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为了解决microRNA模拟物半衰期较短,无法达到长时间研究的问题,实验构建了miR-22真核表达质粒并转染人卵巢癌细胞系SKOV-3,建立稳定表达miR-22的细胞模型,为研究miR-22在肿瘤发生发展过程中的作用提供有效的工具。首先设计并构建过表达miR-22的Psilencer 4.1-miR-22重组质粒,经宿主菌扩增、酶切、测序鉴定后,重组质粒和阴性对照质粒分别转染卵巢癌细胞系SKOV-3,G418筛选建立稳定表达miR-22(实验组)和control-RNA(阴性对照组)的SKOV-3细胞系,用qRT-PCR验证所构建的细胞系中miR-22表达量的差异。最后通过细胞划痕-修复实验和侵袭小室实验观察过表达miR-22对卵巢癌细胞迁移能力的影响。经宿主菌扩增、酶切、测序证实,成功构建miR-22真核表达质粒,插入的DNA片段与设计序列完全一致。与对照组相比,在建立的稳定表达miR-22的SKOV-3细胞中,miR-22 mRNA水平明显上调(P<0.05),细胞的迁移能力也显著下降(P<0.05)。综上所述,成功构建了过表达Pre-miR-22的重组质粒,并获得了稳定表达miR-22的SKOV-3细胞系。
In order to solve the problem that the half-life of microRNA mimics is short and can not be prolonged for a long time, a miR-22 eukaryotic expression plasmid was constructed and transfected into human ovarian cancer cell line SKOV-3 to establish a cell model stably expressing miR-22 To study the role of miR-22 in tumorigenesis and development provides an effective tool. The Psilencer 4.1-miR-22 recombinant plasmid overexpressing miR-22 was designed and constructed. After amplified, digested and sequenced, the recombinant plasmids and negative control plasmids were transfected into ovarian cancer cell lines SKOV-3, G418 The SKOV-3 cell line stably expressing miR-22 (experimental group) and control-RNA (negative control group) was screened. The difference of miR-22 expression in the constructed cell lines was verified by qRT-PCR. Finally, the effects of miR-22 overexpression on migration of ovarian cancer cells were observed by cell scratch-repair experiments and invasion chamber experiments. The host bacteria amplification, digestion, sequencing confirmed that the successful construction of miR-22 eukaryotic expression plasmid, inserted DNA fragment and the design sequence is exactly the same. Compared with the control group, the miR-22 mRNA level was significantly up-regulated (P <0.05) and the cell migration ability was also significantly decreased (P <0.05) in SKOV-3 cells stably expressing miR-22. In conclusion, a recombinant plasmid overexpressing Pre-miR-22 was successfully constructed and a SKOV-3 cell line stably expressing miR-22 was obtained.