食管鳞癌中TGF-β1与上皮—间质转化的研究
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摘要
食管癌是一种高侵袭性的肿瘤，世界范围内，食管癌患者死亡率占肿瘤患者死亡率的第六位，我国是食管癌的高发国家，河南省是高发省份。随着治疗手段的发展，食管癌的预后有所改善，但是其五年生存率仍然不容乐观。浸润转移是引起食管癌患者死亡的主要原因，因此，对食管癌浸润转移机制的探讨已成为研究热点之一。
近来研究发现，在多细胞生物胚胎发育过程中的胚层和组织重建中存在一种现象：上皮-间质转化(epithelial-mesenchymal transition，EMT)。最近研究认为它不仅存在于多细胞生物的胚胎发生过程中，同时也存在于多种慢性疾病以及肿瘤的进展过程中。Thiery这样描述上皮性肿瘤发生发展过程中的EMT：正常上皮细胞沿基底膜生长，通过表型遗传学的改变或基因突变，细胞发生转化生成原位癌(此时基底膜仍然保持完整)；继续发展，癌细胞通过EMT形式局部扩散，基底膜变脆，细胞侵入淋巴管和血管，发生转移；在种植部位，癌细胞还可通过间质-上皮转化形成转移瘤。上皮细胞发生EMT，以间质特性获得、上皮细胞极性丧失及运动能力增强为主要特征。这些特点与肿瘤细胞的原位侵袭和远处转移密切相关。目前关于食管鳞癌中是否存在EMT及EMT在食管鳞癌浸润转移中的作用尚未见报道。
EMT是一个极其复杂的过程，包括：(1)细胞间连接(黏附连接、紧密连接、桥粒连接)解体，细胞分散；(2)细胞骨架重排，形成细胞-基质黏附，细胞运动能力增强。(3)细胞基因型的改变：在EMT过程中表达上调的因子主要包括间叶细胞标志物如纤维连接蛋白(fibronectin)、波形蛋白(Vimentin)、神经钙黏素(N-cadherin)等，以及诱导EMT的细胞因子和转录调节因子，如snail、slug，转化生长因子-β(transforming growth factor-β，TGF-β)，成纤维细胞生长因子(fibroblast growth factor，FGF)；还有对诱导EMT有辅助作用的基质金属蛋白酶-2(MMP-2)，基质金属蛋白酶-9(MMP-9)等。在EMT过程中表达下调的因子主要包括上皮细胞的标志物如上皮钙黏素(E-cadherin)，β-连环素(β-catenin)，γ-连环素(γ-catenin)，细胞角蛋白18(cytokeratin18)等。已有研究发现TGF-β在诱发EMT中起关键作用。
有关食管鳞癌中是否存在EMT以及食管鳞癌中TGF-β1引发的EMT是否与食管癌的浸润转移有关，目前尚未见报道。本研究探讨了食管癌组织及食管鳞癌细胞系EC9706中TGF-β1的表达及其与E-cadherin及Vimentin的关系；采用针对TGF-β1的反义寡核苷酸干扰技术，抑制食管鳞癌细胞EC9706中TGF-β1的转录活性，探讨抑制TGF-β1对E-cadherin及Vimentin表达的影响，观察转染反义寡核苷酸对细胞形态学的影响及对细胞运动能力的影响，从而探讨食管鳞癌细胞中TGF-β1诱导的EMT；并探讨TGF-β1对食管鳞癌细胞EC9706增殖及凋亡的影响。探讨食管鳞癌浸润转移的分子机制，为食管癌浸润转移的阻断治疗提供理论基础。
材料和方法
1．采用免疫组织化学技术检测了100例手术切除的食管鳞癌组织及其癌旁“正常”粘膜中TGF-β1、E-cadherin和Vimentin蛋白的表达情况。
2．采用RT-PCR、免疫细胞化学、流式细胞术对食管鳞癌细胞系EC9706中的TGF-β1、E-cadherin和Vimentin蛋白及mRNA表达进行了检测。
3．针对TGF-β1第一启动子的核苷酸片段设计合成反义寡核苷酸(ASODN)，采用阳离子聚合物瞬时转染培养的人食管鳞癌EC9706细胞。
①观察TGFβ1-ASODN转染后细胞形态学的变化。
②采用RT-PCR、免疫细胞化学方法、流式细胞术方法观察TGFβ1-ASODN转染后细胞中TGF-β1mRNA及蛋白表达的变化。
③采用RT-PCR、免疫细胞化学、流式细胞术方法观察TGFβ1-ASODN转染后细胞中E-cadherine与Vimentin mRNA及蛋白表达的变化。
④采用细胞划痕试验检测TGFβ1-ASODN转染后细胞迁移能力的变化。
4．采用流式细胞术、MTT观察转染TGFβ1-ASODN后，对细胞增殖、凋亡及细胞周期的影响。
5．统计学处理：所有数据均经SPSS13.0软件进行统计分析。计数数据采用阳性率表示，阳性率之间的比较采用χ~2检验(Chi-square)，阳性率间相关性采用Kendall相关进行比较；计量数据采用(?)±S表示，两组均数的比较用t检验(t-test)，两组以上均数的比较用方差分析(ANVOA)。显著性水平α=0.05。
结果
1．TGF-β1蛋白在食管鳞癌中的表达(85.0％)明显高于其在癌旁“正常”粘膜中的表达(27.0％)(P＜0.01)，其在深层浸润组中的表达(90.6％)高于其在浅层浸润组中的表达(75.0％)(P＜0.05)。
2．E-cadherin蛋白在食管鳞癌中的表达(43％)明显低于其在癌旁“正常”粘膜中的表达(85％)(P＜0.01)；Vimentin蛋白在食管鳞癌中的表达(23％)高于其在癌旁“正常”粘膜中的表达(0％)(P＜0.05)。
