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

Effect of Down-regulation of Vascular Endothelial-cadherin on Vasculogenic Mimicry,Proliferation and Apoptosis of Esophageal Squamous Carcinoma Cells

背景:血管内皮钙黏附素(VE-cad)作为血管生成拟态的重要调控分子在多种高侵袭性肿瘤中均存在表达,参与肿瘤的发生、发展过程。前期研究发现食管鳞癌细胞中亦存在VE-cad表达。目的:探讨微小RNA(miRNA)干扰技术下调VE-cad对食管鳞癌细胞血管生成拟态形成、细胞增殖和凋亡的影响及其可能机制。方法:将已成功构建的VE-cad miRNA干扰质粒稳定转染Eca109、TE13细胞,同时设置阴性对照组(转染空质粒)和空白对照组(未转染)。荧光显微镜观察单克隆转染效率,三维培养观察细胞管腔样结构形成的能力,RT-PCR和蛋白质印迹法分别检测VE-cad、EphA2、LN5γ2 mRNA和蛋白表达,MTT法和流式细胞术分别检测细胞增殖和凋亡。结果:荧光显微镜显示Eca109、TE13细胞稳定转染效率均达90%以上。与阴性对照组和空白对照组相比,干扰组细胞管腔样结构数目明显减少(P<0.01),VE-cad、EphA2、LN5γ2 mRNA和蛋白表达明显受抑(P<0.01),细胞增殖能力明显下降(P<0.05),凋亡率明显增高(P<0.05)。结论:miRNA干扰技术能有效抑制食管鳞癌细胞Eca109、TE13的VE-cad表达。VE-cad通过下调EphA2和LN5γ2表达抑制血管生成拟态的体外形成,并影响细胞增殖和凋亡。VE-cad可能成为食管鳞癌分子靶向治疗的新靶点。

Background： Vascular endothelial cadherin （VE-cad）, an important regulatory molecule of vasculogenic mimicry, exists in various highly invasive tumors and is involved in tumor development and progression. Previous studies have shown that VE-cad is expressed in esophageal squamous carcinoma cells. Aims ： To investigate the effect and mechanism of downregulation of VE-cad gene by mieroRNA （miRNA） interference on vasculogenic mimicry, as well as cell proliferation and apoptosis of esophageal squamous carcinoma cells. Methods： Plasmid carrying pcDNATM 6.2-GW/EmGFP-miVE-cad was stablely transfected into Eca109 and TE13 cells; negative control group （transfected with empty plasmid） and blank control group （non-transfection） were served as controls. Transfeetion efficiency was detected by fluorescence microscopy. Capability of lumen-like structure formation was observed by three dimensional culture. RT-PCR and Western blotting were used to detect expressions of VE-cad, EphA2, LN5γ2 mRNA and protein, respectively. Cell proliferation and apoptosis were analyzed by MTT assay and flow cytometry, respectively. Results： Fluorescence microscopy showed that the stable transfection reached 90% both in Eca109 and TE13 cells. Compared with negative control group and blank control group, number of lumen-like structure in interference group was obviously decreased （P 〈0.01 ） , expressions of VE-cad, EphA2 LN5γ2 mRNA and protein were significantly suppressed （ P 〈 0.01 ）, cell proliferation was significantly decreased （ P 〈 0.05）, and apoptosis was significantly increased （ P 〈 0.05 ）. Conclusions： miRNA interference can effectively inhibit the expression of VE-cad in Eca109 and TE13 cells. VE-cad can inhibit the formation of vasculogenic mimicry by suppressing the expressions of EphA2 and LNSγ2, as well as inhibit cell proliferation and induce apoptosis. It may become a novel target for esophageal squamous carcinoma therapy.