Ying Xiao-Yang, Fang Mei-Yun, Wang Yi
Department of Hematology, The Zhongshan Hospital of Dalian University, Dalian 116001, Liaoning Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2008 Feb;16(1):48-53.
The study was supposed to investigate the inhibitory effect of antisense phosphorothioate oligodeoxynucleotide (ASPSODN) targeting hTERT mRNA on gene of interest in K562 cells and influence of ASPSODN on telomerase activity and apoptosis of K562 cells. Human leukemia cell line K562 was transfected by liposome with ASPSODN and SPSODN (sense phosphorothioate oligodeoxynucleotide) at different concentrations (0.2, 0.6, 1.0 micromol/L). At the same time, blank control, liposome control and SPSODN groups were designed for comparison. The transfected cells were collected and detected at 24 and 48 hours; the expression of target gene hTERT mRNA and telomerase activity were detected by RT-PCR and TRAP-ELISA respectively, and cell apoptosis was assayed by flow cytometry. The results showed that after K562 cells were transfected for 24 hours, the expression of hTERT mRNA had no difference between liposome control (0.80+/-0.24), 0.2 micromol/L ASPSODN (0.69+/-0.12), 0.2 micromol/L SPSODN (0.72+/-0.25) and blank control (0.85+/-0.28), but the expression of hTERT mRNA in 0.6 micromol/L ASPSODN group (0.42+/-0.16) remarkably decreased as compared with liposome control group, 0.6 micromol/L SPSODN (0.69 +/- 0.26) had no obvious effect on the expression of hTERT mRNA, the expression of hTERT mRNA in 1.0 micromol/L ASPSODN and SPSODN groups both decreased; mortality of K562 cells transfected by liposome with 1.0 micromol/L ASPSODN and SPSODN remarkably increased. After 24 hours, telomerase relative activity of K562 cells showed no significant difference between blank control (88.9%) and liposome control (77.7%). The telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L ASPSODN were 60.6%, 52%, 58.2% respectively. There was significant difference as compared with blank control; 0.6 micromol/L ASPSODN showed significant difference (p=0.037), as compared with liposome control group. The telomerase relative activities in K562 cells treated with 0.2, 0.6, 1.0 micromol/L SPSODN were 76.1%, 72.2%, 65.7% respectively, but the telomerase relative activities of K562 cells in 0.2, 0.6 micromol/L SPSODN groups was not inhibited obviously. When K562 cells were treated for 48 hours, telomerase relative activity of K562 cells in each ASPSODN groups restored. It showed that telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L ASPSODN were 84.1%, 82.3%, 79.6% respectively, while telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L SPSODN for 48 hours were 74.8%, 74.5%, 67.9% respectively. Telomerase activity of K562 cells could not be inhibited by 0.2 and 0.6 micromol/L SPSODN. After culturing for 48 hours, the cell apoptosis rates of K562 in 0.6 micromol/L ASPSODN, 0.6 micromol/L SPSODN, liposome control and blank control groups were (4.82+/-0.39)%, (1.83+/-0.34)%, 1.84+/-1.04)%, (1.07+/-0.74)% respectively. There was difference between ASPSODN and SPSODN groups (p<0.05), but the significant difference was found in ASPSODN group as compared with liposome control and blank control (p<0.01). It is concluded that the ASPSODN targeting hTERT can specifically inhibit the expression of hTERT mRNA in K562 cells and significantly suppress the telomerase activity of K562 cells at 0.6 micromol/L, which inhibitory time is short. The ASPSODN at high concentration (1.0 micromol/L) shows definite cytotoxicity. 0.6 micromol/L of ASPSODN significantly induces cell apoptosis, while no such effect was seen in SPSODN group.
本研究旨在探讨靶向人端粒酶逆转录酶（hTERT）mRNA的反义硫代磷酸寡脱氧核苷酸（ASPSODN）对K562细胞中目的基因的抑制作用，以及ASPSODN对K562细胞端粒酶活性和凋亡的影响。采用脂质体将不同浓度（0.2、0.6、1.0 μmol/L）的ASPSODN和硫代磷酸正义寡脱氧核苷酸（SPSODN）转染人白血病细胞系K562。同时，设立空白对照、脂质体对照和SPSODN组进行比较。转染后24和48小时收集细胞并进行检测；分别采用逆转录聚合酶链反应（RT-PCR）和端粒重复序列扩增酶联免疫吸附测定（TRAP-ELISA）检测目的基因hTERT mRNA的表达和端粒酶活性，采用流式细胞术检测细胞凋亡。结果显示，K562细胞转染24小时后，脂质体对照组（0.80±0.24）、0.2 μmol/L ASPSODN组（0.69±0.12）、0.2 μmol/L SPSODN组（0.72±0.25）和空白对照组（0.85±0.28）的hTERT mRNA表达无差异，但0.6 μmol/L ASPSODN组（0.42±0.16）的hTERT mRNA表达较脂质体对照组显著降低，0.6 μmol/L SPSODN组（0.69±0.26）对hTERT mRNA表达无明显影响，1.0 μmol/L ASPSODN组和SPSODN组的hTERT mRNA表达均降低；脂质体转染1.0 μmol/L ASPSODN和SPSODN的K562细胞死亡率显著增加。24小时后，K562细胞的端粒酶相对活性在空白对照组（88.9%）和脂质体对照组（77.7%）之间无显著差异。0.2、0.6、1.0 μmol/L ASPSODN处理的K562细胞端粒酶相对活性分别为60.6%、52%、58.2%。与空白对照组相比有显著差异；与脂质体对照组相比，0.6 μmol/L ASPSODN有显著差异（p = 0.037）。0.2、0.6、1.0 μmol/L SPSODN处理的K562细胞端粒酶相对活性分别为76.1%、72.2%、65.7%，但0.2、0.6 μmol/L SPSODN组的K562细胞端粒酶相对活性未明显受到抑制。当K562细胞处理48小时时，各ASPSODN组的K562细胞端粒酶相对活性恢复。结果显示，0.2、0.6、1.0 μmol/L ASPSODN处理的K562细胞端粒酶相对活性分别为84.1%、82.3%、79.6%，而0.2、0.6、1.0 μmol/L SPSODN处理48小时的K562细胞端粒酶相对活性分别为74.8%、74.5%、67.9%。0.2和0.6 μmol/L SPSODN不能抑制K562细胞的端粒酶活性。培养48小时后，0.6 μmol/L ASPSODN组、0.6 μmol/L SPSODN组、脂质体对照组和空白对照组的K562细胞凋亡率分别为（4.82±0.39）%、（1.83±0.34）%、（1.84±1.04）%、（1.07±0.74）%。ASPSODN组和SPSODN组之间有差异（p < 0.05），但ASPSODN组与脂质体对照组和空白对照组相比有显著差异（p < 0.01）。结论：靶向hTERT的ASPSODN能特异性抑制K562细胞中hTERT mRNA的表达，在0.6 μmol/L时能显著抑制K562细胞的端粒酶活性，但其抑制时间较短。高浓度（1.0 μmol/L）的ASPSODN具有一定的细胞毒性。0.6 μmol/L的ASPSODN能显著诱导细胞凋亡，而SPSODN组未见此效应。
