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本研究旨在建立miRNA靶基因高通量筛选系统,筛选出能调控p21NAs,以便试验验证及探索这些miRNA的生物学功能及意义。利用分子克隆技术构建并鉴定2个慢病毒表达载体-pWPXL-Luc及pWPXL-Luc-P21-3’UTR,分别与包装载体psPAX-2及PDM2G共转染HEK 293T细胞;包装病毒颗粒,感染HEK 293细胞,获得稳定单表达荧光素酶及共表达荧光素酶与P21-3’UTR的细胞株,前者在后续实验中作为对照;采用荧光素酶检测试剂检测pWPXL-Luc病毒感染后的293细胞的荧光素酶活性。结果表明:包装出高滴度的病毒颗粒,成功建立稳定株,且在一定范围内稳定株细胞的荧光素酶活性与细胞数目成正比。结论:成功建立了miRNA靶基因高通量筛选系统;利用本筛选系统,成功筛选出靶向细胞周期调控基因P21(CIP1/WAF1)的miRNA。
The purpose of this study was to establish a high-throughput screening system for miRNA target genes and to screen for the ability to regulate p21NAs in order to validate and explore the biological functions and significance of these miRNAs. Two lentiviral expression vectors, pWPXL-Luc and pWPXL-Luc-P21-3’UTR, were constructed and identified by molecular cloning. The recombinant vectors were co-transfected with packaging vector psPAX-2 and PDM2G into HEK 293T cells. The virus particles were packaged and infected with HEK 293 cells to obtain stable expression of single luciferase and co-expression of luciferase and P21-3’UTR cell lines, the former in the follow-up experiment as a control; luciferase detection reagent detection of 293 cells infected with pWPXL-Luc virus Luciferase activity. The results showed that the high titer virus particles were packaged and the stable strains were successfully established. The luciferase activity of the stable strain cells was proportional to the number of cells within a certain range. Conclusion: A high-throughput screening system for miRNA target genes was successfully established. Using this screening system, miRNAs targeting the cell cycle regulatory gene P21 (CIP1 / WAF1) were successfully screened.