短发夹RNA介导的谷胱甘肽S-转移酶P1基因沉默增强雄激素非依赖性细胞系DU145的化学敏感性。
shRNA-mediated GSTP1 gene silencing enhances androgen-independent cell line DU145 chemosensitivity.
作者信息
Jin Peng, Xie Jinliang, Zhu Xiangrong, Zhou Cheng, Ding Xiang, Yang Luoyan
机构信息
Centre of Organ Transplantation, Xiangya Hospital, Central South University, 87 Xiangya Road, Kaifu Area, Changsha, 410008, China,
出版信息
Int Urol Nephrol. 2014 Jun;46(6):1115-21. doi: 10.1007/s11255-013-0616-7. Epub 2013 Dec 11.
PURPOSE
Design short hairpin RNA (shRNA) interference sequence to silence glutathione S-transferase P1 (GSTP1) gene of androgen-independent prostate cancer cell line DU145, and then to explore its effect on sensitivity to chemotherapeutics.
METHODS
Target sequence was picked up to form the shRNA. DU145 cell was divided into five groups according to the shRNA added for transfection: shRNA255, shRNA554, shRNA593, negative-shRNA and blank group. Fluorescence microscope was used to pick up the shRNA with the highest transfection ratio. Western blotting and RT-PCR were taken to pick up the shRNA with the best gene silencing result. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay and terminal de-oxynucleotidyl transferase-mediated dUTP nick end-labeling assay were used to detect survival ratio and apoptosis ratio of DU145 administered of fluorouracil (5-FU) or paclitaxel (PA) at different concentrations before and after shRNA transfection.
RESULTS
Three different shRNA oligonucleotides (shRNA255; shRNA554; shRNA593) targeting the coding sequence of GSTP1 mRNA and one negative control shRNA were constructed. The transfection ratio of shRNA554 (76.2 ± 0.68 %) was higher than that of shRNA255 (63.3 ± 1.04 %) (P < 0.01) or shRNA593 (72.7 ± 0.33 %) (P < 0.01). After transfection of shRNA554, the mRNA and protein of level were the lowest, P < 0.01. The survival ratio of DU145 administered with 5-FU of different concentrations (30, 60, 120, 240 μg/ml) declined after transfection (P < 0.01). Besides, the apoptosis ratio increased after transfection (P < 0.01). Similarly the survival ratio of DU145 administered with PA of different concentrations (0.2, 2, 10, 20 μg/ml) declined (P < 0.01) and the apoptosis ratio increased (P < 0.01) after transfection.
CONCLUSIONS
The gene GSTP1 silence via shRNA transfection to androgen-independent prostate cancer cell line DU145 enhances the sensitivity to chemotherapeutics.
目的
设计短发夹RNA（shRNA）干扰序列，沉默雄激素非依赖性前列腺癌细胞系DU145的谷胱甘肽S-转移酶P1（GSTP1）基因，进而探讨其对化疗药物敏感性的影响。
方法
选取靶序列构建shRNA。将DU145细胞根据添加的shRNA分为五组进行转染：shRNA255组、shRNA554组、shRNA593组、阴性shRNA组和空白组。用荧光显微镜挑选转染率最高的shRNA。采用蛋白质免疫印迹法和逆转录-聚合酶链反应挑选基因沉默效果最佳的shRNA。采用噻唑蓝比色法和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法检测shRNA转染前后不同浓度氟尿嘧啶（5-FU）或紫杉醇（PA）作用下DU145细胞的存活率和凋亡率。
结果
构建了3种针对GSTP1 mRNA编码序列的不同shRNA寡核苷酸（shRNA255、shRNA554、shRNA593）和1种阴性对照shRNA。shRNA554的转染率（76.2±0.68%）高于shRNA255（63.3±1.04%）（P<0.01）或shRNA593（72.7±0.33%）（P<0.01）。转染shRNA554后，mRNA和蛋白水平最低，P<0.01。转染后，不同浓度（30、60、120、240μg/ml）5-FU作用下DU145细胞的存活率下降（P<0.01）。此外，转染后凋亡率升高（P<0.01）。同样，转染后不同浓度（0.2、2、10、20μg/ml）PA作用下DU145细胞的存活率下降（P<0.01），凋亡率升高（P<0.01）。
结论
通过shRNA转染使雄激素非依赖性前列腺癌细胞系DU145的GSTP1基因沉默可增强其对化疗药物的敏感性。
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