S100A6 siRNA的构建及鉴定
Construction and identification of S100A6-siRNA

摘要：目的 利用小片段干扰RNA(small interfering RNA, siRNA)抑制S100A6蛋白的表达，为研究CacyBP/SIP核转位机制提供有利的工具。方法 设计S100A6的siRNA序列，用脂质体LipofectamineTM 2000将siRNA转染至人结肠癌SW480细胞中，在荧光显微镜下观察转染效率，用Real-time PCR及Western blot方法检测S100A6在人结肠癌SW480细胞内的mRNA和蛋白表达。结果 构建了3对S100A6-siRNA，荧光显微镜下检测转染人结肠癌SW480细胞成功；RT-PCR证明了在SW480细胞内S100A6的mRNA表达显著降低(P<0.05)；Western blot证明了SW480细胞内S100A6蛋白表达显著降低(P<0.05)；与S100A6 si-1、S100A6 si-3组相比，S100A6si-2组对S100A6的mRNA和蛋白抑制效果最明显(P<0.05)。结论 成功构建了S100A6-siRNA，为CacyBP/SIP核转位机制研究提供了有利的工具。
ABSTRACT: Objective To inhibit the expression of S100A6 protein with short interfering RNA (siRNA) so as to provide a useful tool for studying calcyclin(S100A6)-binding-protein (CacyBP)/SIP nuclear translocation mechanism. Methods The siRNAs of the S100A6 coding sequence were designed and transiently transfected to human colon cancer SW480 cells by Lipo-fectamine 2000. Western blot and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analyses were used to examine the expression of S100A6 in these transfectants. Results S100A6-siRNA was constructed and transiently transfected successfully to human colon cancer SW480 cells under the fluorescence microscope. Silent expression of S100A6 at the mRNA and protein levels was confirmed by RT-PCR and Western blot, respectively (both P<0.05). The inhibitory effect was the most obvious in S100A6si-2 group (P<0.05). Conclusion The available tool of the CacyBP/SIP nuclear translocation mechanism has been provided by constructing the S100A6-siRNA