3．在食管鳞癌组织中，TGF-β1的表达与E-cadherin的表达呈负相关关系(T_b=-0.257，P＜0.05)；TGF-β1的表达与Vimentin的表达呈正相关关系(T_b=0.163，P＜0.05)；Vimentin的表达与E-cadherin的表达呈负相关关系(T_b=-0.379，P＜0.01)。
4．RT-PCR结果显示EC9706细胞系中可检测到TGF-β1mRNA(0.43±0.09)、E-cadherin mRNA(0.22±0.06)、Vimentin mRNA(0.89±0.09)的表达；免疫细胞化学结果显示在EC9706中存在TGF-β1蛋白(43.57％)、E-cadherin蛋白(12.53％)和Vimentin蛋白(17.97％)的表达；流式细胞术结果也显示在EC9706中存在TGF-β1蛋白(41.4％)、E-cadherin蛋白(12.2％)和Vimentin(17.8％)蛋白的表达。
5．转染TGFβ1-ASODN后，RT-PCR结果表明，TGF-β1mRNA的表达水平(0.25±0.681)明显低于转染前(0.43±0.09)(P＜0.01)；免疫细胞化学结果表明，转染后TGF-β1蛋白的表达(35.07％)明显低于转染前(43.57％)(P＜0.01)，且细胞浆着色减弱；流式细胞术结果表明，转染后TGF-β1蛋白的表达(35.4％)明显低于转染前(41.38％)(P＜0.01)。
6．RT-PCR结果表明，转染TGFβ1-ASODN后，E-cadherin mRNA的表达(0.38±0.09)明显高于转染前(0.22±0.06)(P＜0.01)；Vimentin mRNA的表达(0.73±0.07)低于转染前(0.89±0.09)(P＜0.05)。免疫细胞化学结果表明，转染后，E-cadherin蛋白的表达(17.13％)明显高于转染前(12.53％)(P＜0.01)；Vimentin蛋白的表达(14.15％)明显低于转染前(17.97％)(P＜0.01)。流式细胞术结果表明，转染后E-cadherin蛋白的表达率(17.8％)明显高于转染前(12.2％)(P＜0.01)；Vimentin蛋白的表达(14.9％)明显低于转染前(17.8％)(P＜0.01)。
7．转染后细胞形态变圆，颗粒增多，呈立方形或椭圆形；而对照组细胞贴壁生长良好，细胞轮廓清楚，呈纺锤形或长梭形。
8．划痕试验结果显示，转染TGFβ1-ASODN的EC9706细胞的迁移距离在24h(0.14±0.02)、48h(0.30±0.06)、72h(0.45±0.05)均较转染前24h(0.23±0.02)、48h(0.69±0.12)、72h(0.81±0.11)减少(P＜0.05)。
9．流式细胞术结果表明，转染后细胞凋亡百分率(0.69％)低于转染前(0.96％)(P＜0.05)；转染后，G1期的细胞百分比(62.9％)明显低于转染前(66.5％)(P＜0.01)，S期细胞百分比(21.3％)明显低于转染前(23.7％)(P＜0.01)，G2期的细胞百分比(14.8％)明显高于转染前(9.8％)(P＜0.01)。MTT结果表明，转染TGFβ1-ASODN后72h，细胞存活率(109.4％)高于转染前(100％)(P＜0.05)。
结论
1．TGF-β1蛋白在食管鳞癌组织中及食管鳞癌细胞系EC9706中高表达，且在食管鳞癌中TGF-β1蛋白表达与E-cadherin蛋白表达的负相关关系，与Vimentin蛋白表达的正相关关系提示食管鳞癌发生发展过程中可能存在TGF-β1引发的EMT。
2．转染TGFβ1-ASODN，可特异性地抑制食管鳞癌细胞EC9706中的TGF-β1表达。
3．TGFβ1-ASODN转染EC9706细胞，可导致细胞上皮性标志物E-Cadherin表达上调、间质性标志物Vimentin表达下调、形态发生改变、移动能力减弱，提示阻断TGFβ1信号可抑制食管鳞癌细胞EC9706的EMT过程。
4．TGFβ1-ASODN阻断TGFβ1表达后，可能解除了TGFβ1对食管鳞癌细胞EC9706的生长抑制作用和凋亡促进作用。
Esophageal cancer is a highly aggressive neoplasm. On a global basis, cancer of the esophagus is the sixth leading cause of cancer death worldwide, especially in Henan province of China. With advances in surgical techniques and treatment, the prognosis of esophageal cancer has slowly improved over the past three decades. However, the 5-year overall survival rate remains poor. The most important reason is the mechanism of invasion and metastasis is still unclear. To explore the mechanism of the invasion and metastasis of esophageal carcinoma has become a focus.
