肺腺癌细胞S100A6基因siRNA慢病毒载体构建及鉴定

Construction and identification of lent viral vector of siRNA specific for S100A6 gene

目的:构建肺腺癌细胞S100A6基因siRNA慢病毒载体并鉴定。方法:慢病毒载体(pL enR-GPH)构建靶向S100A6基因的RNA干扰重组体,用以建立S100A6基因稳定沉默的肺腺癌A549细胞株。结果:PCR鉴定结果显示3个扩增的阳性片段均已插入pL enR-GPH载体,测序结果证实三个重组慢病毒载体SH1、SH2、SH3的插入序列完全正确,重组慢病毒载体转染到293T细胞中包装成病毒颗粒,将其感染肺癌A549细胞后,RT-PCR和Western blot检测结果均证实三组重组体感染的细胞内S100A6 mRNA和蛋白的表达受到不同程度的抑制,沉默效率分别为98.29%、84.05%及78.24%。结论:成功构建了S100A6基因RNAi慢病毒重组载体,并建立了其稳定表达的肺腺癌A549细胞株,为探讨S100A6基因对肺腺癌生物学行为的影响提供了可靠的细胞模型。

Objective:To construct a recombinant mediating RNA interference(RNAi)against S100A6 gene and identify.Methods:The lentiviral vector was used for construction of a recombinant mediating RNA interference(RNAi) against S100A6 gene.Recombinant vector plasmid was transfected into non -small cell lung cancer (NSCLC)A549 cells by liposome.Results:Three amplified positive fragments were inserted into pLenR -GPH vectors.DNA sequencing re-sults showed that the 3 recombinant lentiviralplasmids,SH1 /SH2 /SH3 were constructed successfully.After transfec-tion with A549 cells by liposome,RT -PCR and Western blot analysis confirmed that the expression of S100A6 mRNAand protein was inhibited in the 3 recombinant lentiviral plasmids transfected groups,and gene silencing efficacy was 98.29%,84.05% and 78.24%,respectively.Conclusion:The lentiviral vector of RNAi targeting S100A6 gene was successfully constructed,and NSCLC A549 stable cell line with S100A6 gene knockdown was established.This study finally provided a new cell model to explore the biological behavior of the S100A6 gene in NSCLC A549 cells.