靶向hTERT的RNAi载体的构建及其对大肠癌SW-480细胞增殖的影响
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摘要
目的:构建靶向人大肠癌细胞端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)基因的RNAi载体,探讨其对人大肠癌SW480细胞增殖的影响。方法:设计3条靶向hTERT的shRNA序列和阴性对照序列,分别克隆入pGPU6/GFP/Neo载体,构建RNAi载体pGPU6-hTERT-1、2、3和阴性对照载体pGPU6-hTERT-NC,转染SW480细胞。RT-PCR检测各组载体对hTERT mRNA表达的影响,MTT法检测下调hTERT mRNA表达对SW480细胞增殖的影响。结果:成功构建3个携带hTERT mRNA序列的重组载体,3种RNAi载体均能明显抑制SW480细胞hTERT mRNA的表达,pGPU6-hTERT-3组SW480细胞hTERT mRNA表达水平较空白对照组下调最为显著(0.347±0.028 vs 0.513±0.032,P<0.01)。转染3种RNAi载体均能明显抑制SW480细胞增殖,pGPU6-hTERT-3组细胞增殖抑制率较空白对照组、脂质体对照组和pGPU6-hTERT-NC组升高最为显著[(50.08±0.43)%vs(4.11±0.39)%、(3.88±0.35)%、(3.38±0.35)%,P<0.05]。结论:转染RNAi载体pGPU6-hTERT-3能够抑制SW480细胞的增殖,其机制可能与降低hTERT基因的表达从而抑制端粒酶活性有关。
Objective: To construct an optimized human telomerase reverse transcriptase( hTERT) gene-specific RNAi and to evaluate its effect on human colon cancer cell proliferation in vitro. Method: Three hTERT-specific RNAi sequences and a negative control( NC) or scrambled sequence were cloned,respectively,into a pGPU6 /GFP /Neo vector to generate pGPU6-GFP-hTERT-1,pGPU6-GFP-hTERT-2,pGPU6-GFP-hTERt-3 and pGPU6-GFP-NC. Human colon cancer SW480 cells were transfected with these vectors respectively. At 24,48 and 72 h after transfection,hTERT mRNA abundance was assessed by RT-PCR and cell viability by MTT assay. Results: The 3 hTERT-specific RNAi vectors constructed were all effective to silence the hTERT gene; hTERT mRNA abundance in SW480 cells transfected with pGPU6-GFP-hTERT-3 was significantly lower than that in SW480 cells transfected with pGPU6-GFP-NC( 0. 347 ± 0. 028 vs0. 513 ± 0. 032,P < 0. 01). All the three hTERT sequence-specific RNAi vectors were effective to inhibit the proliferation of SW480 cells; cellular proliferation inhibition rate in SW480 cells of pGPU6-GFP-hTERT-3 group was significantly increased than that of blank contro,liposomal and NC group( [50. 08 ± 0. 43]% vs [4. 11 ± 0. 39]%,[3. 88 ±0. 35]% and [3. 38 ± 0. 35]%; P < 0. 05). Conclusion: RNAi-mediated hTERT gene silencing results in colon cancer cell growth inhibition and may offer a novel therapy for colon cancer.
Objective: To construct an optimized human telomerase reverse transcriptase( hTERT) gene-specific RNAi and to evaluate its effect on human colon cancer cell proliferation in vitro. Method: Three hTERT-specific RNAi sequences and a negative control( NC) or scrambled sequence were cloned,respectively,into a pGPU6 /GFP /Neo vector to generate pGPU6-GFP-hTERT-1,pGPU6-GFP-hTERT-2,pGPU6-GFP-hTERt-3 and pGPU6-GFP-NC. Human colon cancer SW480 cells were transfected with these vectors respectively. At 24,48 and 72 h after transfection,hTERT mRNA abundance was assessed by RT-PCR and cell viability by MTT assay. Results: The 3 hTERT-specific RNAi vectors constructed were all effective to silence the hTERT gene; hTERT mRNA abundance in SW480 cells transfected with pGPU6-GFP-hTERT-3 was significantly lower than that in SW480 cells transfected with pGPU6-GFP-NC( 0. 347 ± 0. 028 vs0. 513 ± 0. 032,P < 0. 01). All the three hTERT sequence-specific RNAi vectors were effective to inhibit the proliferation of SW480 cells; cellular proliferation inhibition rate in SW480 cells of pGPU6-GFP-hTERT-3 group was significantly increased than that of blank contro,liposomal and NC group( [50. 08 ± 0. 43]% vs [4. 11 ± 0. 39]%,[3. 88 ±0. 35]% and [3. 38 ± 0. 35]%; P < 0. 05). Conclusion: RNAi-mediated hTERT gene silencing results in colon cancer cell growth inhibition and may offer a novel therapy for colon cancer.