Epithelial-mesenchymal transition (EMT) is an important mechanism for reorganizing germ layers and tissues during embryonic development. Originally EMT was defined by the formation of mesenchymal cells from epithelia in different embryonic territories, but recently, this process is also found reactivated in a variety of diseases including fibrosis and in the progression of carcinoma. Thiery describes EMTs in tumorigenesis like this : Normal epithelial cell grow along the basal membrane, then the phenotype gene change or mutation make the cell transfer to carcinoma in situ (now the basal membrane still maintains integrity); with development , cancer cells proliferation in region by Epithelial-mesenchymal transition, the basal membrane becomes crisply , cells invasion into lymph-vessels and bloodvessels and metastasis; In the planter spot, cancer cell could growth into metastatic tumor by mesenchymal- epithelial transition; For epithelial cells ,EMT involves loss of epithelial phenotype, or de-epithelialization, which would turn itself to non-epithelial, or nominal 'mesenchymal' cells, in place of what was an epithelium, and it involves a lot of remodeling of junctions, polarity, motility, adhesive properties as well. EMT includes changing of phenotypic markers, an increased capacity for migration and three-dimensional invasion, as well as apoptosis. So far, there is noreport about EMT in Esophageal Squamous Cell Carcinoma .
EMT is a very complex process, including: (1) the cell-cell junction (sticking connection, tight junction, desmosome connection) disintegrates, and the cell becomes dispersible; (2) the cell skeleton rearrangement to cell - matrix attaches, cell movement ability enhancement. (3) gene change: In the EMT process, up-regulated factors involves mesenchymal cell marker such as fibronectin、Vimentin、N-cadherin and so on, as well as cell factors and transcription regulaters which can induce EMT, like snail, slug, TGF- beta(transforming growth factor- beta), FGF (fibroblast growthfactor) MMP-2 (matrix metal proteinase -2 ), MMP-9 (matrix metal proteinase -9); down-regulated factors involves E-cadherin、β-catenin、γ-catenin、cytokeratinl 8 and so on. It has found that TGF-βplay a critical role in EMT.
We have not seen any report about EMT and relationship of EMT induced by TGF-P with invasion and metastasis of ESCC. In this study ,we detected the expression of TGF-βin ESCC tissue and ESCC cell line EC9706, and relationship of the expression of TGF-βwith E-cadherin and Vimentin; inhibited the expression of TGF-β1 by TGFβ1-ASODN in EC9706 to detect changes of expressions of E-cadherin and Vimentin、cell morphous, migration ability ,from these aspects to investigate EMT induced by TGF-β1 in ESCC, and investigate the effect of TGF-β1 to cell proliferation and Apoptosis.
Materials and methods:
1. Expressions of TGF-β1、E-cadherin and Vimentin proteins were detected in 100 cases of surgically resected esophageal squamous carcinoma specimens by the method of immunohistochemistry, which including carcinoma tissues and corresponding normal tissues.
2. Expressions of TGF-β1、E-cadherin和Vimentin proteins and mRNA in the ESCC cell lines EC9706 were investigated by reverse transcription-polymerase chain reaction (RT-PCR)、immunocytochemistry and flowcytometry.
3. The antisense oligodeoxynucleotides (ASODN) was complementary to the TGF-β1 sequences. EC9706 was transfected with chemical synthesised TGF-β1 oligodeoxynucleotides by transfection reagents.
(1) Cell morphological changes were observed with inversphase microscope.
(2)The changes of TGF-β1 protein and mRNA were tested by using immunohistochemistry、flowcytometry and RT-PCR.
(3) The changes of E-cadherin and Vimentin were observed by using RT-PCR、 immunohistochemistry and flowcytometry.
(4) Cell migration potentia were tested by scarification test.
4. The apoptosis、cell cycles and proliferation of EC9706 were detected by flowcytometry and MTT.
5. Statistical analysis: All the dates were analyzed by SPSS 13.0 statistical package, the count information calculated the positive rate, enumeration date are expressed by standard deviation [x|-±s]. The comparison of positive rates uses the Chi-square, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable groups is analyzed by the Kendall correlation analysis. The level of significant difference is a=0.05.
