title: Cell-selective gene silencing in prostate cancer LNCap cells using prostate-specific membrane antigen promoter and enhancer in vitro and in vivo.
abstract: RNAi (RNA interference) has been widely used to silence specific genes. However, RNAi may also cause off-target silencing and elicit non-specific side effects. To achieve cell-specific gene silencing, a cell-selective promoter has to be used to drive RNAi expression. Furthermore, different terminators of cell-selective promoters may cause different silencing efficacies. In order to explore the best promoter and terminator combination and prove the cell-selective gene silencing effect of PSMAe/p (prostate-specific membrane antigen enhancer/promoter), we first constructed three plasmids by using PSMAe/p and three different terminators [poly(A), minipoly(A) and poly(U)] to explore the cell-selective driving ability of PSMAe/p by targeting EGFP (enhanced green fluorescent protein) in LNCaP, PC-3, EJ and HEK-293 (human embryonic kidney) cells. Then we chose NS (nucleostemin), an important endogenous gene of prostate cancer, and constructed the NS-targeting shRNA (small-hairpin RNA) expression plasmid by using PSMAe/p-poly(A) combination. Cell proliferation, cell cycle and early apoptosis in vitro and xenograft tumour growth in BALB/c nude mice in vivo were detected after NS knockdown. Results showed that PSMAe/p can drive EGFP silencing in LNCaP, not in PC-3, EJ and HEK-293 cells and PSMAe/p-poly(A) combination achieved the best silencing efficacy. Then PSMAe/p-shNS-poly(A) drives NS knockdown in LNCaP cells, not in PC-3, EJ and HEK-293 cells. Furthermore, RNAi-mediated NS knockdown not only reduces cell proliferation rate, reduces the percentage of S-stage cells and increases the percentage of G1-stage cells and increases the early apoptosis ratio in LNCaP cells in vitro, but also inhibited the LNCaP xenograft tumour growth in BALB/c nude mice in vivo by intratumoural injection. In conclusion, we have demonstrated that PSMAe/p-poly(A) combination is a promising delivery system for targeted RNAi gene therapy of prostate cancer. We showed one effective antitumour strategy by targeting NS protein, an important target in prostate cancer, with PSMAe/p-shNS-poly(A). These results serve as an important step for developing novel strategies to treat prostate cancer.
authors.affiliation: Tianjin Institute of Urology, Second Hospital, Tianjin Medical University, People's Republic of China.
authors.forename: Ranlu
authors.initials: R
authors.lastname: Liu
authors.forename: Jiantao
authors.initials: J
authors.lastname: Sun
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zhang
authors.forename: Yong
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000235:genetics*; D017209:Apoptosis; D002352:Carrier Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019204:GTP-Binding Proteins; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy; D043425:Glutamate Carboxypeptidase II / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000235:genetics*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics / Q000627:therapeutic use; D011401:Promoter Regions, Genetic; D011467:Prostate / Q000378:metabolism / Q000473:pathology; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics* / Q000627:therapeutic use; D016601:RNA-Binding Proteins
chemical_list: D000954:Antigens, Surface; D002352:Carrier Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C470311:nucleostemin protein, mouse; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; D019204:GTP-Binding Proteins