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    基于CRISPR/Cas9技术的miR-374a低表达细胞系的构建及鉴定

    Constructing and identifying of cell line with low-expression miR-374a based on CRISPR/Cas9 system

      摘要
    • 摘要:
      目的运用CRISPR/Cas9技术构建miR-374a低表达细胞系。
      方法设计针对miR-374a成熟体gRNA，合成DNA，插入pSpCas9（BB）-2A-Puro，将鉴定正确的载体转染入肺癌细胞系A549细胞，使用嘌呤霉素筛选转染的细胞；PCR方法扩增，序列测定分析细胞基因组中靶位点区域序列；定量PCR方法检测转染细胞中miR-374a及其靶基因TGFA的表达情况；细胞增殖实验检测miR-374a基因编辑对细胞生长情况的影响。
      结果构建了靶向miR-374a成熟体基因序列的基因编辑载体，转染细胞经过加压筛选，序列分析结果提示靶向位点的基因发生了缺失或插入突变；定量PCR结果显示，转染基因编辑载体的细胞miR-374a表达水平较对照细胞明显下降（P < 0.01），而靶基因TGFA表达则明显上升（P < 0.05）；细胞增殖实验显示miR-374a低表达的细胞增殖能力发生了明显改变（P < 0.05~P < 0.01）。
      结论成功构建了miR-374a低表达细胞系，为后续研究该分子的功能提供了基础。

       

      Abstract:
      ObjectiveTo establish the cell line with low-expression miR-374a using CRISPR/CAS9 gene targeting technology.
      MethodsThe mature gRNA targeting miR-374a was designed to synthesize the DNA, which was inserted into the CRISPR/CAS9 vector to construct the pSpCas9(BB)-2A-Puro.A549 cells were transfected with the constructed vector, and screened using puromycin.The gene sequences of target site were analyzed using PCR and sequencing method.The qPCR was used to detect the relative expression levels of miR-374a and its target gene TGFA in transfected cells.The CCK8 was used to detect the proliferation activity of cells.
      ResultsThe gene targeting miR-374a vector was successfully constructed.The transfected cells were screened under pressure, and the sequencing results showed that the inserting/deletion mutation was found.The results qPCR showed that the expression level of miR-374a significantly decreased, and the expression level of TGFA significantly increased in transfecting gene editing vector compared with control cells(P < 0.01 and P < 0.05).The cell proliferation assay showed that the proliferation activity significantly changed in low-expressed miR-374a cells(P < 0.05 to P < 0.01).
      ConclusionsThe low-expressed miR-374a cell line was successfully established, which provides the basis in studying the molecular function.

       

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