Results
1.The positive rate of expressions of TGF-β1 protein were higher significantly in carcinoma tissues 85.0%than that in normal tissues 27.0% (P <0.01) . The positive rate of expressions of TGF-β1 in deeper invasion group 90.6% were significantly higher than that in superficial invasion group 75.0% (P <0.05).
2. The positive rates of E-cadherin protein were significantly higher in normal mucosa tissues 85.0% than that in cancer tissues43.0% (P <0.01). The positive rates of Vimentin protein were significantly lower in normal mucosa tissues 0 than that in cancer tissues 23.0% (P <0.05).
3.There were negative correlations between the positive rate of TGF-β1 protein expression and that of E-cadherin protein expression in cancer tissues (T_b=-0.257, P<0.05), positive correlations between the positive rate of TGF-β1 protein expression and that of Vimentin protein expression in cancer tissues (T_b=0.163, P<0.05 ) , and negative correlations between the positive rate of E-cadherin protein expression and that of Vimentin protein expression in cancer tissues (T_b=-0.379, P＜0.01) .
4. Results of RT-PCR showed that expressions of mRNA of TGF-pl(0.43±0.09)、E-cadherin (0.22±0.06)and Vimentin(0.89±0.09) can be detected in EC9706.Results of ICC showed that proteins of TGF-β1(43.57%)、E-cadherin(12.53%) and Vimentin(17.97%) express in the ESCC cell line EC9706; results of flow cytometry showed that proteins of TGF-β1(41.4%). E-cadherin(12.2%) and Vimentin(17.8%) express in the ESCC cell line EC9706.
5. After transfection of TGFβ1-ASODN, TGF-β1mRNA/β-actin mRNA half quantitation shows that expressions of TGF-β1 mRNA (0.250±0.681) were obviously lower than control group (0.425±0.093) (P <0.05) . ICC results shows that the positive rates of expressions of TGF-β1 protein were lower significantly in ASODN group (35.07%) than that in control group (43.57%) (P<0.01) , and protein in cytoplasm of transfered group obviously descend. Results of flowcytometry indicated that the positive rates of expressions of TGF-β1 protein were lower significantly in ASODN group (35.4%) than that in control group (41.38%) (P <0.01) .
6. After transfection, results of RT-PCR showed that expressions of E-cadherin mRNA (0.3750±0.0925) were obviously higher than that in control group(0.2183±0.0646) (P <0.05), and expressions of Vimentin mRNA in ASODN group (0.7300±0.0669) were obviously lower than that in control group (0.8933±0.0894) (P<0.05). Results of ICC showed that expressions of E-cadherin protein in ASODN group (17.13%)were obviously higher than that in control group (12.53%) (P<0.01), and expressions of Vimentin protein in ASODN group (14.15%)were obviously lower than that in control group (17.97%) (P<0.01).Results of flowcytometry showed that expressions of epithelial marker E-cadherin protein in ASODN group (17.8%)were obviously higher than that in control group (12.2%) (P<0.01), and expressions of mesenchymal marker Vimentin protein in ASODN group (14.9%)were obviously lower than that in control group (17.8%) (P <0.01).
7. After transfection by TGFβ1- ASODN, transfected cells display distinct biological behaviour, such as shape becoming shrink and round , and more grains . But control group cells were fusiform shape, adherence growth, and had clear layout among cells.
8. Results of scratch test showed that after transfion, migration lenth of ASODN group in24 (0.14±0.02)、48 (0.30±0.06)、72h (0.45±0.05 ) were significantly shorter than control group 24h (0.23±0.02) , 48h (0.69±0.12 ) ,72h (0.81±0.11 ) (P<0.05).
9. After transfection, apoptosis incidence rate in ASODN group (0.69%) was lower than that in control group (0.96%),and the compare has significant difference(P<0.05). Cells in G stage of ASODN group(19.3%) was lower than those of control group (23.7%) (P<0.01); cells in S stage of ASODN group(19.3%)was lower than those of control group (23.7%) (P＜0.01); cells in G2 stage of ASODN group (14.8%) was higher than those of control group ( 9.8% ) (P<0.01); cell survival rate in ASODN group (109.4%) was higher than that in control group
Conclutions
1. Overexpressions of TGF-β1 in esophageal cancer tissue and in ESCC cell lines EC9706 ; in cancer tissues , negative correlations between the expression of TGF-β1 protein and that of E-cadherin protein , and positive correlations between the expression of TGF-(31 protein and that of Vimentin protein indicated that there may have TGF-β1 induced EMT in carcinogenesis and development of ESCC.
2. After transfection by TGFβ1-ASODN , expressions of protein and mRNA of TGF-β1 were suppressed, which indicated that TGFβ1-ASODN can specific inhibit expressions of TGF-β1.
3.After transfection by TGFβ1-ASODN, ESCC cell line EC9706 change in morphous, decrease capacity for migration , and up-regulate of E-cadherine and down-regulate of Vimentin, indicates that blockage of TGFβ1 can inhibit the EMT progress in ESCC cell line EC9706.
