人分化抑制因子3基因靶向微小分子RNA表达载体的构建及筛选

Construction and identification of miRNA expression vectors targeting human Id3 gene

目的人分化抑制因子3(inhibitor of differentination 3,Id3)基因在A549细胞中的外源性表达能抑制细胞生长并诱导细胞凋亡。文中旨在构建靶向人Id3基因的微小分子RNA(miRNA)表达载体,观察miRNA对A549细胞中Id3表达的下调作用,为进一步探讨Id3基因在A549细胞生长调控中的作用机制提供一定实验依据。方法依据miRNA设计原则,针对人Id3基因的mRNA序列,设计并合成编码miRNA的2条寡核苷酸序列,经退火成互补双链,再克隆至pcDNATM6.2-GW/EmGFP-miR真核表达载体中,构建含靶向人Id3基因的miRNA表达质粒(pcDNA6.2-GW/EmGFP-miR-Id3),DNA测序验证后,采用Lipofectamine2000TM脂质体转染技术将该质粒导入A549细胞。荧光倒置显微镜下观察miRNA干扰质粒转染A549细胞后EGFP的表达情况,应用RT-PCR和Western blot方法分别检测miRNA干扰后Id3mRNA及蛋白水平的表达。结果DNA测序表明重组质粒pcDNA6.2-GW/EmGFP-miR-Id3构建正确,将其成功导入A549细胞后,在Id3mRNA水平和蛋白表达水平均有不同程度的抑制作用。结论成功构建了针对人Id3基因的4组pcDNA6.2-GW/EmGFP-miR-Id3miRNA表达载体,其中,SRId3-1干扰组在mRNA水平和蛋白表达水平均能有效抑制A549细胞Id3的表达。

Objective The exogenous expression of the gene inhibitor of differentiation 3 （Id3） in A549 cells can induce cell growth inhibition and apoptosis. This study aimed to construct miRNA expression vectors targeting the human Id3 gene, observe the down-regulatory effect of miRNA-mediated RNA interference （RNAi） on the Id3 expression, and provide some evidence for further study of cell growth regulation mechanism of Id3 in A549 cells. Methods According to the encoding sequence of Id3 mRNA, two oligonucleotide sequences were designed and synthesized, and the annealed oligonucleotide fragments subcloned into the pcDNATM6.2-GW/EmGFP-miR expression vector. After identified by DNA sequencing, the recombinant plasmids pcDNA6.2-GW/EmGFP-miR-Id3 were transfected into A549 cells by Lipofectamine2000TM. Transfection efficiencies were monitored by inverted fluorescence microscopy, and the expressions of Id3 mRNA and proteins measured by reverse transcription polymerase chain reaction （RT-PCR） and Western blot respectively. Results DNA sequencing results indicated that the recombinant expression plasmids pcDNA6.2-GW/EmGFP-miR-Id3 were correctly constructed and successfully expressed in A549 cells. The expressions of Id3 mRNA and proteins in A549 cells were inhibited by pcDNA6.2-GW/EmGFP-miR-Id3 at different levels. Conclusion We successfully constructed 4 groups of pcDNA6.2-GW/EmGFP-miR-Id3 expression vectors targeting the human Id3 gene, among which SRId3-1 could effectively inhibit the expressions of Id3 mRNA and proteins in A549 cells.