title: shRNA-mediated GSTP1 gene silencing enhances androgen-independent cell line DU145 chemosensitivity.
abstract: OBJECTIVE
  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.
authors: Centre of Organ Transplantation, Xiangya Hospital, Central South University, 87 Xiangya Road, Kaifu Area, Changsha, 410008, China, jinpeng_421@aliyun.com.
authors: Peng
authors: P
authors: Jin
authors: Jinliang
authors: J
authors: Xie
authors: Xiangrong
authors: X
authors: Zhu
authors: Cheng
authors: C
authors: Zhou
authors: Xiang
authors: X
authors: Ding
authors: Luoyan
authors: L
authors: Yang
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005472:Fluorouracil / Q000494:pharmacology; D020868:Gene Silencing; D051549:Glutathione S-Transferase pi / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D017239:Paclitaxel / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
chemical_list: D000964:Antimetabolites, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D017239:Paclitaxel; D005472:Fluorouracil