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目的构建神经元表达发育下调基因9(NEDD9)特异性微小核糖核酸(miRNA)干扰质粒,检测其在胃癌SGC-7901细胞中的干扰效果。方法根据GenBank中提供的NEDD9基因序列,设计4种NEDD9靶向干扰序列(MR0112-1、MR0112-2、MR0112-3和MR0112-4)及1种阴性对照干扰序列(MR0112-N),连接到pcDNA6.2-GW/EmGFP-miR载体中,构建重组质粒。重组质粒转染人胃癌细胞株SGC-7901;采用FQ-PCR和Western blot法检测转染前后SGC-7901细胞中NEDD9mRNA及蛋白的表达。结果测序表明,4种干扰质粒的干扰序列及读框完全正确。FQ-PCR和Western blot结果显示,转染MR0112-1、MR0112-2、MR0112-3和MR0112-4质粒后其NEDD9表达较未处理组分别下降88.01%、96.70%、78.55%和81.34%(P<0.05),以MR0112-2干扰效果最为明显。阴性质粒组NEDD9mRNA表达与未处理组比较无明显变化(P>0.05)。结论成功构建了NEDD9干扰质粒,为进一步研究NEDD9基因表达在胃癌增殖、迁移、侵袭过程中的作用提供实验基础。
Objective To construct a specific miRNA interference plasmid targeting neurodegenerative development-9 (NEDD9) gene in gastric cancer SGC-7901 cells. Methods Four NEDD9 targeted interference sequences (MR0112-1, MR0112-2, MR0112-3 and MR0112-4) and one negative control interference sequence (MR0112-N) were designed based on the NEDD9 gene sequence provided in GenBank. pcDNA6.2-GW / EmGFP-miR vector to construct a recombinant plasmid. The recombinant plasmids were transfected into human gastric cancer cell line SGC-7901. The expression of NEDD9 mRNA and protein in SGC-7901 cells was detected by FQ-PCR and Western blot. Results Sequencing showed that the interference sequences and reading frames of the four kinds of interference plasmids were completely correct. The results of FQ-PCR and Western blot showed that the expression of NEDD9 in MR0112-1, MR0112-2, MR0112-3 and MR0112-4 transfected cells decreased by 88.01%, 96.70%, 78.55% and 81.34% (P <0.05), the effect of MR0112-2 interference is the most obvious. Negative plasmid NEDD9mRNA expression compared with the untreated group had no significant change (P> 0.05). Conclusion The NEDD9 interference plasmid was successfully constructed and provided an experimental basis for further study of the role of NEDD9 gene in the proliferation, migration and invasion of gastric cancer.