4. After TGF-β1 inhibited by TGFβ1-ASODN , the effect of growth inhibition and Apoptosis promotion by TGF-β1 might be relieved.
近来研究发现，在多细胞生物胚胎发育过程中的胚层和组织重建中存在一种现象：上皮-间质转化(epithelial-mesenchymal transition，EMT)。最近研究认为它不仅存在于多细胞生物的胚胎发生过程中，同时也存在于多种慢性疾病以及肿瘤的进展过程中。Thiery这样描述上皮性肿瘤发生发展过程中的EMT：正常上皮细胞沿基底膜生长，通过表型遗传学的改变或基因突变，细胞发生转化生成原位癌(此时基底膜仍然保持完整)；继续发展，癌细胞通过EMT形式局部扩散，基底膜变脆，细胞侵入淋巴管和血管，发生转移；在种植部位，癌细胞还可通过间质-上皮转化形成转移瘤。上皮细胞发生EMT，以间质特性获得、上皮细胞极性丧失及运动能力增强为主要特征。这些特点与肿瘤细胞的原位侵袭和远处转移密切相关。目前关于食管鳞癌中是否存在EMT及EMT在食管鳞癌浸润转移中的作用尚未见报道。
EMT是一个极其复杂的过程，包括：(1)细胞间连接(黏附连接、紧密连接、桥粒连接)解体，细胞分散；(2)细胞骨架重排，形成细胞-基质黏附，细胞运动能力增强。(3)细胞基因型的改变：在EMT过程中表达上调的因子主要包括间叶细胞标志物如纤维连接蛋白(fibronectin)、波形蛋白(Vimentin)、神经钙黏素(N-cadherin)等，以及诱导EMT的细胞因子和转录调节因子，如snail、slug，转化生长因子-β(transforming growth factor-β，TGF-β)，成纤维细胞生长因子(fibroblast growth factor，FGF)；还有对诱导EMT有辅助作用的基质金属蛋白酶-2(MMP-2)，基质金属蛋白酶-9(MMP-9)等。在EMT过程中表达下调的因子主要包括上皮细胞的标志物如上皮钙黏素(E-cadherin)，β-连环素(β-catenin)，γ-连环素(γ-catenin)，细胞角蛋白18(cytokeratin18)等。已有研究发现TGF-β在诱发EMT中起关键作用。
有关食管鳞癌中是否存在EMT以及食管鳞癌中TGF-β1引发的EMT是否与食管癌的浸润转移有关，目前尚未见报道。本研究探讨了食管癌组织及食管鳞癌细胞系EC9706中TGF-β1的表达及其与E-cadherin及Vimentin的关系；采用针对TGF-β1的反义寡核苷酸干扰技术，抑制食管鳞癌细胞EC9706中TGF-β1的转录活性，探讨抑制TGF-β1对E-cadherin及Vimentin表达的影响，观察转染反义寡核苷酸对细胞形态学的影响及对细胞运动能力的影响，从而探讨食管鳞癌细胞中TGF-β1诱导的EMT；并探讨TGF-β1对食管鳞癌细胞EC9706增殖及凋亡的影响。探讨食管鳞癌浸润转移的分子机制，为食管癌浸润转移的阻断治疗提供理论基础。
材料和方法
1．采用免疫组织化学技术检测了100例手术切除的食管鳞癌组织及其癌旁“正常”粘膜中TGF-β1、E-cadherin和Vimentin蛋白的表达情况。
2．采用RT-PCR、免疫细胞化学、流式细胞术对食管鳞癌细胞系EC9706中的TGF-β1、E-cadherin和Vimentin蛋白及mRNA表达进行了检测。
3．针对TGF-β1第一启动子的核苷酸片段设计合成反义寡核苷酸(ASODN)，采用阳离子聚合物瞬时转染培养的人食管鳞癌EC9706细胞。
①观察TGFβ1-ASODN转染后细胞形态学的变化。
②采用RT-PCR、免疫细胞化学方法、流式细胞术方法观察TGFβ1-ASODN转染后细胞中TGF-β1mRNA及蛋白表达的变化。
③采用RT-PCR、免疫细胞化学、流式细胞术方法观察TGFβ1-ASODN转染后细胞中E-cadherine与Vimentin mRNA及蛋白表达的变化。
④采用细胞划痕试验检测TGFβ1-ASODN转染后细胞迁移能力的变化。
4．采用流式细胞术、MTT观察转染TGFβ1-ASODN后，对细胞增殖、凋亡及细胞周期的影响。
5．统计学处理：所有数据均经SPSS13.0软件进行统计分析。计数数据采用阳性率表示，阳性率之间的比较采用χ~2检验(Chi-square)，阳性率间相关性采用Kendall相关进行比较；计量数据采用(?)±S表示，两组均数的比较用t检验(t-test)，两组以上均数的比较用方差分析(ANVOA)。显著性水平α=0.05。
结果
1．TGF-β1蛋白在食管鳞癌中的表达(85.0％)明显高于其在癌旁“正常”粘膜中的表达(27.0％)(P＜0.01)，其在深层浸润组中的表达(90.6％)高于其在浅层浸润组中的表达(75.0％)(P＜0.05)。
2．E-cadherin蛋白在食管鳞癌中的表达(43％)明显低于其在癌旁“正常”粘膜中的表达(85％)(P＜0.01)；Vimentin蛋白在食管鳞癌中的表达(23％)高于其在癌旁“正常”粘膜中的表达(0％)(P＜0.05)。
3．在食管鳞癌组织中，TGF-β1的表达与E-cadherin的表达呈负相关关系(T_b=-0.257，P＜0.05)；TGF-β1的表达与Vimentin的表达呈正相关关系(T_b=0.163，P＜0.05)；Vimentin的表达与E-cadherin的表达呈负相关关系(T_b=-0.379，P＜0.01)。
4．RT-PCR结果显示EC9706细胞系中可检测到TGF-β1mRNA(0.43±0.09)、E-cadherin mRNA(0.22±0.06)、Vimentin mRNA(0.89±0.09)的表达；免疫细胞化学结果显示在EC9706中存在TGF-β1蛋白(43.57％)、E-cadherin蛋白(12.53％)和Vimentin蛋白(17.97％)的表达；流式细胞术结果也显示在EC9706中存在TGF-β1蛋白(41.4％)、E-cadherin蛋白(12.2％)和Vimentin(17.8％)蛋白的表达。
5．转染TGFβ1-ASODN后，RT-PCR结果表明，TGF-β1mRNA的表达水平(0.25±0.681)明显低于转染前(0.43±0.09)(P＜0.01)；免疫细胞化学结果表明，转染后TGF-β1蛋白的表达(35.07％)明显低于转染前(43.57％)(P＜0.01)，且细胞浆着色减弱；流式细胞术结果表明，转染后TGF-β1蛋白的表达(35.4％)明显低于转染前(41.38％)(P＜0.01)。
6．RT-PCR结果表明，转染TGFβ1-ASODN后，E-cadherin mRNA的表达(0.38±0.09)明显高于转染前(0.22±0.06)(P＜0.01)；Vimentin mRNA的表达(0.73±0.07)低于转染前(0.89±0.09)(P＜0.05)。免疫细胞化学结果表明，转染后，E-cadherin蛋白的表达(17.13％)明显高于转染前(12.53％)(P＜0.01)；Vimentin蛋白的表达(14.15％)明显低于转染前(17.97％)(P＜0.01)。流式细胞术结果表明，转染后E-cadherin蛋白的表达率(17.8％)明显高于转染前(12.2％)(P＜0.01)；Vimentin蛋白的表达(14.9％)明显低于转染前(17.8％)(P＜0.01)。
7．转染后细胞形态变圆，颗粒增多，呈立方形或椭圆形；而对照组细胞贴壁生长良好，细胞轮廓清楚，呈纺锤形或长梭形。
8．划痕试验结果显示，转染TGFβ1-ASODN的EC9706细胞的迁移距离在24h(0.14±0.02)、48h(0.30±0.06)、72h(0.45±0.05)均较转染前24h(0.23±0.02)、48h(0.69±0.12)、72h(0.81±0.11)减少(P＜0.05)。
9．流式细胞术结果表明，转染后细胞凋亡百分率(0.69％)低于转染前(0.96％)(P＜0.05)；转染后，G1期的细胞百分比(62.9％)明显低于转染前(66.5％)(P＜0.01)，S期细胞百分比(21.3％)明显低于转染前(23.7％)(P＜0.01)，G2期的细胞百分比(14.8％)明显高于转染前(9.8％)(P＜0.01)。MTT结果表明，转染TGFβ1-ASODN后72h，细胞存活率(109.4％)高于转染前(100％)(P＜0.05)。
结论
1．TGF-β1蛋白在食管鳞癌组织中及食管鳞癌细胞系EC9706中高表达，且在食管鳞癌中TGF-β1蛋白表达与E-cadherin蛋白表达的负相关关系，与Vimentin蛋白表达的正相关关系提示食管鳞癌发生发展过程中可能存在TGF-β1引发的EMT。
2．转染TGFβ1-ASODN，可特异性地抑制食管鳞癌细胞EC9706中的TGF-β1表达。
3．TGFβ1-ASODN转染EC9706细胞，可导致细胞上皮性标志物E-Cadherin表达上调、间质性标志物Vimentin表达下调、形态发生改变、移动能力减弱，提示阻断TGFβ1信号可抑制食管鳞癌细胞EC9706的EMT过程。
4．TGFβ1-ASODN阻断TGFβ1表达后，可能解除了TGFβ1对食管鳞癌细胞EC9706的生长抑制作用和凋亡促进作用。
Esophageal cancer is a highly aggressive neoplasm. On a global basis, cancer of the esophagus is the sixth leading cause of cancer death worldwide, especially in Henan province of China. With advances in surgical techniques and treatment, the prognosis of esophageal cancer has slowly improved over the past three decades. However, the 5-year overall survival rate remains poor. The most important reason is the mechanism of invasion and metastasis is still unclear. To explore the mechanism of the invasion and metastasis of esophageal carcinoma has become a focus.
