survivin 启动子调控肿瘤干细胞标记 CD133基因 siRNA增殖型溶瘤腺病毒的构建及对肝癌细胞生长的抑制作用

摘要

目的：构建 survivin 启动子调控的靶向 CD133基因的 siRNA 增殖型溶瘤腺病毒，研究其对肝癌细胞生长的影响。方法RT－PCR 法扩增 survivin 启动子，测序鉴定，双酶切连接，获得 pH－XC2－survivin。酶切 pH－XC2－survivin、pZD55－CD133－siRNA 获得 survivin 启动子表达框的亚克隆和CD133－siRNA 基因表达框的亚克隆，连接获得 survivin 启动子调控的 siRNA 增殖型溶瘤腺病毒表达载体质粒 pT－ZD55－CD133－siRNA。增殖型溶瘤腺病毒 survivin－T－ZD55－CD133－siRNA 经 PCR 和测序鉴定。 qRT－PCR 法检测 CD133表达， Western blot 法检测 E1A，CCK－8法检测细胞生长，流式细胞术检测细胞凋亡。结果成功构建增殖型溶瘤腺病毒 sur－vivin－T－ZD55－CD133－siRNA。 qRT－PCR 法检测 CD133 mRNA明显下降， Western blot 证实 survivin－T－ZD55－CD133－siRNA在肿瘤细胞中表达 E1A 能抑制肝癌细胞 CD133表达及生长。结论构建的增殖型溶瘤腺病毒可有效降低肝癌细胞CD133的表达，用于肝癌基因治疗的进一步研究。%Objective To construct a replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and investigate its inhibitory effect on Hep 3B cells.Methods The fragment of the survivin promoter was amplified by PCR and inserted into pH -XC2 to reconstruct a recombinant plasmid pH -XC2-survivin.Complete digestion pH-XC2-survivin and pZD55-CD133-siRNA, combinational joining the subclones, then getting replication-competent adenovirus expressing short interference RNA targeting CD 133 gene regulated by survivin promoter, replication-competent adenovirus was constructed .The recombined adenoviruses ( T-ZD55-CD133-siRNA) were verified by PCR and sequencing .The effect of T-ZD55-CD133-siRNA on CD133 expression in Hep3B cells was detected by qRT-PCR.The expression of E1A was detected by Western blot.The antitumor po-tential of replication-competent adenovirus in Hep3B cells were evaluated by CCK-8 assay and cell apoptosis was detected by Flow cytometry.Results Replication-competent adenovirus were constructed successfully .Western blot analyses indicated that T-ZD55-CD133-siRNA might express E1A in adenovirus-infected Hep3B cells.T-ZD55-CD133-siRNA were more effective to inhibit CD133 mRNA expression and Hep3B cells proliferation.Apop-tosis was significantly increased in the interference group compared with the control group .Conclusion Survivin-T-ZD55-CD133-siRNA expressing CD133-siRNA can inhibit CD133 expression and may be used for further investi -gation of gene therapy for liver cancer.