Tumor-targeted efficiency of shRNA vector harboring chimera hTERT/U6 promoter.
Tumor-targeted efficiency of shRNA vector harboring chimera hTERT/U6 promoter.
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DOI：
10.3892/or_00000765
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发表时间：
2010-05
期刊：
影响因子：
4.2
通讯作者：
Peng-hui Zhang;Yaqin Chen;Xixin Jiang;Z. Tu;Lin Zou
中科院分区：
文献类型：
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作者：
Peng-hui Zhang;Yaqin Chen;Xixin Jiang;Z. Tu;Lin Zou
端粒酶与肿瘤关系密切，hTERT是端粒酶活性的限速因子。hTERT启动子具有锚定端粒酶阳性细胞的能力，决定hTERT的转录和表达。RNA干扰技术在功能基因组学和基因治疗中具有广阔的应用前景。然而，RNA干扰不确定性和安全性的局限性阻碍了其广泛应用。为了克服这些局限性，我们构建了针对EGFP和hTERT基因的U6启动子和hTERT/U6嵌合启动子的shRNA载体（shRNA-EGFP-U6，shRNA-EGFP-hTERT/U6，shRNA-hTERT-U6和shRNA-hTERT-hTERT/U6），分别在端粒酶阴性的人正常成纤维细胞HELF细胞和端粒酶阳性的人肝癌细胞SMMC-7721和HepG 2中抑制GFP和hTERT的表达。构建稳定表达EGFP或hTERT的HELF-EGFP和SMMC-7721-EGFP细胞。GFP表达在表达shRNA-EGFP-U6的HELF-EGFP和SMMC-7721-EGFP细胞中均受到抑制。进一步的结果显示，GFP的表达仅在端粒酶阳性的SMMC-7721细胞和HepG 2细胞中受到抑制，而在端粒酶阴性的HELF细胞中不表达shRNA-EGFP-hTERT/U6。进一步的结果发现，无论在体外还是体内，hTERT的表达都被有效地抑制自表达shRNA-hTERT-U6或shRNA-hTERT-hTERT/U6的肝癌细胞。我们的研究表明，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性，这将有助于肿瘤的治疗。
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.