Antisense human telomerase reverse transcriptase could partially reverse malignant phenotypes of gastric carcinoma cell line in vitro
Antisense human telomerase reverse transcriptase could partially reverse malignant phenotypes of gastric carcinoma cell line in vitro
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DOI：
10.1097/cej.0b013e3282b71f0d
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发表时间：
2008-06-01
影响因子：
2.4
通讯作者：
Liang, Guang-Ping
中科院分区：
文献类型：
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作者：
Yang, Shi-Ming;Fang, Dian-Chun;Liang, Guang-Ping
端粒酶活性在90%以上的肿瘤中检测到，但在大多数正常体细胞中检测不到。端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）是端粒酶活性的限速酶。因此，靶向hTERT代表了用于减少端粒酶功能的有希望的方法，其可能不会对端粒酶阴性体细胞造成实质性副作用。为探讨反义hTERT（ahTERT）对人胃癌细胞株SGC-7901恶性表型的影响，采用基因重组技术构建hTERT反义真核表达载体。采用端粒重复序列扩增-ELISA法检测转染前后SGC-7901细胞端粒酶活性，逆转录-PCR法检测细胞端粒酶亚基、c-myc和bcl-2的mRNA表达，Southern blot法检测细胞末端限制性片段（TRF），流式细胞术检测细胞周期分布，Western blot法检测hTERT、c-myc和bcl-2蛋白表达。并对上述细胞进行了软琼脂克隆形成率测定和裸鼠成瘤实验。结果表明，ahTERT基因转染后，SGC-7901细胞的增殖明显受到抑制。进一步研究发现，转染ahTERT的细胞端粒酶活性、端粒长度、hTERT、bcl-2和c-myc的mRNA和蛋白表达均降低。转染和未转染的细胞对人端粒酶RNA（hTR）和人端粒酶相关蛋白1（TP 1）的转录无明显影响。流式细胞术分析显示，在ahTERT转染的细胞中，G（0）/G（1）期的积累和增殖指数的降低（131）。此外，在皮下注射ahTERT转染的细胞后在裸鼠中未发现致瘤性，而在注射对照细胞的小鼠中观察到可触知的肿瘤。我们的研究表明外源性ahTERT可通过抑制端粒酶活性、hTERT、c-myc和bcl-2的表达，抑制胃癌细胞的增殖，部分逆转胃癌细胞的恶性表型。反义技术靶向hTERT的策略可能成为胃癌治疗的一种潜在途径。
Telomerase activity is detected in more than 90% of examined tumors but not in most normal somatic cells. Among three subunits of human telomerase, human telomerase reverse transcriptase (hTERT) is the rate-limiting component for telomerase activity. Therefore, targeting hTERT represents a promising approach for diminishing telomerase function that will probably not cause substantial side effects on telomerase negative somatic cells. To explore the effects of antisense hTERT (ahTERT) on the malignant phenotypes of human SGC-7901 gastric cancer cell line in vitro, an antisense eukaryotic expression vector of hTERT was constructed by gene recombinant technology. Telomerase activity by telomeric repeat amplification protocol-ELISA, mRNA of telomerase subunits, c-myc and bcl-2 by reverse transcript-PCR, terminal restriction fragment (TRF) by Southern blot, cell cycle distribution by flow cytometry and protein expression of hTERT, c-myc and bcl-2 by Western blot were analyzed in SGC-7901 cells before and after transfection. Cloning efficiency assay in soft agar and tumorigenesis in nude mice were also examined and evaluated in the above cells. The results demonstrated that after ahTERT transfection, the proliferation of SGC-7901 cells was significantly inhibited. Further study showed that telomerase activity, telomere length, the mRNA and protein expression of hTERT, bcl-2 and c-myc were decreased in ahTERT-transfected cells. There were, however, no obvious effects on transcription of human telomerase RNA (hTR) and human telomerase associated protein1 (TP1) in both transfected and untransfected cells. Flow cytometric analysis displayed an accumulation of G(0)/G(1) phase and a decreasing proliferation index (131) in ahTERT-transfected cells. Moreover, no tumorigenicity was found after subcutaneous injection of ahTERT-transfected cells in nude mice, whereas palpable tumors were observed in mice injected with control cells. Our study indicates that exogenous ahTERT can inhibit proliferation and partially reverse malignant phenotypes of SGC-7901 cells via the suppression of telomerase activity, hTERT, c-myc and bcl-2 expression. Antisense technology targeted hTERT strategy might be a potential approach for gastric cancer therapy.