摘要
背景:研究表明大量高度恶性肿瘤组织中存在血管生成拟态(VM),其分子机制已形成相关假说,但确切机制和关键信号通路尚未明确。目的:探讨缺氧诱导因子(HIF)-1α在食管鳞癌VM形成中的作用。方法:设计合成3种HIF-1α-siRNA质粒,测序后瞬时转染293T细胞,蛋白质印迹法鉴定质粒的干扰效果。将筛选出的pGCsi-HIF3质粒稳定转染食管鳞癌细胞株Eca109和TE13,以蛋白质印迹法检测干扰效果,三维培养法观察VM形成情况,蛋白质印迹法检测VE-cadherin、EphA2、LN5γ2和MMP2蛋白表达。结果:成功构建了3个靶位的质粒,其中pGCsiHIF3的干扰效果最好。pGCsi-HIF3稳定转染食管鳞癌细胞株Eca109和TE13后,与对照组相比,HIF-1α表达明显降低,细胞体外管道形成能力被明显抑制(P<0.05)。常氧下转染组细胞中VE-cadherin、EphA2、LN5γ2均显著下调(P<0.05),MMP2表达无明显差异,且缺氧条件下上述指标蛋白表达无明显增加。结论:Eca109和TE13细胞能形成管状结构,HIF-1α可能通过调节VE-cadherin、EphA2、LN5γ2等的表达而调节食管鳞癌VM的形成。
Background:It has been demonstrated that vasculogenic mimicry was found in many malignant tumor tissues.Some hypothesis concerning the molecular mechanism of vasculogenic mimicry has been evolved,however,the exact mechanism and key signaling pathways of vasculogenic mimicry are not completely understood.Aims:To investigate the role of hypoxiainducible factor-1α (HIF-1α) in the process of vasculogenic mimicry in esophageal squamous cell carcinoma.Methods:Three HIF-1 α-siRNA plasmids were designed,constructed,sequenced,and transiently transfected into embryonic kidney cell 293T,and the effects of these plasmids were assayed by Western blotting.Two esophageal squamous cell carcinom cell lines Eca109 and TE13 were stably transfected with pGCsi-HIF3,and the interfering effect on HIF-1α was detected by Western blotting.The formation of tubular networks in Eca109 and TE13 cells was observed by three-dimension culture in vitro.The expressions of VE-cadherin,EphA2,LN5γ2 and MMP2 were measured by Western blotting.Results:DNA sequencing showed that recombinant plasmids were successfully constructed,pGCsi-HIF3 showed higher interfering efficiency than pGCsi-HIF1 and pGCsi-HIF2.Compared with controls,the expression of HIF-1α was markedly inhibited in pGCsi-HIF3 stably transfected Eca109 and TE13 cells,and the number of tubular networks remarkably decreased (P < 0.05).The expressions of VE-cadherin,EphA2 and LN5γ2 were inhibited dramatically in the stably transfected Eca109 and TE13 cells than in controls under normoxia (P < 0.05),no significant difference in expression of MMP2 was found ; above-mentioned indices did not increase under hypoxia.Conclusions:Esophageal squamous cell carcinoma cell lines Eca109 and TE13 both can form typical tubular networks.HIF-1α may regulate the ability of forming vasculogenic mimicry in esophageal squamous cell carcinoma by regulating the expressions of VE-cadherin,EphA2 and LN5γ2.
出处
《胃肠病学》
2014年第2期78-82,共5页
Chinese Journal of Gastroenterology
关键词
食管肿瘤
缺氧诱导因子1
Α亚基
RNA干扰
血管生成拟态
Esophageal Neoplasms
Hypoxia-Inducible Factor 1, alpha Subunit
RNA Interference
Vasculogenic Mimicry