Epithelial-mesenchymal transition (EMT) is an important mechanism for reorganizing germ layers and tissues during embryonic development. Originally EMT was defined by the formation of mesenchymal cells from epithelia in different embryonic territories, but recently, this process is also found reactivated in a variety of diseases including fibrosis and in the progression of carcinoma. Thiery describes EMTs in tumorigenesis like this : Normal epithelial cell grow along the basal membrane, then the phenotype gene change or mutation make the cell transfer to carcinoma in situ (now the basal membrane still maintains integrity); with development , cancer cells proliferation in region by Epithelial-mesenchymal transition, the basal membrane becomes crisply , cells invasion into lymph-vessels and bloodvessels and metastasis; In the planter spot, cancer cell could growth into metastatic tumor by mesenchymal- epithelial transition; For epithelial cells ,EMT involves loss of epithelial phenotype, or de-epithelialization, which would turn itself to non-epithelial, or nominal 'mesenchymal' cells, in place of what was an epithelium, and it involves a lot of remodeling of junctions, polarity, motility, adhesive properties as well. EMT includes changing of phenotypic markers, an increased capacity for migration and three-dimensional invasion, as well as apoptosis. So far, there is noreport about EMT in Esophageal Squamous Cell Carcinoma .
EMT is a very complex process, including: (1) the cell-cell junction (sticking connection, tight junction, desmosome connection) disintegrates, and the cell becomes dispersible; (2) the cell skeleton rearrangement to cell - matrix attaches, cell movement ability enhancement. (3) gene change: In the EMT process, up-regulated factors involves mesenchymal cell marker such as fibronectin、Vimentin、N-cadherin and so on, as well as cell factors and transcription regulaters which can induce EMT, like snail, slug, TGF- beta(transforming growth factor- beta), FGF (fibroblast growthfactor) MMP-2 (matrix metal proteinase -2 ), MMP-9 (matrix metal proteinase -9); down-regulated factors involves E-cadherin、β-catenin、γ-catenin、cytokeratinl 8 and so on. It has found that TGF-βplay a critical role in EMT.
We have not seen any report about EMT and relationship of EMT induced by TGF-P with invasion and metastasis of ESCC. In this study ,we detected the expression of TGF-βin ESCC tissue and ESCC cell line EC9706, and relationship of the expression of TGF-βwith E-cadherin and Vimentin; inhibited the expression of TGF-β1 by TGFβ1-ASODN in EC9706 to detect changes of expressions of E-cadherin and Vimentin、cell morphous, migration ability ,from these aspects to investigate EMT induced by TGF-β1 in ESCC, and investigate the effect of TGF-β1 to cell proliferation and Apoptosis.
Materials and methods:
1. Expressions of TGF-β1、E-cadherin and Vimentin proteins were detected in 100 cases of surgically resected esophageal squamous carcinoma specimens by the method of immunohistochemistry, which including carcinoma tissues and corresponding normal tissues.
2. Expressions of TGF-β1、E-cadherin和Vimentin proteins and mRNA in the ESCC cell lines EC9706 were investigated by reverse transcription-polymerase chain reaction (RT-PCR)、immunocytochemistry and flowcytometry.
3. The antisense oligodeoxynucleotides (ASODN) was complementary to the TGF-β1 sequences. EC9706 was transfected with chemical synthesised TGF-β1 oligodeoxynucleotides by transfection reagents.
(1) Cell morphological changes were observed with inversphase microscope.
(2)The changes of TGF-β1 protein and mRNA were tested by using immunohistochemistry、flowcytometry and RT-PCR.
(3) The changes of E-cadherin and Vimentin were observed by using RT-PCR、 immunohistochemistry and flowcytometry.
(4) Cell migration potentia were tested by scarification test.
4. The apoptosis、cell cycles and proliferation of EC9706 were detected by flowcytometry and MTT.
5. Statistical analysis: All the dates were analyzed by SPSS 13.0 statistical package, the count information calculated the positive rate, enumeration date are expressed by standard deviation [x|-±s]. The comparison of positive rates uses the Chi-square, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable groups is analyzed by the Kendall correlation analysis. The level of significant difference is a=0.05.
Results
1.The positive rate of expressions of TGF-β1 protein were higher significantly in carcinoma tissues 85.0%than that in normal tissues 27.0% (P <0.01) . The positive rate of expressions of TGF-β1 in deeper invasion group 90.6% were significantly higher than that in superficial invasion group 75.0% (P <0.05).
