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目的构建稳定敲低E6AP表达的胃癌细胞BGC-823,并检测对胃癌细胞增殖和迁移等恶性生物学行为的影响。方法采用Western印迹方法检测E6AP在不同胃癌细胞系及胃正常黏膜上皮细胞系中的表达情况;将含有E6AP的慢病毒短发夹RNA(short hairpin RNA,shRNA)的干扰载体与慢病毒包装辅助质粒共转染人胚肾293T细胞后,收集病毒上清感染胃癌细胞BGC-823,经嘌罗霉素(puromycin)筛选后,获得慢病毒介导的E6AP shRNA的稳定表达细胞,用实时(real-time)PCR和Western印迹方法检测E6AP干扰效果;并利用细胞生长曲线实验、划痕愈合(wound healing)和Transwell实验检测E6AP shRNA对胃癌细胞增殖和迁移的影响。结果 E6AP在不同胃癌细胞系及胃正常黏膜上皮细胞系中均有表达;建立了稳定敲低E6AP的胃癌细胞BGC-823;E6AP shRNA可有效抑制胃癌细胞增殖和迁移。结论 E6AP的生物学功能在胃癌恶性转化过程中发挥重要作用。
Objective To construct stable gastric cancer cell line BGC-823 knocked down by E6AP, and to detect the effect on malignant biological behavior such as proliferation and migration of gastric cancer cells. Methods Western blotting was used to detect the expression of E6AP in different gastric cancer cell lines and gastric normal mucosa epithelial cell lines. The interference vectors of E6AP-containing lentivirus short hairpin RNA (shRNA) and lentivirus packaging helper plasmid After co-transfection of human embryonic kidney 293T cells, the virus supernatant was collected to infect gastric cancer cell line BGC-823. After being screened by puromycin, stable expression cells of lentivirus-mediated E6AP shRNA were obtained. Real- time PCR and Western blotting were used to detect the effect of E6AP on the proliferation and migration of gastric cancer cells. Cell growth curve, wound healing and Transwell assay were used to detect the effect of E6AP shRNA on the proliferation and migration of gastric cancer cells. Results E6AP was expressed in different gastric cancer cell lines and normal mucosa epithelial cell lines. E6AP stable gastric cancer cell line BGC-823 was established. E6AP shRNA could effectively inhibit the proliferation and migration of gastric cancer cells. Conclusion The biological function of E6AP plays an important role in the malignant transformation of gastric cancer.