Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, 30 Yeongudanjiro, Cheongju, 28116, Republic of Korea; Ractigen Therapeutics, Nantong, Jiangsu, 226400, China.
Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, 30 Yeongudanjiro, Cheongju, 28116, Republic of Korea.
Int J Biochem Cell Biol. 2018 Apr;97:36-42. doi: 10.1016/j.biocel.2018.02.002. Epub 2018 Feb 6.
Recent studies have reported that chemically synthesized double-stranded RNAs (dsRNAs), also known as small activating RNA (saRNAs), can specifically induce gene expression by targeting promoter sequences by a mechanism termed RNA activation (RNAa). In the present study, we designed 4 candidate saRNAs targeting the Von Hippel-Lindau (VHL) gene promoter. Among these saRNAs, dsVHL-821 significantly inhibited cell growth by up-regulating VHL at both the mRNA and protein levels in renal cell carcinoma 769-P cells. Functional analysis showed that dsVHL-821 induced apoptosis by increasing p53, decreasing Bcl-xL, activating caspase 3/7 and poly-ADP-ribose polymerase in a dose-dependent manner. Chromatin immunoprecipitation analysis revealed that dsVHL-821 increased the enrichment of Ago2 and RNA polymerase II at the dsVHL-821 target site. In addition, Ago2 depletion significantly suppressed dsVHL-821-induced up-regulation of VHL gene expression and related effects. Single transfection of dsVHL-821 caused long-lasting (14 days) VHL up-regulation. Furthermore, the activation of VHL by dsVHL-821 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 4 (H4ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) and lysine 27 (H3K27me2) in the dsVHL-821 target region. Taken together, these results demonstrate that dsVHL-821, a novel saRNA for VHL, induces the expression of the VHL gene by epigenetic changes, leading to inhibition of cell growth and induction of apoptosis, and suggest that targeted activation of VHL by dsVHL-821 may be explored as a novel treatment of renal cell carcinoma.
最近的研究报告称，化学合成的双链 RNA（dsRNA），也称为小激活 RNA（saRNA），可以通过一种称为 RNA 激活（RNAa）的机制，特异性地靶向启动子序列来诱导基因表达。在本研究中，我们设计了 4 种针对 Von Hippel-Lindau（VHL）基因启动子的候选 saRNA。在这些 saRNA 中，dsVHL-821 可在 mRNA 和蛋白水平上显著上调 VHL，从而显著抑制肾癌细胞 769-P 细胞的生长。功能分析表明，dsVHL-821 通过增加 p53、减少 Bcl-xL、激活 caspase 3/7 和多聚 ADP-核糖聚合酶，呈剂量依赖性诱导细胞凋亡。染色质免疫沉淀分析显示，dsVHL-821 增加了 Ago2 和 RNA 聚合酶 II 在 dsVHL-821 靶位点的富集。此外，Ago2 耗尽显著抑制了 dsVHL-821 诱导的 VHL 基因表达上调及其相关作用。单独转染 dsVHL-821 可导致 VHL 长时间（14 天）上调。此外，dsVHL-821 激活 VHL 伴随着 dsVHL-821 靶区组蛋白 H3 赖氨酸 4 二甲基化（H3K4me2）和组蛋白 H4 乙酰化（H4ac）增加，以及组蛋白 H3 赖氨酸 9 二甲基化（H3K9me2）和赖氨酸 27 二甲基化（H3K27me2）减少。综上所述，这些结果表明，dsVHL-821 是一种新型的 VHL saRNA，通过表观遗传变化诱导 VHL 基因表达，抑制细胞生长并诱导细胞凋亡，提示靶向激活 VHL 可能作为肾细胞癌的一种新的治疗方法。
