沉默cIAP-1基因对膀胱癌细胞表柔比星化疗敏感性的影响

Effect of cIAP-1 gene silence on sensitivity of bladder cancer cells to pharmorubicin chemotherapy in vitro

目的探讨沉默细胞凋亡蛋白抑制剂1(cellular inhibitor of apoptosis protein 1,c IAP-1)基因对膀胱癌细胞株T24及EJ细胞化疗敏感性的影响。方法体外培养T24及EJ细胞,分别接受4.6、9.2、18.4、36.8、73.6μmol/L的表柔比星处理。构建p GCsi-H1-shRNA(沉默c IAP-1基因)及p GCsi-H1-control(对照)两种质粒载体,并分别对处于对数生长期的T24及EJ细胞进行转染。未转染的细胞作为阴性对照。采用荧光标记法及CCK8试验检测细胞凋亡情况,Western blot及实时定量聚合酶链反应(qRT-PCR)分别检测c IAP-1蛋白及mRNA的相对表达量,流式细胞术检测细胞周期的变化情况。结果转染p GCsi-H1-shRNA的T24及EJ细胞较转染p GCsi-H1-control的细胞及未转染的细胞c IAP-1 mRNA和蛋白的表达水平显著降低,差异均具有统计学意义(P均<0.05)。不同浓度表柔比星处理条件下,与未沉默c IAP-1的细胞比较,沉默c IAP-1的细胞生存率均明显降低,其细胞周期明显被阻滞在G1/G0期,细胞凋亡率显著增加(P均<0.05)。结论沉默c IAP-1基因表达可提高膀胱癌细胞对表柔比星化疗敏感性。

Objective To explore the effect of silencing cellular inhibitor of apoptosis protein1（ c IAP-1） gene on the sensitivity of T24 and EJ bladder cancer cell lines to pharmorubicin chemotherapy. Methods T24 and EJ cells were cultured in vitro and were respectively treated with 4. 6-9. 2-,18. 4-,36. 8-,73. 6 μmol / L pharmorubicin. The plasmid vectors of PGCsi-H1-shRNA（ for silencing c IAP-1 gene） and p GCsi-H1-control（ for control） were constructed and respectively transfected to T24 and EJ cells in the logarithmic growth phase. The non transfected cells were served as negative control. Fluorescence labeling method and cell Counting Kit-8（ CCK-8） test were used to detect cell apoptosis. Western blot method and real-time quantitative polymerase chain reaction（ qRT-PCR） were used to respectively detect relative expression quantities of c IAP-1 protein and c IAP-1 mRNA. Flow cytometry was used to detect cell cycle changes. Results Compared with the cells transfected by p GCsi-H1-control plasmid and non transfected cells,the expression levels of c IAP-1 mRNA and protein in the cells transfected by p GCsi-H1-shRNA plasmid significantly decreased（ all P〈0. 05）. After being treated with different concentrations of pharmorubicin,the survival rates of the silencing c IAP-1 gene cells significantly decreased,and the cell cycle was significantly blocked in the G1 / G0 phase,as well as the cell apoptosis rate significantly increased compared with the non silencing IAP-1 gene cells（ all P〈0. 05）. Conclusion Silencing c IAP-1 gene expression can enhance the sensitivity of bladder cancer cells to pharmorubicin chemotherapy.