3种卵巢癌细胞中hTERT转录水平及其与端粒酶活性的相关性研究

Relationship between Human Telomerase Reverse Transcriptase Transcriptional Level and Telomerase Activity in Three Ovarian Cancer Cell Lines

背景与目的:端粒酶在约90%的肿瘤细胞中有活性而在绝大多数正常细胞中受抑,催化亚单位端粒酶逆转录酶(humantelomerasereversetranscriptase,hTERT)是端粒酶的重要组成部分,其表达的调控主要发生在转录水平。端粒酶、hTERT、hTERT启动子三者之间密切相关。本研究探讨卵巢癌细胞系中hTERT启动子的活性及其与hTERTmRNA表达和端粒酶活性之间的关系。方法:应用脂质体转染法将hTERT核心启动子基因转入3种卵巢癌细胞OVCAR3、SKOV3、3AO及正常卵巢上皮细胞中,并用荧光素酶检测实验检测其中hTERT启动子的活性;应用RT-PCR半定量法检测这4种细胞中hTERTmRNA的表达水平;应用PCR-ELISA定量检测这4种细胞中端粒酶的活性。同时以端粒酶阳性的永生化人胚肾成纤维细胞HEK293作为阳性对照,以端粒酶阴性的人胚肺成纤维细胞HELF作为阴性对照。结果:OVCAR3、SKOV3、3AO细胞及正常卵巢上皮细胞中的hTERT启动子活性(视每种细胞中pGL3-control的启动子活性为100%)分别为31.4%、20.3%、17.7%和0.3%;hTERTmRNA的相对表达水平(视SKOV3的表达水平为1.00)分别为1.30、1.00、0.63和0;端粒酶活性分别为0.580、0.414、0.386和0.103(>0.200时定为阳性)。3种卵巢癌细胞中hTERT启动子活性、hTERTmRNA表达水平和端粒酶活性均特异性增高。

BACKGROUND &OBJECTIVE: Approximately 90%of tumors have telomerase activity, whereas most normal cells do not express telomerase. Telomerase catalytic subunit or human telomerase reverse transcriptase (hTERT) is the main component of telomerase. Telomerase expression is predominantly regulated at the transcriptional level of hTERT. Telomerase, hTERT, and hTERT promoter are closely related. The aim of this study was to investigate the relationship among human telomerase reverse transcriptase (hTERT) promoter activity, hTERT mRNA expression, and telomerase activity in three ovarian cancer cell lines. METHODS: hTERT promoter activity was determined by luciferase assay after the plasmids of pBTdel 279 containing hTERT core promoter were transfected into three ovarian carcinoma derived cell lines of OVCAR3, SKOV3, 3AO and normal human ovarian epithelial cells. hTERT mRNA expression levels of these cells were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT PCR). Telomerase activity was determined by PCR ELISA (enzyme linked immunosorbent assay) in above four cells. Immortalized human embryonic kidney cell line HEK293 and the human embryonic lung fibroblasts HELF were used as positive control and negative control, respectively. RESULTS: The relative luciferase activities of OVCAR3, SKOV3, 3AO and normal ovarian epithelial cells by the hTERT promoter were 31.4%, 20.3%, 17.7%, and 0.3%, respectively, as the luciferase activity of pGL3 control plasmid in each cell line was considered as 100%. The hTERT mRNA relative expression levels of above four cells were 1.30, 1.00, 0.63, and 0, respectively, as SKOV3 expression level was considered as 1. The telomerase activities were 0.580, 0.414, 0.386, and 0.103, respectively, as >0.2 was considered as positive. The hTERT promoter activity, hTERT mRNA expression level, and telomerase activity were specially raised in three ovarian cancer cell lines. The highest levels of hTERT promoter activity, hTERT mRNA expression, and telomerase activity were observed in OVCAR3 cell line, whereas negative in normal ovarian epithelial cells. hTERT promoter activity was closely associated with hTERT mRNA expression level and telomerase activity (P< 0.01,P< 0.02). CONCLUSION: hTERT promoter is specially activated in ovarian cancer cells which expresses telomerase. hTERT promoter activity is positively correlated with telomerase activity. Acting as a targeting promoter, hTERT promoter may be applied in gene therapy of ovarian cancer.