靶向GCSsi RNA表达载体与胃癌细胞耐药性

Construction of siRNA expression vector for glueosylceramide synthase and its effect on drug resistance of gastric carcinoma cells

目的构建葡萄糖神经酰胺合成酶(GCS)小干扰RNA(SiRNA)表达载体,观察其对胃癌耐药细胞(SGC7901/VCR)GCS基因表达和耐药性的影响。方法根据siRNA的设计原则,针对人GCS的mRNA序列设计并合成编码siRNA的寡核苷酸序列,并将其克隆入SiRNA的表达载体,构建重组质粒,将重组质粒转染胃癌长春新碱(VCR)耐药细胞SGC7901/VCR,通过蛋白印迹实验(WB)等方法检测转染细胞GCS的表达水平,采用四甲基偶氮噻唑蓝(MTT)法检测细胞耐药情况。结果构建的GCS siRNA表达载体经限制性酶切分析,证实释放出的片段大小与预期结果一致;转染SGC7901/VCR细胞后,可使细胞中GCS的表达受抑制,并使细胞对VCR的敏感性增加。结论GCS siRNA表达载体的成功构建及其对GCS表达的抑制和细胞耐药的逆转,使其可能成为肿瘤基因治疗的一种新手段。

Objective To construct a glucosylceramide synthase（GCS） specific small interfering RNA（siRNA） expression vector and to investigate its effect on GCS expression and multidrug resistance in gastric carcinoma cells. Methods According to the encoding sequence of mRNA of GCS, oligonucleotide sequences were designed and synthesized, and then cloned into siRNA expression vector to generate the plasmid pSUPER GCS. The recombinant plasmid was transfected into vincristine（VCR） resistant gastric carcinoma cells SGC7901/VCR. The expression levels of GCS were detected by western blot. The drug resistance of SGC7901/VCR was assayed by MTT. Results The expected fragment was obtained by restriction endonuclease digestion; The constructed vector effectively inhibited the expression of GCS in SGC7901/VCR cells. The sensitivity to VCR of the transfected cells was increased. Conclusion GCSsiRNA expression vector is successfully constructed, which suppresses GCS expression and reverses drug resistance of SGC7901/VCR. It will be hdpful for the gene therapy of tumors.