文档介绍：siRNA沉默Annexin1基因对膀胱癌T24细胞顺铂耐药的影响
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作者:高琳杨远航徐万海林相国李建章杨德君王晓民
【摘要】目的观察RNA干扰沉默膜联蛋白1(Annexin1)基因表达技术对膀胱癌T24细胞Annexin1基因表达和顺铂耐药的影响。方法针对Annexin1基因不同部位设计并化学合成3对不同的靶向小分子干扰RNA(siRNA),脂质体介导瞬时转染膀胱癌T24细胞,转染后应用半定量RTPCR和Western印迹检测Annexin1 mRNA 和蛋白的改变,用MTT法检测沉默Annexin1表达在膀胱癌T24细胞对顺铂敏感性的变化。结果转染siRNA后膀胱癌T24细胞Annexin1的mRNA和蛋白水平显著下降(P<),增强细胞对顺铂的敏感性(P<)。结论 RNA 干扰Annexin1基因后其mRNA 和蛋白水平显著下降,并且增强T24细胞对顺铂的敏感性。
【关键词】膀胱癌;膜联蛋白1;siRNA;顺铂
【Abstract】 Objective To explore the effects of annexin1 gene silencing by small interfering RNA(siRNA) on bladder cancer cells T24 and the sensitivity change of DDP. Methods Three siRNA targeting three different domains of annexin
1 gene were designed, synthesized, and transfected into bladder cancer T24 cells by M2000. After transfection, expression of annexin1 was detected by RTPCR and Westblotting. The cellular sensitivity to DDP was evaluated by MTT assay. pared with the negative control (siRNANC) groups, transfection of three targeting siRNA in T24 cells decreased the expression of annexin1 mRNA and protein. Annexin1 gene silencing increased the sensitivity of T24 cells to DDP. Conclusions The chemically synthesized specific siRNA targeting annexin1 could effectively reduce the expression of annexin1 gene, increase the sensitivity of T24 cells to DDP.
【Key words】Bladder cancer; Annexin1 ; siRNA; DDP
膜联蛋白1(Annexin1)是上皮生长因子(EGFR)激酶的主要底酶,糖皮质激素刺激其产生和活性。Annexin1增强感染局部的凋亡表明它对肿瘤的诊断和治疗有重要意义,因此提出该蛋白质与肿瘤细胞的活性调节相关〔1,2〕。Annexin1与膀胱癌发生、发展密切相关。本研究针对Annexin1基因mRNA序列设计合成与之互补的双链小分子干扰RNA(siRNA),在脂质体介导下转染膀胱癌T24细胞系,观察对Annexin1基因mRNA和蛋白表达及对顺铂敏感性的变化。
1 材料与方法
材料
细胞株
膀胱癌T24细胞株。
主要试剂
RNA干扰试剂盒(Silencer
TM siRNA Construction Kit)购自美国Ambion公司。M2000、Trizol、MMLV购自Invitrogen 生命技术公司。RPMI1640、IMDM、胰蛋白酶购自Gibco公司。
方法
siRNA的设计及合成
根据基因库中Annexin1基因序列(GenBank Accession )和siRNA设计原则,应用Ambion生物公司的设计软件(siRNA Target Finder and Design Tools),自Annexin