慢病毒载体介导E6AP基因敲低乳腺癌细胞株的构建及其功能检测

Lentiviral vector for E6AP small-interfering RNA and its effect on breast cancer growth

目的构建E6AP基因的小干扰RNA(siRNA)真核表达载体,并检测其对乳腺癌细胞ZR75-1生长的影响。方法利用RNA干扰(RNAi)技术设计并合成两条E6AP基因的siRNA,并将其与siRNA表达载体p SIH-H1连接。经过酶切和测序鉴定成功后,人胚肾细胞293T包装E6AP siRNA的慢病毒,然后感染乳腺癌细胞ZR75-1,建立敲低E6AP表达的稳定细胞株。稳定细胞株建立后通过实时定量PCR(qRT-PCR)和Western印迹等方法检测RNAi的干扰效果,并利用细胞生长实验检测E6AP siRNA对乳腺癌细胞生长的影响。结果 DNA测序证明,成功构建了E6AP siRNA的表达载体。实时定量PCR和Western印迹实验证明,构建的siRNA确能有效抑制E6AP基因的表达,并建立了敲低E6AP表达的稳定细胞株。生长实验表明,E6AP siRNA可有效抑制细胞的生长。结论成功构建E6AP基因的siRNA真核表达载体,转染细胞后能有效地抑制E6AP基因的表达,且抑制细胞ZR75-1的生长。

Objective To construct the lentiviral vector（ pSIH-H1 ） for E6AP small-interfering RNA（siRNA） and to detect its effect on breast cancer ZR75-1 cell growth. Methods E6AP siRNA was designed and constructed based on hu- man papillomavirus E6-associated protein（E6AP） cDNA sequence. The expression of E6AP was examined by real-time quantitative PCR（qRT-PCR） and Western blotting. The effect of E6AP on ZR75-1 cell growth was determined by cck-8 kit. Results DNA sequencing indicated that E6AP siRNA expression vector was constructed successfully, qRT-PCR and Western blotting experiments showed that pSIH-H1-E6AP siRNA could suppress the E6AP gene expression. Suppression of E6AP could markedly inhibit the growth of ZR75-1. Conclusion A lentivirus RNA interference （RNAi） vector targeting E6AP gene is successfully constructed,which inhibits the cell growth of ZR75-1.