RNA干扰对神经母细胞瘤细胞VEGFA的mRNA表达的影响

目的 构建表达VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰作用,研究对人神经母细胞瘤细胞株IGR-N-91细胞中VEGF-A的mRNA表达的影响.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒.对重组质粒进行PCR、测序鉴定验证后,经Western Blot筛选RNA干扰效果最佳的质粒,然后将该质粒转染人神经母细胞瘤细胞株IGR-N-91.转染48 h后提取总RNA,逆转录为CDNA,经RT-PCR半定量检测VEGF-A mRNA的表达水平.结果 成功构建含有VEGF-A的RNA干扰片段真核表达载体,经Western Blot筛选后分别得到RNA干扰效果最佳的片段.RT-PCR结果表明质粒转染人神经母细胞瘤后VEGF-A的mRNA的表达升高.结论 成功构建含有人VEGF-A的RNA干扰片段的真核表达载体,该重组质粒转染人神经母细胞瘤IGR-N-91细胞后VEGF-A的mRNA表达升高.

The effect of VEGF-A mRNA expression in neuroblastoma with VEGF-A RNA interference

Objetive To construct the expression VEGF-A of the short hairpin RNA eukaryotic expres- sion vector, and mediated through its role in RNA interference research on human neuroblastoma cell line IGR -N-91 cells in the VEGF - A the mRNA expression. Methods According to VEGF - A cDNA sequences synthesis two pairs of oligonucleotide; then annealed respectively, inserted into the eukaryotic expression vector comprising the recombinant plasmid. Recombinant plasmid for PCR, sequencing and identification of validated screening by Western Blot, and then will be screened from the best RNA interference plasmid transfected hu- man neuroblastoma cell line IGR-N-91. The total RNA extracted when the plasmid was transfected for 48h, then reversed to CDNA. By semi - quantitative RT-PCR of VEGF-A mRNA expression levels were detected. Results We successfully constructed eukaryotic expression vector containing VEGF-A RNA interference frag- ments. The recombinant plasmids transfected into human neuroblastoma cell line IGR-N-91, which VEGF-A mRNA was overexpressed measuring by semi-quantitative RT-PCR. Conclusions We successfully constructed eukaryotic expression vector containing human VEGF-A RNA interference fragments. The recombinant plas- mids transfected into human neuroblastoma cell line IGR-N-91 significantly overexpressed VEGF-A mRNA.