miR-20a海绵载体构建及在白血病细胞株Jurkat中的表达

Construction of miRNA Sponge Targeting miR-20a and Stable Expression in Jurkat Leukemia Cell Line

本研究构建miR-20a基因的miRNA海绵载体,建立Jurkat-S稳定细胞株,为进一步研究的miR-20a功能及应用干扰技术治疗奠定基础。设计、合成1对含有2个重复针对miR-20a成熟序列第9-12碱基错配的序列,并带有酶切位点,退火后连接到pCDNA3.0-L表达载体上;载体双酶切后再与退火产物连接,重复4次后进行酶切及荧光素酶活性鉴定;亚克隆到慢病毒表达载体,与psPAX2、PMD2G共转染HEK 293T细胞,包装产生慢病毒颗粒并测定病毒滴度,感染Jurkat细胞,建立稳定细胞株;应用实时PCR和Western blot技术分别检测Jurkat-S稳定细胞中P21及E2F1 mRNA和蛋白的表达,并与对照组进行比较。结果表明,成功构建了针对miR-20a基因的海绵载体,病毒滴度为5×107TU/ml;建立了稳定转染的Jurkat-S细胞株。有效干扰验证显示,miR-20a海绵能明显上调P21及E2F1的mRNA及蛋白表达水平,差异有统计学意义(P<0.05)。结论:成功构建miR-20a基因的miRNA海绵载体,建立稳定干扰miR-20a表达的Jurkat-S细胞株。

This study was aimed to construct miRNA sponge targeting miR-20a and to establish a stable cell line Jurkat-S,paving the way for further research on function of miR-20a and application of RNAi in gene therapy.One pair of two-repeated oligonucleotide sequences containing bulged sites that are mispaired opposite miR-20a positions 9-12 was designed and synthesized with enzyme cutting sites.The annealed oligonucleotide fragments were subcloned into pCDNA3.0-L expressing vector.After double-enzyme cutting,the vector was ligated to the annealed oligonucleotide fragments again.Enzyme cutting and luciferase activity assay were performed for identification after four repeats.Then the ligated fragment was subcloned to lentivirus expressing vector.Virus particles were collected after the control or sponge vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pMD2.G into HEK-293T cells using Lipofectamine2000.The Jurkat cells were transdused with recombinant lentivirus-transdusing units plus 6 μg/ml of Polybrene.Real-time PCR and Western blot were used to detect the mRNA and protein expression of P21 and E2F1 after lentivirus transdusion respectively.As a result,luciferase activity assay demonstrated that the sponge targeting miR-20a was constructed successfully and the virus was packaged in 293T.The titer of virus was 5×107 TU /ml.Stable transfected Jurkat-S cell line was established.As was expected,the mRNA and protein level of P21 and E2F1 was upregulated significantly in Jurkat-S cells.It is concluded that the miR-20a sponge is constructed successfully,and Jurkat-S stable cell line is established,in which the expression of miR-20a is inhibited stably.