PMID- 22954916 OWN - NLM STAT- MEDLINE DCOM- 20140227 LR - 20120907 IS - 1672-7347 (Print) IS - 1672-7347 (Linking) VI - 37 IP - 8 DP - 2012 Aug TI - [Effect of gene GSTP1 silencing via shRNA transfection on androgen independent prostate cancer cell line Du145]. PG - 807-16 LID - 10.3969/j.issn.1672-7347.2012.08.009 [doi] AB - OBJECTIVE: To design short hairpin RNA (shRNA) interference sequence to silence glutathione S-transferase P1 (GSTP1) gene of androgen independent prostate cancer cell line DU145, and to explore its effect on proliferation and sensitivity to chemotherapeutics. METHODS: The target sequence was picked up to form the shRNA, and the 3 shRNA expression vectors were shRNA255, shRNA554 and shRNA593. The DNA template was cloned to plasmid pGPU6/GFP/Neo. The shRNA was identified by enzyme digesting and gene sequencing. The screening experiment was done to pick up the shRNA expression vector with the highest transfection ratio and best gene silencing results. DU145 cells were divided into a blank plasmid group and a shRNA transfected group. According to the chemotherapeutics the DU145 cells were divided into a fluorouracil (FU) group and a paclitaxel (PA) group, and the 2 groups were subdivided into 4 subsets according to the chemotherapeutic concentrations (FU: 30, 60, 120, and 240 mug/mL; PA: 0.2, 2, 10, and 20 mug/mL), meanwhile a blank control group was included respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the proliferation after the transfection. MTT and terminal de-oxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were used to detect the inhibition effect of different concentrations of 5-FU or PA on the proliferation and induction of apoptosis of DU145. RESULTS: The transfection ratio of the 3 shRNA expression vectors (shRNA255, shRNA554, and shRNA593) was (63.30+/-1.04)%, (76.20+/-0.68)%, and (72.70+/-0.33)%, and the transfection ratio of shRNA554 was the highest. there was significant difference among the above 3 shRNA expression vectors (P<0.01). After the transfection, the mRNA was 128.31+/-2.50, 43.24+/-4.30 and 85.62+/-6.30, the GSTP1 protein was 163.92+/-12.40, 65.38+/-9.30 and 114.25+/-16.70. After the transfection of shRNA554, the mRNA and protein of GSTP1 were the lowest level. there was significant difference among the above 3 shRNA expression vector (P<0.01). MTT analysis showed that before the transfection, the survival ratio of cells under different concentrations of FU (30, 60, 120, and 240 mug/mL) was (95.60+/-2.11)%, (90.20+/-0.86)%, (83.10+/-3.12)% and (74.60+/-1.32)%; however after the transfection, the survival ratio of cells was (91.30+/-1.43)%, (84.60+/-2.13)%, (73.20+/-1.52)%, and (65.5+/-0.942)%. TUNEL assay showed that before the transfection, the apoptosis ratio of cells under different concentrations of FU (30, 60, 120, and 240 mug/mL) was (5.50+/-0.88)%, (10.20+/-1.64)%, (15.20+/-2.39)%, and (25.10+/-2.59)%; however after the transfection, the apoptosis ratio of cells was (10.8+/-0.62)%, (15.7+/-1.32)%, (20.4+/-1.89)%, and (34.9+/-2.54)%. After the transfection, the cell survival ratio decreased under the same concentration of FU, and the apoptosis ratio increased, with statistical significance (both P<0.01). MTT analysis showed that before the transfection, the survival ratio of cells under different concentrations of PA (0.2, 2, 10, and 20 mug/mL) was (98.50+/-2.34)%, (95.20+/-1.32)%, (89.40+/-0.68)%, and (82.70+/-1.73)%; after the transfection the survival ratio of cells was (94.20+/-0.78)%, (86.50+/-2.13)%, (78.70+/-1.34)%, and (70.10+/-0.76)%. TUNEL assay showed that before the transfection, the apoptosis ratio of cells under different concentrations of PA (0.2, 2, 10, and 20 mug/mL) were (2.40+/-1.07)%, (5.20+/-1.33)%, (10.50+/-2.41)%, (20.70+/-1.92)%; after the transfection the apoptosis ratio of cells was (5.46+/-2.13)%, (13.80+/-1.24)%, (21.20+/-2.39)%, and (29.20+/-2.21)%. After the transfection, the cell survival ratio decreased under the same PA concentration, and the apoptosis ratio increased, with statistical significance (both P<0.01). CONCLUSION: gene GSTP1 silence via shRNA transfection to androgen independent prostate cancer cell line DU145 can inhibit its proliferation in time dependent manner, and induce apoptosis and raise its sensitivity to chemotherapeutics. FAU - Jin, Peng AU - Jin P AD - Centre of Organ Transplantation, Xiangya Hospital, Central South University, Changsha 410008, China. jinpeng_421@yahoo.cn FAU - Xie, Jinliang AU - Xie J FAU - Zhu, Xiangrong AU - Zhu X FAU - Zhou, Cheng AU - Zhou C FAU - Ding, Xiang AU - Ding X FAU - Yang, Luoyan AU - Yang L LA - chi PT - English Abstract PT - Journal Article PL - China TA - Zhong Nan Da Xue Xue Bao Yi Xue Ban JT - Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences JID - 101230586 RN - 0 (Androgens) RN - 0 (Antineoplastic Agents) RN - 0 (RNA, Small Interfering) RN - EC 2.5.1.18 (GSTP1 protein, human) RN - EC 2.5.1.18 (Glutathione S-Transferase pi) SB - IM MH - Androgens/metabolism MH - Antineoplastic Agents/pharmacology MH - Apoptosis/*genetics MH - Cell Line, Tumor MH - Cell Proliferation MH - Gene Silencing MH - Glutathione S-Transferase pi/*genetics MH - Humans MH - Male MH - Prostatic Neoplasms/*genetics/pathology MH - RNA Interference MH - RNA, Small Interfering/*genetics MH - Transfection EDAT- 2012/09/08 06:00 MHDA- 2014/02/28 06:00 CRDT- 2012/09/08 06:00 PHST- 2012/09/08 06:00 [entrez] PHST- 2012/09/08 06:00 [pubmed] PHST- 2014/02/28 06:00 [medline] AID - 10.3969/j.issn.1672-7347.2012.08.009 [doi] PST - ppublish SO - Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2012 Aug;37(8):807-16. doi: 10.3969/j.issn.1672-7347.2012.08.009.