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浙江大学学报（医学版）
Journal of ZheJiang University(Medical Science)  2012, Vol. 41 Issue (1): 81-88    DOI: 10.3785/j.issn.1008-9292.2012.01.014
    
Construction of miRNA expression vector dual-targeting on HIF-1α/survivin genes and its effects on proliferation of pancreatic cancer Panc-1 cell line
XU Sun-bing，ZHU Yi-ping，MOU Yi-ping，ZHU Ling-hua
Sir Run Run Shaw Hospital，Zhejiang University School of Medicine，Hangzhou 310016，China
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Abstract  Objective： To design and construct miRNA expression vector dual-targeting on HIF-1α and 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-1α 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-1α 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.

Key words： Pancreatic neoplasms/genetics； Microtubule-associated proteins/genetics； Hypoxia-inducible factor 1/genetics； Gene expression； Genetic vectors； Plasmids/genetics； Transfection； Tumor cells      cultured； RNA interference； MicroRNAs     
Published: 25 January 2012
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. Construction of miRNA expression vector dual-targeting on HIF-1α/survivin genes and its effects on proliferation of pancreatic cancer Panc-1 cell line. Journal of ZheJiang University(Medical Science), 2012, 41(1): 81-88.

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https://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2012.01.014     OR     https://www.zjujournals.com/med/Y2012/V41/I1/81


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

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)，72 h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)。

关键词： 胰腺肿瘤/遗传学,  微管相关蛋白质类/遗传学,  缺氧诱导因子1/遗传学,  基因表达,  遗传载体,  质粒/遗传学,  转染,  肿瘤细胞,  培养的,  RNA干扰,  微RNAs 
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