TSG101 siRNA表达载体的构建及其作用研究

Construction and identification of TSG101 siRNA eukaryotic expression vectors

目的构建TSG101(tumor susceptibility gene 101,TSG101)小干扰RNA(TSG101 siRNA)的真核表达载体并鉴定,初步研究其与胃癌多药耐药的相关性。方法根据小干扰RNA的设计原则由计算机软件辅助设计TSG101的靶序列,应用DNA重组技术将目的序列克隆入小干扰RNA的表达载体;将TSG101 siRNA的真核表达载体转染胃癌长春新碱(VCR)耐药细胞SGC7901/VCR,通过Western blot鉴定TSG101 siRNA的抑制效率;以MTT实验检测细胞生长曲线和对VCR、阿霉素(ADM)的药物敏感性。结果所构建的载体经酶切后释放出预期大小的片断并经测序证实;TSG101 siRNA真核表达载体瞬时转染胃癌耐药细胞SGC7901/VCR后,发现TSG101的表达被显著抑制;细胞生长减慢,且对VCR、ADR的敏感性增加。结论成功构建了TSG101 siRNA的真核表达载体,并初步提示其参与了胃癌的多药耐药,为下一步工作奠定了基础。

Objective To construct and identify the eukaryotic expression plasmids of TSG101 siRNA and examine the role in multidrug resistance of gastric cancer. Methods The targeting fragments specifically against TSG101 were designed according to the principle of small interfering RNA designation using computer software. The sequences were cloned into siRNA expression plasmids （mU6pro） through DNA recombinant technology. TSG101 siRNA was introduced into vincristine （VCR） resistant gastric cancer cells （SGC7901/VCR） with lipofectamineTM2000 as directed by manufacturer. The expression levels of TSG101 in SGC7901/VCR and the transfectants were detected with Western blot, Growth curve and drug sensitivity of ceils for vincristine and adriamycin （ADR） were assayed by MTT assay. Results The expected fragments were obtained by digestion identification and further confirmed by DNA sequencing. The expression of TSG101 was dramatically silenced in TSG101 siRNA transfectants with Western blot. MTT assay showed that transfectants proliferated slowly and were more sensitive to VCR and ADR than non-transfectants. Conclusion The eukaryotic expression vector of TSG101 siRNA is successfully constructed. The preliminary study suggests that TSG101 may participate in muhidrug resistance of gastric carcinoma. The study establishes a foundation for further study.