hTERT启动子克隆及其肿瘤细胞特异性的研究

Tumor-selective effect mediated by hTERT promoter on cancer cells

目的:探讨克隆人端粒酶逆转录酶(hTERT)启动子对肿瘤细胞特异性的意义。方法:全基因合成hTERT启动子核心序列TP258,克隆入T载体,构建pUCmT-TP258测序;SalⅠ+BamHⅠ酶切pUCmT-TP258,将其片断插入pGL3-Basic/XhoⅠ+BglⅡ位点,构建pGL3-TP258质粒载体。将pGL3-Basic、pGL3-control和pGL3-TP258转染培养的人结肠癌细胞株(CaCo-2)、人乳腺癌细胞株(MCF-7)、原代肾癌细胞(RCC)和人正常成纤维细胞(UFC、BJ、MRC-5),测定Luciferase活性,计算其相对活性。结果:在端粒酶阳性的肾癌细胞RCC、结肠癌细胞CaCo-2和乳腺癌细胞MCF-7中,TP258启动子相对活性很高,分别为32·40±15·32、37·70±6·38和12.80±7.28;而在正常的UFC、BJ和MRC-5细胞中,TP258活性很低,分别是0.40±0.14、0.26±0.13和0.38±0.05,两组间差异有统计学意义,P=0·0035。结论:hTERT核心启动子TP258在肿瘤细胞中活性明显高于在正常细胞中的活性,具有肿瘤特异性。TP258可以介导基因在肿瘤中的定向表达,实现基因表达的靶向性。

OBJECTIVE： To clone the promoter of human telomerase reverse transcriptase （hTERT） and investigate its selective effect in cancer cells. METHODS： The core sequence of hTERT promoter （TP258） was synthesized and subcloned into T-vector to construct the pUCmT-TP258. After sequencing, TP258 fragment was released from pUCmT-TP258 by Sal Ⅰ ＋BamH Ⅰ digestion and inserted into pGL3-Basic/Xho Ⅰ＋Bgl Ⅱ , and then generated the plasmid pGL3-TP258. The vectors of pGL3-Basic,pGL3-control,pGL3-TP258 were transferred into CaCo-2, RCC, MFC-7 cancer cells, and MRC-5, UFC,BJ normal cells. The activity of TP258 was caculated by the detection of relative luciferase activity. RESULTS： The activities of the hTERT promoter TP258 were higher in the RCC,CaCo-2, MCF-7 cancer cells （32.40 ± 15.32, 37.70 ±6.38, 12.80±7.28, respectively）, but the activities of TP258 were lower in UFC, MRC-5, BJ cells （0. 40 ±0.14,0.26 ± 0.13,0.38 ±0.05, respectively）. There was a significant difference between the two groups, P= 0. 003 5. CONCLUSIONS：The activity of the hTERT promoter is higher in cancer cells than that in normal cells,demonstrating its tumor specificity. TP258 may mediate the anti-tumor gene expressed in cancer cells and achieve the target gene therapy of cancer.