Liu Lei, Li Chen-rong, Sun Lai-bao, Wang Guo-bing, Wang Bing
Department of Surgery, Shenzhen Children's Hospital, Shenzhen, 518026 China.
Zhonghua Er Ke Za Zhi. 2004 Jul;42(7):481-5.
Telomerase, a complex of ribose and nucleoprotein, is a specific marker of tumor, which expresses in 98% infinite cell lines and 90% malignant tumor organizations and whose function is to maintain the length of telomere. Human telomerase reverse-transcript protein subunit (hTERT) is the key element and rate-limiting factor of telomerase activity. Our study was to investigate the effects of antisense hTERT gene on biological characteristics of hepatoblastoma cell line in vitro.
The sense and antisense hTERT eukaryotic expression vectors that we had constructed before were transfected into hepatoblastoma cell line HepG2 by using the SuperFect transfection reagent (Qiagen) according to the manufacturer's instructions, then the HepG2-s and HepG2-as of G418-resistant colonies were obtained with G418 and identified for the presence of hTERT insert by PCR with T7 and pcDNA3.1/BGH reverse primers. After that, we have detected the endogenous hTERT mRNA expression and telomerase activity by quantitative real-time RT-PCR and TRAP-silver staining assay in cells from each group. Meanwhile, MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were employed to analyze if the proliferation capacity of liver cancer cells was affected in vitro and the tumor cells could be induced to apoptosis by antisense hTERT.
Antisense hTERT significantly down-regulated the endogenous hTERT mRNA expression (15.35 +/- 1.72/HepG2-as, 43.8 +/- 2.89/HepG2-s, 45.2 +/- 3.46/HepG2) (n = 10, t = 7.61, P < 0.01) and telomerase activity in HepG2, compared to blank control and sense hTERT. After 20 passages of three group cells, a 7-day cell growth curve and the numbers (size) of soft agar colony formation showed the proliferation and the anchorage-independent growth in HepG2-as were significantly suppressed (50.6 +/- 4.8/HepG2-as, 113.52 +/- 8.15/HepG2-s, 119.12 +/- 10.82/HepG2) (n = 10, t = 4.54, P < 0.01 and n = 10, t = 3.96, P < 0.01), compared to HepG2 and HepG2-s. However there was a significant increase in apoptosis percentage of HepG2-as by flow cytometry (n = 10, t = 9.24, P < 0.01 and n = 10, t = 8.37, P < 0.01), compared to control group.
Antisense hTERT could significantly suppress the hepatoblastoma cell growth and reverse its malignant phenotypes in vitro and cause the increase in apoptosis percentage of HepG2, thus it might be applied in malignant tumor gene therapy through the telomerase-targeted molecular mechanism.
端粒酶是一种核糖核蛋白复合体，是肿瘤的特异性标志物，在98%的永生化细胞系和90%的恶性肿瘤组织中表达，其功能是维持端粒长度。人端粒酶逆转录蛋白亚基（hTERT）是端粒酶活性的关键成分和限速因子。本研究旨在探讨反义hTERT基因对肝母细胞瘤细胞系体外生物学特性的影响。
按照制造商的说明，使用SuperFect转染试剂（Qiagen）将我们之前构建的正义和反义hTERT真核表达载体转染至肝母细胞瘤细胞系HepG2中，然后用G418筛选出G418抗性克隆的HepG2-s和HepG2-as，并通过使用T7和pcDNA3.1/BGH反向引物进行PCR鉴定hTERT插入片段的存在。之后，我们通过定量实时RT-PCR和TRAP-银染法检测了每组细胞中内源性hTERT mRNA表达和端粒酶活性。同时，采用MTT细胞增殖试验、软琼脂集落形成试验和流式细胞术分析反义hTERT是否会影响体外肝癌细胞的增殖能力以及是否能诱导肿瘤细胞凋亡。
与空白对照和正义hTERT相比，反义hTERT显著下调了HepG2中的内源性hTERT mRNA表达（HepG2-as为15.35±1.72，HepG2-s为43.8±2.89，HepG2为45.2±3.46）（n = 10，t = 7.61，P < 0.01）和端粒酶活性。三组细胞传代20次后，7天细胞生长曲线以及软琼脂集落形成的数量（大小）显示，HepG2-as中的增殖和非锚定依赖性生长受到显著抑制（HepG2-as为50.6±4.8，HepG2-s为113.52±8.15，HepG2为119.12±10.82）（n = 10，t = 4.54，P < 0.01；n = 10，t = 3.96，P < 0.01），与HepG2和HepG2-s相比。然而，通过流式细胞术检测发现，与对照组相比，HepG2-as的凋亡百分比显著增加（n = 10，t = 9.24, P < 0.01；n = 10，t = 8.37，P < 0.01）。
反义hTERT可显著抑制肝母细胞瘤细胞生长并在体外逆转其恶性表型，导致HepG2凋亡百分比增加，因此可能通过端粒酶靶向分子机制应用于恶性肿瘤基因治疗。
