Department of General Surgery, Children Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China.
World J Surg Oncol. 2012 Jan 13;10:11. doi: 10.1186/1477-7819-10-11.
Recent studies have reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a process termed RNA activation. The present study was conducted to evaluate the potential of WT1 induction by small activating RNA targeting the WT1 promoter (dsWT1) in the treatment of hepatocellular carcinoma.
The human hepatocellular carcinoma cell line HepG2 was transfected with dsRNA by liposomes. The expression of mRNA and protein in cells were investigated using real-time reverse real-time quantitative PCR and Western blot, respectively. Cell viability and clonogenicity were determined by MTT assay and clonogenicity assay, respectively. Cell apoptosis was evaluated by flow-cytometric analysis.
Expressions of WT1 mRNA and protein in dsWT1 treated HepG2 cells were significantly elevated. Inhibition of cell viability by dsWT1 was dose-dependent and time-dependent. Reduction of the number and size of colonies formed were found in dsWT1 treated cells. dsWT1 induced significant apoptosis in HepG2 cells. The decreased anti-apoptotic protein Bcl-2 and elevated pro-apoptotic protein Bak expression were detected in dsWT1 treated cells. The level of pro-caspase-3 remarkably decreased and cleaved caspase-3 and PARP fragment were also detected in dsWT1 treated cells.
These data show that RNAa-mediated overexpression of WT1 may have therapeutic potential in the treatment of hepatocellular carcinoma.
最近的研究报告称，双链 RNA（dsRNA）可以通过靶向启动子序列来激活基因表达，这一过程称为 RNA 激活。本研究旨在评估针对 WT1 启动子的小激活 RNA（dsWT1）靶向诱导 WT1 表达在肝细胞癌治疗中的潜力。
用脂质体将 dsRNA 转染入人肝癌细胞系 HepG2。分别采用实时反转录定量 PCR 和 Western blot 检测细胞中 mRNA 和蛋白的表达。采用 MTT 法和集落形成实验分别检测细胞活力和集落形成能力。采用流式细胞术评估细胞凋亡。
dsWT1 处理的 HepG2 细胞中 WT1 mRNA 和蛋白的表达明显升高。dsWT1 对细胞活力的抑制呈剂量和时间依赖性。dsWT1 处理的细胞中形成的菌落数量和大小减少。dsWT1 诱导 HepG2 细胞发生明显的细胞凋亡。dsWT1 处理的细胞中检测到抗凋亡蛋白 Bcl-2 减少和促凋亡蛋白 Bak 升高。dsWT1 处理的细胞中 pro-caspase-3 水平显著降低，cleaved caspase-3 和 PARP 片段也被检测到。
这些数据表明，RNAa 介导的 WT1 过表达可能在肝细胞癌的治疗中有治疗潜力。
