【摘要】 目的研究半胱氨酸蛋白酶抑制剂A在胃癌中可能存在的作用及机制。方法建立体外HGC-27胃癌细胞模型。构建过表达质粒PCDNA3.1-CSTA和合成RNA干扰抑制剂siRNA-CSTA,并转染至细胞株中,综合使用MTT,Transwell和流式细胞技术评估细胞增殖,迁移和凋亡。同时,使用PCR(qRT-PCR)和Western-Blot检测半胱氨酸蛋白酶抑制剂A和PI3K/Akt/mTOR通路相关蛋白的表达。结果半胱氨酸蛋白酶抑制剂A在HGC-27细胞株中的表达与其在胃正常上皮细胞中的表达相比显著下调,半胱氨酸蛋白酶抑制剂A在pc-CSTA转染细胞后过表达,同时在si-CSTA转染细胞中抑制。与对照组相比,细胞活力及细胞迁移在pc-CSTA组显着减弱,用si-CSTA组明显增强,细胞凋亡在pc-CSTA组显著诱导,在si-CSTA组明显抑制。与对照组相比,p-PI3K,p-Akt和p-mTOR蛋白的表达,在pc-CSTA组明显低表达,而在si-CSTA组中明显高表达。结论半胱氨酸蛋白酶抑制剂A可作为治疗胃癌的潜在靶点。半胱氨酸蛋白酶抑制剂A下调能影响HGC-27细胞增殖和迁移,同时激活PI3K/Akt/mTOR信号通路促进胃癌的进展。更多还原

【Abstract】 Objective To study the possible mechanism of CSTA in the process of gastric cancer. Methods HGC-27, one of the human gastric cancer cell lines was chosen to establish in vitro. Both the plasmids PCDNA3.1-CSTA and RNA interference inhibitor si RNA-CSTA were constructed and transfected into cells. The MTT, transwell assay, and flow cytometry were used to assess the cell counts of cell proliferation, migration and apoptosis. The expression of Cystain A and PI3K/Akt/mTOR pathway-related proteins was detected by PCR(qRT-PCR) and Western-Blot. Results Compared with normal gastric epithelial cells, the expression of Cystatin A in the HGC-27 cell was significantly down-regulated. Cystatin A was overexpressed after transfection with pc-CSTA cells and inhibited in cells transfected with si-CSTA. In addition, compared with the control group, cell viability and migration were significantly lower after cells were transfection with pc-CSTA, but increased significantly after transfection with si-CSTA. The apoptotic control group was significantly induced in the pc-CSTA transfection group and was significantly inhibited in the si-CSTA transfection group.In addition, the expression levels of p-PI3K, p-Akt and p-mTOR protein were significantly lower in pc-CSTA transfected group than in control group, but were significantly higher in si-CSTA transfection group. Conclusion Cystatin A downregulation may active the PI3K/Akt/mTOR signaling pathway, at the same time it can promote the progression of gastric cancer by affecting cell proliferation and migration. Cystatin A can also be used as a potential target for the treatment of gastric cancer.更多还原