Cell-selective gene silencing in prostate cancer LNCap cells using prostate-specific membrane antigen promoter and enhancerin vitroandin 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.