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[目的]构建靶向COX-2基因的microRNA真核表达载体,评估其转染人喉癌细胞株Hep-2细胞后对COX-2基因的干扰效果。[方法]根据COX-2 mRNA序列设计合成4对microRNA片段,定向克隆到pcDNA6.2GW/EmGFPmiR真核表达载体上,采用测序分析鉴定插入序列的完整性;并将其转染至Hep-2细胞株中。采用实时荧光定量PCR检测其对COX-2基因表达的干扰效果以得到最佳干预效果的RNAi片段。[结果]构建的4组重组体插入片段的碱基序列完全正确。4组microRNA片段对Hep-2细胞中COX-2基因mRNA表达水平均有下降。其中COX-2 mi-1组的表达水平最低。[结论]从构建的4组COX-2 microRNA重组体中成功的筛选出最佳干扰靶点质粒,即COX-2 mi-1组,为应用microRNA靶向COX-2的基因治疗的研究奠定了基础。
[Objective] To construct the eukaryotic expression vector of microRNA targeting COX-2 gene and evaluate its effect on COX-2 gene transfection in human laryngeal carcinoma cell line Hep-2. [Method] Four pairs of microRNA fragments were designed and synthesized based on the COX-2 mRNA sequence. The fragments were cloned into the eukaryotic expression vector pcDNA6.2GW / EmGFPmiR. The integrity of the inserted sequence was identified by sequencing analysis. The inserted sequence was then transfected into Hep-2 cells Strain. Real-time fluorescent quantitative PCR was used to detect the interference effect of COX-2 gene expression to get the best RNAi fragment. [Result] The constructed 4 sets of recombinants had complete correct nucleotide sequence. Four microRNA fragments of COX-2 mRNA expression levels in Hep-2 cells were decreased. Among them, COX-2 mi-1 group had the lowest expression level. [Conclusion] The best interference target plasmid (COX-2 mi-1) was successfully screened from the four COX-2 microRNA recombinants constructed, which laid the foundation for the study of gene therapy using microRNA targeting COX-2 basis.