抗HIF-1α和survivin基因双靶位miR-RNAi载体的构建及其对胰腺癌细胞的作用

摘要

目的:构建抗HIF-1α单靶位(anti-H)、抗Survivin单靶位(anti-S)以及抗HIF-1α和Survivin基因双靶位微小RNA表达载体(anti-H+S),观察和比较这3种载体对胰腺癌Panc-1细胞增殖的影响.方法:设计4组靶向HIF-1α和Survivin基因的寡核苷酸序列,连接至pcDNA6.2-GW/EmGFP-miR质粒,构建两组共8个载体,依次为anti-H(Ⅰ)、anti-H(Ⅱ)、anti-H(Ⅲ)、anti-H(Ⅳ)和anti-S(Ⅰ)、anti-S(Ⅱ)、anti-S(Ⅲ)、anti-S(Ⅳ).实时定量RT-PCR检测靶基因mRNA的表达水平,将两组中mRNA下调作用最强的质粒串联,构建anti-H+S.用anti-H+S及mRNA下调作用最强的anti-H和anti-S转染胰腺癌Panc-1细胞,Western blot测定靶蛋白表达情况,MTT法检测细胞增殖活性.结果:经测序鉴定,所有anti-H、anti-S及双靶位质粒anti-H+S装载位点核苷酸序列与设计序列完全相符.anti-H的靶基因mRNA沉默效率依次为48％、2％、44％和30％；anti-S的靶基因mRNA沉默效率依次为72％、75％、58％和59％.由anti-H(Ⅰ)和anti-S(Ⅱ)串联得到双靶位质粒anti-H+S.anti-H+S的靶基因mRNA沉默效率为53％ (HIF-1α)和42％(survivin).anti-H(Ⅰ)、anti-S(Ⅱ)及anti-H+S与对照质粒相比均使靶基因蛋白的表达明显下降,细胞增殖受到明显抑制(P＜0.05).其中anti-H+S对细胞的增殖抑制作用强于anti-H(Ⅰ)和anti-S(Ⅱ),72h后差异有统计学意义(P＜0.05).结论:本研究成功构建了抗HIF-1α和Survivin的单靶位质粒和双靶位质粒；其中,anti-H+S抑制胰腺癌Panc-1细胞增殖作用强于anti-H(Ⅰ)和anti-S(Ⅱ).%Objective:To design and construct miRNA expression vector dual-targeting on HIF-1αand survivin genes and to investigate its effects on proliferation of human pancreatic cancer cells. Methods; The specific pre-miRNA single strand DNA oligos for HIF-1α and survivin genes were designed and synthesized,then via annealing and ligating with pcDNA6. 2-GW/EmGFP-miR plasmids in order,two kinds (eight in total) of miRNA expression vectors for HIF-1 a and survivin genes were constructed. The vectors,which were most effective to knockdown target genes,were screened with real-time RT-PCR and combined by chaining technology to construct dual-targeting plasmid. The recombined dual-targeting plasmid,mono-targeting plasmids and negative plasmid were transfected into Panc-1 cells,the suppression effect on two genes was identified by real-time RT-PCR, Western blot and MTT assays. Results; The miRNA expression plasmids anti-H, anti-S and anti-H + S were successfully constructed by identification of sequencing analysis,and they were able to effectively inhibit the target genes expressing. MTT assays showed that the inhibition effect of dual-targeting vector anti-H + S was higher than that of mono-targeting vectors anti-H and anti-S 72 h after transfection ( P < 0. 05). Conclusions; The effective miRNA expression vector dual-targeting on HIF-1 α and survivin genes has been successfully constructed. The inhibition effect on proliferation of pancreatic cancer Panc-1 cells by dual-targeting plasmid was higher than that by mono-target plasmids.