引文
[1]崔晶,崔丽萍,孙青.负载hTERT基因片段的DC肿瘤疫苗制备及其抗肿瘤作用的研究[J].中华肿瘤防治杂志,2010,17(16):1257-1261.
[2]王国玉,夏伟,张玉娜,等.端粒酶逆转录酶小干扰RNA对结肠癌SW480细胞体外抑制作用的观察[J].中华肿瘤防治杂志,2011,18(21):1676-1680.
[3]刘爱群,葛莲英,罗小玲,等.靶向hTERT基因RNAi慢病毒载体的构建与鉴定[J/CD].中华临床医师杂志:电子版,2011,5(1):241-245.
[4]Fraser A.RNA interference human genes hit the big screen[J].Nature,2004,428(6981):375-378.
[5]季加忠,周鹤同,陆万明,等.hTERT启动子克隆及其肿瘤细胞特异性的研究[J].中华肿瘤防治杂志,2008,15(7):491-493.
[6]卫立辛,吴孟超.端粒酶:肿瘤治疗研究的新希望[J].中国肿瘤生物治疗杂志,2006,13(5):325-328.
[7]蔡艳玲,罗小玲,葛连英,等.RNAi沉默hTERT基因诱导大肠癌SW480细胞凋亡[J].中国肿瘤生物治疗杂志,2011,18(1):46-50.
[8]Jemal A,Siegel R,Ward E,et al.Cancer statistics[J].CA Cancer J Clin,2009,59(4):225-249.
[9]Ferlay J,Autier P,Boniol M,et al.Estimates of the cancer incidence and mortality in Europe in 2006[J].Ann Oncol,2007,18(3):581-592.
[10]黎银燕,王云南,Chen Hong,等.检测端粒酶及其亚基在肺癌诊断中的临床价值[J].国际医药卫生导报,2008,14(14):5-8.
[11]Willers H,McCarthy EE,Alberti W,et al.Loss of wild-type P53function is responsible for upregulated homologous recombination in immortal rodent fibroblasts[J].Int J Radiat Biol,2007,76(8):1055-1062.
[12]Opitz OG,Suliman Y,Hahn WC,et al.Cyclin D1overexpression and p53 inactivation immortalize primary oral keratinacytes by a telomerase-independent mechanism[J].J Clin Invest,2001,108(5):725-732.
[13]Luzar B,Poljak M,Marin IJ,et al.Quantitative measurement of telomerase catalytic subunit(hTERT)mRNA in laryngeal squamous cell carcinomas[J].Anticancer Res,2001,21(6A):4011-4015.
[14]Lehner R,Enomoto T,McGregor JA,et al.Quantitative analysis of telomerase hTERT mRNA and telomerase activity in endometrioid adenocarcinoma and in normal endometrium[J].Gynecol Oncol,2002,84(1):120-125.
[15]Hara H,Yamashita K,Shinada J,et al.Clinicopathologic significance of telomerase activity and hTERT mRNA expression in nonsmall cell lung canaer[J].Lung Cancer,2001,34(2):219-226.
[16]张娇,陈爱平,戚玉言,等.靶向EGFR的干扰RNA对卵巢癌耐药细胞凋亡的影响[J].中国肿瘤生物治疗杂志,2009,16(6):619-623.
[17]de Fougerolles A,Vornlocher HP,Maraganore J,et al.Interferedwith disease:A progress report on siRNA-based therapeutics[J].Nat Rev Drug Discovery,2007,6(6):4432-4453.
[18]尤振兵,何敬东,喻晓娟.生存素siRNA表达质粒对结肠癌细胞侵袭和增殖能力的抑制作用[J].肿瘤防治研究,2008,35(7):471-475.
