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中国癌症防治杂志

中国癌症防治杂志 ›› 2011, Vol. 3 ›› Issue (3): 187-191.doi: 10.3969/j.issn.1674-5671.2011.03.02

• 基础研究 • 上一篇    下一篇

TIZ基因mRNA在卵巢癌细胞系中的表达及其RNA干扰载体构建

赵冰冰, 张玮, 李力, 阳志军   

  • 出版日期:2011-09-25 发布日期:2011-10-17
  • 通讯作者: 阳志军 E-mail:yzj7528@126.com
  • 基金资助:

    广西医学科学实验中心开放基金资助项目（编号：kfjj2010-10）

Expression of TIZ mRNA in ovarian cancer cell lines and the construction of RNA interference vectors

ZHAO Bing-Bing, ZHANG Wei, LI Li, YANG Zhi-Jun   

  • Online:2011-09-25 Published:2011-10-17

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被引次数

3

摘要： 目的 检测卵巢癌细胞株中TIZ基因的mRNA表达水平，并构建TIZ基因的特异性RNA干扰(RNAi)载体。方法 通过RT-PCR方法检测卵巢癌细胞系（SKOV3、SKOV3顺铂耐药细胞、SKOV3卡铂耐药细胞、A2780、A2780卡铂耐药细胞、HO8910）中TIZ mRNA的表达；根据Genebank上TIZ的mRNA序列，设计5对siRNA干扰片段，采用脂质体转染法介导siRNA干扰片段并下调卵巢癌细胞系中TIZ mRNA的表达。采用pTG19-T质粒构建TIZ 和β-actint mRNA表达重组质粒并制备其标准品。采用实时定量PCR（QRT-PCR）检测siRNA干扰片段对TIZ基因的下降效率，并筛选最佳siRNA干扰片段。以pGPU6/GFP/Neo载体和质粒DNA测序方法构建pGPU6/GFP/Neo-siRNA-TIZ-573重组质粒。结果 ①除卵巢癌细胞HO8910细胞无TIZ基因mRNA表达外，其余细胞均有TIZ基因mRNA表达。②细胞siRNA干扰片段筛选结果显示介导TIZ-573片段后可下调60%细胞TIZ mRNA表达。③成功构建了TIZ基因RNAi载体pGPU6/GFP/Neo-TIZ-573。结论 多数卵巢癌细胞系表达TIZ mRNA。siRNA-TIZ-573为最佳下调细胞TIZ mRNA片段。构建的pGPU6/GFP/Neo-TIZ-573重组质粒可用于下一步的实验研究。

关键词: TIZ基因, 卵巢癌细胞, siRNA

Abstract: Objective To detect the level of TIZ mRNA in ovarian cancer cell lines and to construct the vector of TIZ mRNA interference (RNAi).Methods The expression levels of TIZ mRNA in several ovarian cancer cell lines were detected.The cell lines included SKOV3，SKOV3-cisplatin resistance，SKOV3-carboplatin resistance，A2780，A2780-cisplatin resistance and HO8910.Five pairs of TIZ siRNA were designed according to the sequence of TIZ mRNA in Genebank.The siRNAs were transfected by liposome to down-regulate the expression of TIZ mRNA in ovarian cancer cell lines.TIZ mRNA and β-actin mRNA expression plasmids were construct red by using pTG19-T plasmid，and their standards were prepared.The real-time quantitative polymerase chain reaction (QRT-PCR) was used to detect the efficiency of TIZ siRNA and then to select the best one.The pGPU6/GFP/Neo vector and plasmid DNA sequencing method was used to build pGPU6/GFP/Neo-siRNA-TIZ-573 restructured plasmid.Results TIZ mRNA expressed in all ovarian cancer cells except HO8910.siRNA TIZ-573 down regulated 60% of TIZ mRNA expression.The vector TIZ RNAi pGPU6/GFP/Neo-TIZ-573 was constructed successfully.Conclusions Most ovarian cancer cell lines express TIZ mRNA.siRNA-TIZ-573 is the best segments that can down regulate TIZ mRNA.The recombinanted plasmid GPU6/GFP/Neo-TIZ-573 can be used for the next experiments.

Key words: TIZ gene, Ovarian cancer cells, siRNA