Effects of Bcl-2 Interference on Esophageal Cancer Cell Ec9706 Vasculogenic Mimicry in Hypoxic Microenvironment
-
摘要: 目的 探讨缺氧微环境对食管癌Ec9706细胞血管生成拟态的影响及其可能的分子机制。方法 应用CoCl2化学法诱导人食管癌细胞形成缺氧微环境，在缺氧微环境中以Bcl-2-siRNA干扰人食管鳞癌细胞系Ec9706，三维培养体系模拟人体内环境并置于荧光倒置显微镜下观察管道结构形成数量及形态。流式细胞术检测干扰前后细胞的增殖情况，采用RT-PCR法和Western blot法检测Bcl-2、VEcadherin(VE-cad)及MMP2的mRNA及蛋白表达情况。结果 缺氧对照组比常氧对照组形成更多的网络样管状结构（P＜0.000），凋亡减少（P＜0.05），Bcl-2、VE-cad和MMP2的mRNA和蛋白表达较常氧下显著增多（P＜0.05）。而在缺氧培养下进行Bcl-2-siRNA干扰后，缺氧实验组对比缺氧对照组网络样管状结构显著减少（P＜0.000），凋亡增多（P＜0.05）， 而Bcl-2、VE-cad及MMP2的mRNA和蛋白表达也较缺氧对照组细胞的表达显著降低（P＜0.05）。缺氧空载体组与缺氧对照组网络样管状结构未见明显差异（P＞0.05），凋亡率差异未见统计学意义（P＞0.05），Bcl-2、VE-cad和MMP2的mRNA 和蛋白表达两组之间差异没有统计学意义（P＞0.05）。结论 缺氧能够诱导食管癌Ec9706细胞血管生成拟态的形成，且Bcl-2依赖的VE-cad过表达可能是缺氧诱导血管生成拟态形成的重要分子机制。Abstract: Objective To investigate hypoxic microenvironment on esophageal cancer cells Ec9706 vasculogenic mimicry and its possible molecular mechanism. Methods Human esophageal cancer cells were induced by CoCl2 chemical method to form hypoxia-microenvironment. While in hypoxic microenvironment, human esophageal squamous cell carcinoma cell lines Ec9706 was interfered by Bcl-2-siRNA, and human body environment was simulated by three-dimensional culture system and placed in the inverted fluorescence microscope to observe the number of pipeline structure formation and morphology. Flow cytometry was applied to detect cell proliferation. RT-PCR and Western blot were used to assay mRNA and protein expression of Bcl-2, VE-cad and MMP2. Results Hypoxia group formed more network-like tubular structure (P<0.000) than normoxic control group, decreased apoptosis (P<0.05), the mRNA and protein expression of Bcl-2, VE-cad and MMP2 compared with normoxic significantly increased (P<0.05) lower. After training in hypoxia carried out under Bcl-2-siRNA interference, the tubular network-like structure of experimental group significantly reduced than hypoxic hypoxia control group (P<0.000), apoptosis increased (P<0.05), whereas the mRNA and protein expression of Bcl-2, VE-cad and MMP2 was also significantly lower (P<0.05). The network-like tubular structures between hypoxia and anoxia empty vector control was no significant difference (P>0.05), apoptosis rate was no statistically significant (P>0.05), the mRNA and protein of Bcl-2, VE-cad and MMP2 was not statistically significant between groups(P>0.05). Conclusion Hypoxia could induce the formation of esophageal cancer cells Ec9706 vasculogenic mimicry, and the important molecular mechanism may be related to Bcl-2-dependent VE-cad overexpression.
-
Key words:
- Esophageal cancer /
- Vascular mimicry /
- Hypoxia /
- RNA interference /
- Bcl-2 /
- VE-cadherin
-
-
[1] Zhang SW, Zhang M, Li GL,et al. An analysis of incidence and mortality of esophageal cancer in China, 2003-2007[J]. Zhongguo Zhong Liu,2012,21(4):241-7.[张思维,张敏,李光 琳,等.2003～2007年中国食管癌发病与死亡分析[J].中国肿 瘤,2012,21(4):241-7.] [2] Maniotis AJ, Folberg R, Hess A, et al. Vascular channel formation by human melanomoa cells in vivo and in vitro: vasculogenic mimicry[J].Am J Pathol,1999,155(3):739-52. [3] Guzman G, Cotler SJ, Lin AY, et al. A pilot study of vasculogenic mimicry immunohistoche mical expression in hepatocellular carcinoma[J].Arch Pathol Lab Med,2007,131(12):1776-81. [4] Baeten CI, Hillen F, Pauwels P, et al. Prognostic role of vasculogenic mimicry in colorectal cancer[J]. Dis Colon Rectum, 20 09,52(12):2028-35. [5] Clemente M, Pérez-Alenza MD, Illera JC, et al. Histological, immunohistological and ultrastructural description of vasculogenic mimicry in canine mammary cancer[J]. Vet Pathol,2010, 47 (2):265-74. [6] Liu F, Niu J. Investigation of vacuole membrane proteins 1 and relations with carcinomas[J].Zhong Liu Fang Zhi Yan Jiu,2013,40(1):102-4.[刘芬,牛昀.空泡膜蛋白1及其与肿瘤关系 的研究进展[J].肿瘤防治研究,2013,40(1):102-4.] [7] Zhao J,Huang JS,Yang AJ,et al.Three-dimensional cell culture and hustology of vasculogenic mimicry in heatocellular carcinoma[J]. Ai Zheng,2007,26(2):123-6.[赵静,黄江生,杨爱军,等.肝细 胞癌中血管生成拟态的三维细胞培养及细胞学研究[J].癌 症,2007,26(2):123-6.] [8] Zhao N,Sun BC,Sun T,et al. Hypoxia-induced vasculogenic mimicry formation via VE-cadherin regulation by Bcl-2[J]. Med Oncol,2012,29(5):3599-607. [9] Rezaei M, Friedrich K, Wielockx B, et al. Interplay between neural-cadherin and vasular endothelial-cadherin in breast cancer progression[J].Breast Cancer Res, 2012,14(6):R154. [10] Wang L, Shi RH, Wang P, et al. Effect of down-regulation of vascular endothelial-cadherin on vasculogenic mimicry, proliferation and apoptosis of esophageal squamous carcinoma cells[J]. Wei Chang Bing Xue,2012,17(5):260-5.[王璐,施瑞华,王 频等.血管内皮钙黏附素下调对食管鳞癌细胞血管生成拟态、 增殖和凋亡的影响[J].胃肠病学,2012,17(5):260-5.] [11] Liu Y, Shen Y, Qin C, et al. Growth inhibition and apoptosis of human B-cell lymphoma in vitro and in vivo by Bcl-2 short hairpin RNA[J]. Oncol Rep,2013,29(1):244-52. [12] Dong Z, Venkatachalam MA, Wang J, et al. Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia. Hif-1-independent mechanisms[J]. J Biol Chem,2001,276(22):18702-9. [13] Fan YZ, Sun W. Molecular regulation of vasculogenic mimicry in tumors and potential tumor-target therapy[J]. World J Gastrointest Surg,2010,2(4): 117-27.
计量
- 文章访问数: 1275
- HTML全文浏览量: 382
- PDF下载量: 484
下载:
