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.

Lung endothelial HO‐1 targetingin vivousing lentiviral miRNA regulates apoptosis and autophagy during oxidant injury

“…These studies are the first to demonstrate endothelial‐targeted gene silencing in vivo using silencing RNA constructs. Previous reports of cell‐specific silencing in vivo have targeted leukocytes and prostate or breast tissue (28–30). Our laboratory has achieved lung specificity using an intranasal route, and we previously reported siRNA delivery to multiple lung cells in vivo (7).…”

A novel regulation of PSMA and PSA expression by Q640X AR in 22Rv1 and LNCaP prostate cancer cells

“…On the basis of the above results, upregulation of PSMA involving CTE‐truncated mutated AR may explain the escape of prostate cancer cells to androgen deprivation therapy. These features support the concept to make it one of the most promising biomarkers in the diagnosis and treatment of prostate cancer (Liu et al, ; Yang et al, ). Androgen dependence of prostate epithelium in vivo requires paracrine activity of stromal AR, similar to the requirement for mesenchymal AR in epithelial–mesenchymal interactions during early prostate organogenesis (Abate‐Shen and Shen, ).…”