Up-regulation of VEGF by Small Activator RNA in Human Corpus Cavernosum Smooth Muscle Cells

Abstract

Introduction Functional failure of smooth muscle cells and endothelial cells in corpus cavernosum contributes to erectile dysfunction (ED) in aging men. Given that vascular endothelial growth factor (VEGF) may improve the function of smooth muscle cells and endothelial cells through different mechanisms, it is thus expected that increasing the expression of VEGF may have beneficial effects on erectile function. Aim The aim of this article is to explore the possibility that VEGF can be induced by ribonucleic acid activation (RNAa) technology, and VEGF induction by RNAa has the potential of treating ED. Methods Primary human corpus cavernosum smooth muscle cells (CCSMCs) were isolated and cultured in vitro. The expression of α-smooth muscle actin was detected by immunohistochemistry to identify CCSMCs. A previously identified VEGF promoter-targeted small activator RNA (saRNA, double-stranded [ds]VEGF-706) and a negative control dsRNA were chemically synthesized. Cultured human CCSMCs were transfected with the saRNAs. The expression of VEGF messenger RNA (mRNA) and protein in transfected CCSMCs was evaluated by real-time polymerase chain reaction (RT-PCR) and Western blotting assay, respectively. Immunofluorescent staining was also used to confirm VEGF protein expression in cultured CCSMCs. Main Outcome Measure The expression of VEGF was assessed by RT quantitative PCR, Western blotting, and immunofluorescence assays. Results After transfection, RT quantitative PCR analysis showed that the expression of VEGF mRNA was significantly induced in dsVEGF-706 transfected cells compared with cells receiving control treatments (P < 0.05). Consistent with mRNA induction, Western blotting and immunofluorescence analysis showed that VEGF protein expression was also induced by dsVEGF-706. Conclusion VEGF expression can be activated by RNAa in primary human CCSMCs, suggesting a potential application of RNAa-mediated VEGF activation for the treatment of ED.

MALAT1 promotes colorectal cancer cell proliferation/migration/invasion via PRKA kinase anchor protein 9

“…RNA activation-induced gene expression by targeting promoter sequence allowed us to physiologically and directly test the function of a given gene [23–26] . In fact, lentiviral-based delivery of dsRNAs has been reported to activate VEGF expression by targeting promoter sequences in an ischemic mouse model [27] . We found for the first time that RNA activation can up-regulate MALAT1 expression in CRC cells.…”

Adipose‐derived stem cells improve erectile function partially through the secretion of IGF‐1, bFGF, and VEGF in aged rats

“…In our previous study, we successfully isolated and cultured CCSMCs in vitro (Chen et al ., ). Briefly, corpus cavernosum tissues were obtained from 8‐week‐old rats.…”

Inducing gene expression by targeting promoter sequences using small activating RNAs

“…RNAa has been performed in cell lines [8,9,39] and primary cells [18,39] utilizing synthetic short dsRNAs [8,9,18] or expressed short hairpin RNA (shRNA) in vivo [14]. Any method that can introduce nucleic acids effectively into cells (especially into the nucleus) with limited cytotoxicity should be adequate to facilitate RNAa.…”