Stat3靶向RNA干扰重组体构建及鉴定.docVIP

Stat3靶向RNA干扰重组体构建及鉴定 　 作者：吕伟 张超 郝迎学 刘伟 余佩武 【摘要】 目的 设计针对Stat3的发夹样RNA干扰重组体，通过脂质体转染结肠癌细胞HCT116观察其干扰效果，为探索肿瘤基因治疗的新途径奠定基础。方法 设计针对Stat3编码区的有短发夹结构的两条DNA序列，经退火成互补双链，克隆到载体Pavu6＋27中构建重组体Pavu6＋27Stat3，再对重组质粒进行酶切鉴定、DNA测序分析。脂质体法转染重组体至大肠癌HCT116细胞株中，以空质粒(Pavu6＋27)转染为对照；RTPCR法观察Stat3基因表达改变。结果 酶切鉴定和测序分析均提示Stat3靶向RNA干扰重组体的构建成功，Pavu6＋27Stat3转染后HCT116细胞Stat3基因表达较空载体Pavu6＋27转染的对照组显著下降。结论 Stat3靶向RNA干扰重组体成功构建，并能有效抑制HCT116细胞中Stat3基因表达。本实验为下一步利用RNAi技术沉默肿瘤细胞中Stat3基因的表达，诱导肿瘤细胞凋亡或抑制其增生，为探索肿瘤基因治疗的新途径打下基础。 【关键词】 Stat3 RNA干扰 HCT116细胞系 【Abstract】 Objective To construct RNA interference expression vector of targeting Stat3 and test the silencing effect by using liposome transfecting colon cancer cell line HCT116 to make foundation for exploring new way of gene therapy for cancer. Methods Two DNA sequences containing small hairpin structure were designed and synthesized. The complement form obtained by annealing was inserted into vector Pavu6＋27 to construct the recombinant Pavu6＋27Stat3, the plasmid of which was then identified by enzyme digestion and DNA sequence analysis. The recombinant plasmid was transfected into human colon cancer cell line HCT116 by DOTAP method to assay the suppression effect of Stat3 by RTPCR 48 hours after transfection. Empty plasmid (Pavu6＋27) transfected into the same cell line was set as control group. Results The enzyme digestion analysis and DNA sequencing showed that RNA interference expression vector of targeting Stat3 was constructed successfully. The expression of Stat3mRNA in HCT116 cell line transfected with Pavu6＋27Stat3 was weaker than that transfected with Pavu6＋27. Conclusions RNA interference expression vector of targeting Stat3 is successfully constructed and can effectively inhibit expression of Stat3 in colon cancer cell line HCT116. Our study makes a foundation for gene therapy for cancer by using RNAi technique to silence expression of gene Stat3 in cancer cells and induce apoptosis or inhibit proliferation of cancer cells. 【Key words】 Stat3