Children's Hospital, Chongqing Medical University, Chongqing 400014, P.R. China.
Oncol Rep. 2010 May;23(5):1309-16. doi: 10.3892/or_00000765.
Telomerase is closely related to tumor, and hTERT is the rate-limiting factor for telomerase activity. The transcription and expression of hTERT is determined by hTERT promoter, which has the ability of anchoring telomerase positive cells. RNA interference (RNAi) has been potentially used in the functional genomics and gene therapy recently. However, the limitations of RNAi uncertain interference and safety hamper its wide applications. To overcome these limitations, we constructed shRNA vectors harboring either U6 promoter or chimera hTERT/U6 promoter aiming at EGFP and hTERT genes (shRNA-EGFP-U6, shRNA-EGFP-hTERT/U6, shRNA-hTERT-U6 and shRNA-hTERT-hTERT/U6), to suppress the expression of GFP and hTERT in telomerase negative human normal fibroblast HELF cells and telomerase positive human hepatocarcinoma SMMC-7721 and HepG2 cells, respectively. HELF-EGFP and SMMC-7721-EGFP cells stably expressing EGFP or hTERT were constructed. GFP expression was inhibited in both HELF-EGFP and SMMC-7721-EGFP cells expressing shRNA-EGFP-U6. Further results showed that GFP expression was suppressed only in telomerase positive SMMC-7721 cells and HepG2 cells, but not in telomerase negative HELF cells expressing shRNA-EGFP-hTERT/U6. Further results found that hTERT expression was effectively inhibited from liver cancer cells expressing shRNA-hTERT-U6 or shRNA-hTERT-hTERT/U6 both in vitro and in vivo. Our study illustrates the tumor-targeted efficiency of shRNA vectors harboring chimera hTERT/U6 promoter in telomerase positive cells, which will benefit tumor therapy.
端粒酶与肿瘤密切相关，端粒酶逆转录酶（hTERT）是端粒酶活性的限速因素。hTERT 的转录和表达受 hTERT 启动子调控，该启动子具有锚定端粒酶阳性细胞的能力。RNA 干扰（RNAi）最近已被潜在用于功能基因组学和基因治疗。然而，RNAi 的不确定性干扰和安全性限制了其广泛应用。为了克服这些限制，我们构建了含有 U6 启动子或嵌合 hTERT/U6 启动子的 shRNA 载体，针对 EGFP 和 hTERT 基因（shRNA-EGFP-U6、shRNA-EGFP-hTERT/U6、shRNA-hTERT-U6 和 shRNA-hTERT-hTERT/U6），以抑制端粒酶阴性人正常成纤维细胞 HELF 细胞和端粒酶阳性人肝癌 SMMC-7721 和 HepG2 细胞中 GFP 和 hTERT 的表达。构建了稳定表达 EGFP 或 hTERT 的 HELF-EGFP 和 SMMC-7721-EGFP 细胞。在表达 shRNA-EGFP-U6 的 HELF-EGFP 和 SMMC-7721-EGFP 细胞中，GFP 表达受到抑制。进一步的结果表明，仅在端粒酶阳性的 SMMC-7721 细胞和 HepG2 细胞中抑制 GFP 表达，而在端粒酶阴性的表达 shRNA-EGFP-hTERT/U6 的 HELF 细胞中不抑制 GFP 表达。进一步的结果发现，shRNA-hTERT-U6 或 shRNA-hTERT-hTERT/U6 表达载体在体外和体内均能有效抑制肝癌细胞 hTERT 的表达。本研究表明，含有嵌合 hTERT/U6 启动子的 shRNA 载体在端粒酶阳性细胞中具有肿瘤靶向效率，这将有利于肿瘤治疗。