2. The positive rates of E-cadherin protein were significantly higher in normal mucosa tissues 85.0% than that in cancer tissues43.0% (P <0.01). The positive rates of Vimentin protein were significantly lower in normal mucosa tissues 0 than that in cancer tissues 23.0% (P <0.05).
3.There were negative correlations between the positive rate of TGF-β1 protein expression and that of E-cadherin protein expression in cancer tissues (T_b=-0.257, P<0.05), positive correlations between the positive rate of TGF-β1 protein expression and that of Vimentin protein expression in cancer tissues (T_b=0.163, P<0.05 ) , and negative correlations between the positive rate of E-cadherin protein expression and that of Vimentin protein expression in cancer tissues (T_b=-0.379, P＜0.01) .
4. Results of RT-PCR showed that expressions of mRNA of TGF-pl(0.43±0.09)、E-cadherin (0.22±0.06)and Vimentin(0.89±0.09) can be detected in EC9706.Results of ICC showed that proteins of TGF-β1(43.57%)、E-cadherin(12.53%) and Vimentin(17.97%) express in the ESCC cell line EC9706; results of flow cytometry showed that proteins of TGF-β1(41.4%). E-cadherin(12.2%) and Vimentin(17.8%) express in the ESCC cell line EC9706.
5. After transfection of TGFβ1-ASODN, TGF-β1mRNA/β-actin mRNA half quantitation shows that expressions of TGF-β1 mRNA (0.250±0.681) were obviously lower than control group (0.425±0.093) (P <0.05) . ICC results shows that the positive rates of expressions of TGF-β1 protein were lower significantly in ASODN group (35.07%) than that in control group (43.57%) (P<0.01) , and protein in cytoplasm of transfered group obviously descend. Results of flowcytometry indicated that the positive rates of expressions of TGF-β1 protein were lower significantly in ASODN group (35.4%) than that in control group (41.38%) (P <0.01) .
6. After transfection, results of RT-PCR showed that expressions of E-cadherin mRNA (0.3750±0.0925) were obviously higher than that in control group(0.2183±0.0646) (P <0.05), and expressions of Vimentin mRNA in ASODN group (0.7300±0.0669) were obviously lower than that in control group (0.8933±0.0894) (P<0.05). Results of ICC showed that expressions of E-cadherin protein in ASODN group (17.13%)were obviously higher than that in control group (12.53%) (P<0.01), and expressions of Vimentin protein in ASODN group (14.15%)were obviously lower than that in control group (17.97%) (P<0.01).Results of flowcytometry showed that expressions of epithelial marker E-cadherin protein in ASODN group (17.8%)were obviously higher than that in control group (12.2%) (P<0.01), and expressions of mesenchymal marker Vimentin protein in ASODN group (14.9%)were obviously lower than that in control group (17.8%) (P <0.01).
7. After transfection by TGFβ1- ASODN, transfected cells display distinct biological behaviour, such as shape becoming shrink and round , and more grains . But control group cells were fusiform shape, adherence growth, and had clear layout among cells.
8. Results of scratch test showed that after transfion, migration lenth of ASODN group in24 (0.14±0.02)、48 (0.30±0.06)、72h (0.45±0.05 ) were significantly shorter than control group 24h (0.23±0.02) , 48h (0.69±0.12 ) ,72h (0.81±0.11 ) (P<0.05).
9. After transfection, apoptosis incidence rate in ASODN group (0.69%) was lower than that in control group (0.96%),and the compare has significant difference(P<0.05). Cells in G stage of ASODN group(19.3%) was lower than those of control group (23.7%) (P<0.01); cells in S stage of ASODN group(19.3%)was lower than those of control group (23.7%) (P＜0.01); cells in G2 stage of ASODN group (14.8%) was higher than those of control group ( 9.8% ) (P<0.01); cell survival rate in ASODN group (109.4%) was higher than that in control group
Conclutions
1. Overexpressions of TGF-β1 in esophageal cancer tissue and in ESCC cell lines EC9706 ; in cancer tissues , negative correlations between the expression of TGF-β1 protein and that of E-cadherin protein , and positive correlations between the expression of TGF-(31 protein and that of Vimentin protein indicated that there may have TGF-β1 induced EMT in carcinogenesis and development of ESCC.
2. After transfection by TGFβ1-ASODN , expressions of protein and mRNA of TGF-β1 were suppressed, which indicated that TGFβ1-ASODN can specific inhibit expressions of TGF-β1.
3.After transfection by TGFβ1-ASODN, ESCC cell line EC9706 change in morphous, decrease capacity for migration , and up-regulate of E-cadherine and down-regulate of Vimentin, indicates that blockage of TGFβ1 can inhibit the EMT progress in ESCC cell line EC9706.
4. After TGF-β1 inhibited by TGFβ1-ASODN , the effect of growth inhibition and Apoptosis promotion by TGF-β1 might be relieved.
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