siRNA对SGC7901/VCR细胞mdr1基因沉默效果的影响因素分析
Analysis of Influence Factors on siRNA Efficacy Silencing mdr1 Gene in SGC7901/VCR Cells
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摘要: 目的分析siRNA沉默人胃癌SGC7901/VCR细胞mdr1基因效果的相关因素。方法设计并体外转录合成4条靶向mdr1的siRNA(mdr1si326、mdr1si1513、mdr1si2631和mdr1si3071),转染SGC7901/VCR细胞,用RTPCR和免疫印迹检测mdr1mRNA和Pgp的表达、流式细胞仪检测细胞内阿霉素的蓄积和MTT法检测细胞对阿霉素的敏感性,综合这4方面结果评价4条siRNA的沉默效果情况;用分子生物学软分析siRNA沉默效果的影响因素。结果沉默效果最好的mdr1si326和较好的mdr1si2631靶序列编码Pgp的跨膜区而且自身无茎和袢;沉默效果较差的mdr1si3071和最差的mdr1si1513靶序列编码Pgp的胞内区,前者自身成茎和成袢。沉默效果最好的mdr1si326和较好的mdr1si2631靶序列在靶位点和靶位点外间有较少的碱基配对和氢键。siRNA的沉默效果与siRNA3'5'端3个碱基中的A/U数量、N1和N19、GC含量间无规律可循。结论siRNA沉默SGC7901/VCR细胞mdr1的效果与靶序列的结构关系密切。Abstract: Objective To analyze the influence factors on siRNA efficacy silencing mdr1 gene in human gastric cancer SGC7901/VCR cells. Methods Four siRNAs(mdr1si326,mdr1si1513,mdr1si2631 and mdr1si3071)specifically targeting mdr1 gene were designed and synthesized by in vitro transcription. The silencing efficacy of the four siRNAs was evaluated with RT-PCR for mdr1 mRNA expression, immunoblotting for P-glycoprotein (P-gp, MDR1) expression, flowcytometry for adriamycin (ADR) accumulation and MTT for sensitivity to ADR...
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