靶向血管内皮生长因子基因的微小RNA真核表达载体的构建

Construction of a microRNA expressing eukaryotic vector targeting vascular endothelial growth factor

背景:有研究表明微小RNA及潜在靶基因在肝癌中起重要作用,但具体机制仍不清楚。目的:构建靶向血管内皮生长因子基因微小RNA真核表达载体,评估其转染人肝癌细胞株HepG2后血管内皮生长因子基因的干扰效果。方法:根据血管内皮生长因子序列设计合成4对微小RNA不同干扰片段,克隆到pcDNA6.2-GW/EmGFP-微小RNA真核表达载体上,测序分析鉴定插入序列的完整性;并将其转染至HepG-2细胞株中。采用实时荧光定量聚合酶链式反应分析血管内皮生长因子微小RNA干扰效果,蛋白印迹技术测定重组体对血管内皮生长因子基因蛋白表达情况。结果与结论:构建的4组重组体插入片段的碱基序列完全正确,重组体能干扰肝癌细胞HepG2细胞血管内皮因子基因的表达,4组重组体基因的mRNA的表达水平和蛋白表达水平与阴性对照组相比明显降低(P<0.05),其中血管内皮生长因子微小RNA-3表达水平最低,抑制率87%。结果证实,实验成功构建血管内皮生长因子微小RNA表达载体,在体外能有效抑制HepG2细胞血管内皮生长因子基因表达。

BACKGROUND：Studies have shown that the microRNA（miRNA） and its potential target gene play an important role in hepatocellular carcinoma,but the exact mechanism is still unclear.OBJECTIVE：To construct a vascular endothelial growth factor（VEGF） miRNA expressing eukaryotic vector and to identify biological activity of VEGF miRNA transfected into HepG-2 cells.METHODS：According to the sequence of VEGF mRNA,the VEGF miRNA was designed and synthesized,and then cloned into the pcDNA6.2-GW/EmGFP-miRNA vector and transfected into HepG-2 cell lines.The integrity of the insert fragment was detected using colony PCR and sequencing analysis.The biological activity of VEGF miRNA by way of real-time PCR and Western blot was determined.RESULTS AND CONCLUSION：Sequences of the inset fragment in the four miRNA expressing recombinants were correct.VEGF mRNA expression of the four miRNA recombinants were significantly decreased（P 0.05）,especially in the VEGF-miRNA-3 with an inhibitory rate of 87%.Four VEGF-targeting miRNA expressing recombinants were successfully constructed which can significantly inhibit VEGF gene expression in HepG2 cells.