端锚酶反义寡核苷酸联合反义端粒酶催化亚单位对人肺腺癌A549细胞端粒动力学的影响  

Effect of Tankyrase Antisense Oligonucleotide Combined Human Telomerase Reverse Transcriptase Antisense Oligonucleotide on Telomere Dynamics in Human Lung Adenocarcinoma A549 Cells

作　　者：卢宏达[1] 黄涛[2] 申雯竹[3] 甄燕[3] 孔庆志[2] 

机构地区：[1]湖北中医学院中医系,湖北武汉430060 [2]武汉市普爱医院肿瘤科,湖北武汉430033 [3]武汉市普爱医院检验科,湖北武汉430033

出　　处：《癌症》2007年第11期1164-1169,共6页Chinese Journal of Cancer

基　　金：湖北省卫生厅科研基金(No.JX1B029);武汉市人民政府"晨光计划"基金(No.20035002016-20)~~

摘　　要：背景与目的:端锚酶是真核生物体内的一种重要的功能蛋白,对调节细胞端粒长度及参与细胞衰老和永生化过程起着重要的作用。本研究探讨端锚酶及端粒酶反义寡核苷酸联合作用对人肺腺癌A549细胞端粒相关蛋白表达和翻译的影响,以及对端粒缩短效能和细胞周期的作用。方法:将培养的A549细胞分为空白对照组、端锚酶正义寡核苷酸对照组(tankyrase sense oligonucleotide,sTANKS)、端粒酶催化亚单位正义寡核苷酸对照组(human telomerase reverse transcriptase sense oligonucleotide,shTERT)、端锚酶反义寡核苷酸实验组(tankyrase antisense oligonucleotide,asTANKS)、端粒酶催化亚单位反义寡核苷酸实验组(human telomerase reverse transcriptase antisense oligonucleotide,ashTERT)、端锚酶及端粒酶催化亚单位反义寡核苷酸联合实验组(asTANKS+ashTERT)。分别与不同的正、反义寡核苷酸作用,采用RT-PCR(reverse transcription-polymerase chain reaction)法观察端粒酶催化亚单位的mRNA表达,ELISA-PCR法测定端粒酶活性,Western blot法观察端锚酶活性,Q-FISH法检测各组端粒的平均长度;并通过传代实验观察不同正、反义寡核苷酸对A549细胞传代寿命的影响。结果:ashTERT能明显抑制端粒酶催化亚单位的mRNA表达及蛋白质活性,不影响端锚酶活性;作用48h后细胞平均端粒长度明显缩短[(7.59±0.07)kb];细胞在经过56.92±0.46个倍增时间(population double,PD)连续培养终止。asTANKS不影响端粒酶催化亚单位mRNA表达及蛋白质活性,却能明显抑制端锚酶活性;作用48h后细胞平均端粒长度明显缩短[(7.33±0.09)kb];细胞在经过(53.33±0.57)PD连续培养终止。ashTERT+asTANKS联合作用同时抑制端粒酶和端锚酶,其细胞平均端粒长度缩短更为明显[(3.55±0.08)kb],流式直方图上FITC荧光"左偏"更显著,与ashTERT、asTANKS比较差异有显著性(t=37.33、32.50,P<0.001);ashTERT+asTANKS组细胞在经(24.53±0.40)PD后培养�BACKGROUND ＆ OBJECTIVE： Tankyrase （TANKS） regulates telomerase-mediated telomere elongation and plays an impotant role in cellular senescence and immortalization. This study was to determine the effect of tankyrase antisense oligonucleotide （asTANKS） combined human telomerase reverse transcriptase antisense oligonucleotide （ashTERT） on telomere dynamics in human lung adenocarcinoma A549 cells. METHODS. A549 ceils were transfected with ashTERT, asTANKS, ashTERT combined asTANKS, and human telomerase reverse transcriptase sense oligonucleotide （shTERT）, tankyrase sense oligonucleotide （sTANKS）, or blank vector as control. The expression of hTERT mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）. Telomerase activity was detected by enzyme-linked immunosorbent assay-PCR （ELISA-PCR）. Tankyrase activity was detected by Western blot. Telomere length was analyzed by quantitative fluorescence in situ hybridization （Q-FISH）. Cell proliferation was measured by population doubling test. RESULTS. The mRNA level and telomerase activity of hTERT in A549 ceils were strongly suppressed by ashTERT, but not by asTANKS： while tankyrase activity was significantly inhibited by asTANKS, not by ashTERT. Telomere length was significantly shorter in the cells treated with ashTERT combined asTANKS than in the cells treated with either ashTERT or asTANKS [（3.55±0.08） kb vs. （7.59±0.07） kb and （7.33±0.09） kb, t=37.33, t=32.50, P〈0.0011. The generational activity of the A549 cells continuously treated with ashTERT combined asTANKS was significantly weaker than those treated with either ashTERT or asTANKS [（24.53±0.40） population double times （PD） vs. （56.92±0.46） PD and （53.33±0.57） PD, t= 53.38, t= 43.39, P〈 0.0011. CONCLUSIONS; The combination of ashTERT and asTANKS can enhance the efficacy of telomere shortening and hasten early tumor cellular crisis. Tankyrase might be a potential target of telomere-based molecular cancer therapy.

关 键 词：端粒长度 端锚酶 端粒酶催化亚单位 反义寡核苷酸 A549细胞 

分 类 号：R734.2[医药卫生—肿瘤]