表达的升高相关;对细胞迁移的影响可能与E-cadherin表达的上调有关。     

血管内皮生长因子、血管生成素在食管鳞状细胞癌组织中的表达及其意义

目的 研究血管内皮生长因子(VEGF)、血管生成素(Ang)-1及Ang-2在食管鳞状细胞癌(ESCC)中的表达及其与预后的关系.方法 用免疫组织化学二步法检测60份ESCC、20份食管癌旁组织及10份正常食管黏膜上皮组织中VEGF、Ang-1及Ang-2的表达,计数微血管密度(MVD).结果 ESCC与食管癌旁组织及食管正常鳞状上皮组织比较,Ang-1表达率显著降低,而VEGF及Ang-2表达率则显著升高(P＜0.05).MVD在Ang-1阴性表达组显著高于阳性表达组(P=0.000);MVD在VEGF、Ang-2阳性表达组显著高于阴性表达组(P＜0.05).Ang-1表达与肿瘤分化程度显著相关(P=0.013).T3、T4期VEGF和Ang-2表达率显著高于T1、T2期(P＜0.05);VEGF及Ang-2在有淋巴结转移者显著高于无淋巴结转移者(P＜0.001).VEGF及Ang-2表达阳性组术后3年生存率低于阴性表达组(P＜0.05),Ang-1表达阳性和阴性组比较,差异无统计学意义(P=0.749).结论 Ang-1表达的降低及Ang-2、VEGF表达的增高可能与食管癌的发生、发展密切相关;VEGF及Ang-2在食管癌中表达提示预后不良.