过表达Beclin 1对食管癌Eca109细胞株裸鼠成瘤的影响

The Effect of Overexpressed Beclin 1 on Esophageal Cancer (Eca109) Cells in vivo

自噬是一个进化上保守的溶酶体降解途径,激活自噬可以降解有缺陷的细胞器而发挥抑制肿瘤的作用,Beclin 1是关键的自噬和肿瘤抑制基因。该研究检测了Beclin 1蛋白在食管癌组织中的表达情况,构建Beclin 1的重组质粒并转染人食管癌Eca109细胞株。分别以电子显微镜观察自噬体的形成和Eca109细胞的自噬性死亡情况,RT-PCR检测Beclin 1基因的mRNA表达,Westernblot法检测Beclin 1及Bcl-2、P53的表达水平,裸鼠成瘤实验检测Beclin 1基因对Eca109细胞体内增殖的影响。结果显示,Beclin 1蛋白在食管癌组织中相对于癌旁正常食管组织低表达(P<0.05);成功构建了Beclin 1重组质粒并将其转染到Eca109细胞中,电子显微镜观察到自噬体形成,RT-PCR和Western blot显示Beclin 1基因过表达。升高Beclin 1蛋白的表达水平能降低Bcl-2表达和上调P53蛋白,使Eca109细胞的体内致瘤性减弱。过表达Beclin 1基因对食管癌有很好的抑制作用,这将为食管癌的靶向治疗奠定基础。

Autophagy is an evolutionarily conserved lysosomal degradation pathway, and activation of autophagy may function as a tumor suppressor by degrading defective organelles and other cellular components. Beclin ! is a main actor of autophagy, also known as a tumor suppressor gene. In the present study, we examined the expression of Beclin 1 in esophageal cancer by immunohistochemical SP method, Beclinl recombinant plasmid pIRES2-ZsGreenl-hBeclin 1 was constructed and transfected into Ecal09 cells. Autophagy was observed under electron microscopy. The expression of Beclin at mRNA level was detected by the reverse transcription- polymerase chain reaction （RT-PCR）, and Western blot was used to examine the protein expression of Beclin 1, Bcl-2 and P53. Moreover, in vivo, the proliferation of the cancer cells was evaluated in xenotransplant nude mice model. The results showed that reduced Beclin 1 expression was observed in esophageal cancer cells than in normal tissues（P〈0.05）. We successfully constructed Beclin 1 recombinant plasmid and transfected it into Ecal09 cells, autophagosomes were widely formated. The results of RT-PCR and Western blot showed that the expression of Beclin 1 was remarkably higher in transfected group ECA109 cells at both mRNA and protein levels than that of control group and untransfected group. In addition, the protein expression of Bcl-2 was down-regulated but P53 up-regulated after transfected. In vivo, the average weight and volume of tumor in Beclin 1 transfection group xenografts mice were significantly lower than those in the control group （P〈0.05）. The result suggested that Beclin 1 gene can well inhibit esophageal cancer, and this lay the foundation for targeted therapy for esophageal cancer.