microRNA-22真核表达载体的构建及其在卵巢癌细胞系SKOV-3中的稳定表达  

Construction of MicroRNA-22 Eukaryotic Expression Vector and Stable Expression in Ovarian Cancer Cell Line SKOV-3

作　　者：张蕊[1] 汤锋[1] 邓琳[1] 邢莹莹[1] 奚涛[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2012年第3期209-212,共4页Pharmaceutical Biotechnology

摘　　要：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 resolve the problem of short half life of microRNA mimics for long-term research, a miR-22 eukaryotic expres- sion vector was constructed and transfected into human ovarian cancer cell line SKOV-3 to establish a eel1 model of stable expression of miR-22 ,which would provide an effective tool for the study of the effect of miR-22 on carcinogenesis and tumor progression. Firstly, Psilencer4.1-miR-22 expression plasmid and the negative control Psilencer4.1-control were designed and constructed according to the sequence of Pre-miR-22. Then the vectors were amplified in host cells, digested with restrictive endonuclease, identified by sequencing,transfected into human ovarian cancer cell line SKOV-3, at last the cell line stably expressing miR-22 （experimental group） and control-RNA （negative control group） were established by G418 selection. Then, cell wound healing assay and transwell migration assay were utilized to detect the migration potential of the cell lines. Sequencing result showed that the miR-22 eukaryotic expression vector with correct sequence was successfully constructed, miR-22 mRNA expression level of SKOV-3 cells transfected with Psilencer-4.1-miR-22 was significantly up-regulated （ P 〈 O. 05 ） and the migration potentiality was significantly decreased （ P 〈 0.05 ） compared with the control group. In conclusion, a recombinant plasmid carrying Pre-miR-22 was successfully constructed and SKOV-3 cell line of stable expression of miR-22 after transfection was obtained. Our study could be applied for further research on cancer metastasis.

关 键 词：microRNA-22 肿瘤转移 真核表达质粒 人卵巢癌细胞系SKOV-3 

分 类 号：Q7[生物学—分子生物学]