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目的:探讨siRNA干扰Slug基因表达对BxPC-3细胞增殖、凋亡和对5-FU化疗敏感性的影响。方法:用脂质体转染法将表达siRNA-Slug的真核表达载体pGenesil-1-Slug-siRNA(pSlug-siRNA)和空载体pGen-esil-1-Neg-siRNA(pNeg-siRNA)导入胰腺癌BxPC-3细胞,G418筛选阳性细胞,获得稳定转染的阳性克隆。将转染载体的BxPC-3阳性克隆细胞和未转染载体的BxPC-3细胞分别暴露于系列浓度(0.01～100μmol/L)的5-FU72h。RT-PCR和蛋白质印迹法检测不同组细胞经5-FU作用72h后的Slug表达;MTT法测定各组细胞生存率并计算IC50,Hoechst33258和PI双染色和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测细胞的凋亡。结果:实验组细胞内Slug基因被有效沉默;BxPC-3、Bx-PC-3/pSlug-siRNA和BxPC-3/pNeg-siRNA细胞的5-FUIC50分别为(11.0±2.1)、(1.5±0.4)和(9.7±1.3)μmol/L(P<0.05).BxPC-3/pSlug-siRNA细胞对5-FU作用的敏感性增加了7.33倍。5-FU以剂量依赖方式诱导BxPC-3细胞凋亡,但对BxPC-3/pSlug-siRNA细胞所诱导的凋亡比BxPC-3和BxPC-3/pNeg-siRNA更明显。结论:靶向抑制Slug基因表达促进胰腺癌BxPC-3细胞凋亡,并增强BxPC-3对5-FU的化疗敏感性。
AIM: To investigate the effect of Slug gene silencing siRNA on proliferation, apoptosis and chemosensitivity to 5-FU in BxPC-3 cells. Methods: The eukaryotic expression vector pGenesil-1-Slug-siRNA (pSlug-siRNA) and empty vector pGen-esil-1-Neg-siRNA Cancer BxPC-3 cells, G418 positive cells were screened to obtain stable transfected positive clones. BxPC-3 positive cloned cells transfected with vector and untransfected BxPC-3 cells were exposed to 5-FU72h with serial concentration (0.01-100μmol / L). Slug expression in different groups of cells was detected by RT-PCR and Western blot 72h after treatment with 5-FU. Cell viability was measured by MTT assay and IC50, Hoechst33258 and PI double staining and terminal deoxynucleotidyl transferase mediated Cell apoptosis was detected by dUTP nick end labeling (TUNEL). Results: The Slug gene in the experimental group was effectively silenced. The 5-FUIC50 of BxPC-3, Bx-PC-3 / pSlug-siRNA and BxPC-3 / pNeg- siRNA were (11.0 ± 2.1) ) And (9.7 ± 1.3) μmol / L, respectively (P <0.05) .BxPC-3 / pSlug-siRNA cells increased the sensitivity to 5-FU by 7.33 times. 5-FU induced apoptosis in BxPC-3 cells in a dose-dependent manner, but more pronounced in BxPC-3 / pSlug-siRNA cells than BxPC-3 and BxPC-3 / pNeg-siRNA. CONCLUSIONS: Targeted inhibition of Slug gene expression promotes apoptosis of pancreatic cancer BxPC-3 cells and enhances chemosensitivity of BxPC-3 to 5-FU.