[19]葛莲英.端粒酶与大肠癌相关性及RNAi沉默hTERT基因治疗大肠癌的研究[D].南宁:广西医科大学,2010.
[20]Ui-Tei K,Naito Y,Takahashi F,et al.Guidelines for the selection of highly effective siRNA sequences for mammalian and click RNA interference[J].Nucleic Acids Res,2004,32(3):936-948.
[21]Schwarz DS,Hutvágner G,Du T,et al.Asymmetry in the assembly of the RNAi enzyme complex[J].Cell,2003,115(2):199-208.
[2]王国玉,夏伟,张玉娜,等.端粒酶逆转录酶小干扰RNA对结肠癌SW480细胞体外抑制作用的观察[J].中华肿瘤防治杂志,2011,18(21):1676-1680.
[3]刘爱群,葛莲英,罗小玲,等.靶向hTERT基因RNAi慢病毒载体的构建与鉴定[J/CD].中华临床医师杂志:电子版,2011,5(1):241-245.
[4]Fraser A.RNA interference human genes hit the big screen[J].Nature,2004,428(6981):375-378.
[5]季加忠,周鹤同,陆万明,等.hTERT启动子克隆及其肿瘤细胞特异性的研究[J].中华肿瘤防治杂志,2008,15(7):491-493.
[6]卫立辛,吴孟超.端粒酶:肿瘤治疗研究的新希望[J].中国肿瘤生物治疗杂志,2006,13(5):325-328.
[7]蔡艳玲,罗小玲,葛连英,等.RNAi沉默hTERT基因诱导大肠癌SW480细胞凋亡[J].中国肿瘤生物治疗杂志,2011,18(1):46-50.
[8]Jemal A,Siegel R,Ward E,et al.Cancer statistics[J].CA Cancer J Clin,2009,59(4):225-249.
[9]Ferlay J,Autier P,Boniol M,et al.Estimates of the cancer incidence and mortality in Europe in 2006[J].Ann Oncol,2007,18(3):581-592.
[10]黎银燕,王云南,Chen Hong,等.检测端粒酶及其亚基在肺癌诊断中的临床价值[J].国际医药卫生导报,2008,14(14):5-8.
[11]Willers H,McCarthy EE,Alberti W,et al.Loss of wild-type P53function is responsible for upregulated homologous recombination in immortal rodent fibroblasts[J].Int J Radiat Biol,2007,76(8):1055-1062.
[12]Opitz OG,Suliman Y,Hahn WC,et al.Cyclin D1overexpression and p53 inactivation immortalize primary oral keratinacytes by a telomerase-independent mechanism[J].J Clin Invest,2001,108(5):725-732.
[13]Luzar B,Poljak M,Marin IJ,et al.Quantitative measurement of telomerase catalytic subunit(hTERT)mRNA in laryngeal squamous cell carcinomas[J].Anticancer Res,2001,21(6A):4011-4015.
[14]Lehner R,Enomoto T,McGregor JA,et al.Quantitative analysis of telomerase hTERT mRNA and telomerase activity in endometrioid adenocarcinoma and in normal endometrium[J].Gynecol Oncol,2002,84(1):120-125.
[15]Hara H,Yamashita K,Shinada J,et al.Clinicopathologic significance of telomerase activity and hTERT mRNA expression in nonsmall cell lung canaer[J].Lung Cancer,2001,34(2):219-226.
[16]张娇,陈爱平,戚玉言,等.靶向EGFR的干扰RNA对卵巢癌耐药细胞凋亡的影响[J].中国肿瘤生物治疗杂志,2009,16(6):619-623.
[17]de Fougerolles A,Vornlocher HP,Maraganore J,et al.Interferedwith disease:A progress report on siRNA-based therapeutics[J].Nat Rev Drug Discovery,2007,6(6):4432-4453.
[18]尤振兵,何敬东,喻晓娟.生存素siRNA表达质粒对结肠癌细胞侵袭和增殖能力的抑制作用[J].肿瘤防治研究,2008,35(7):471-475.
[19]葛莲英.端粒酶与大肠癌相关性及RNAi沉默hTERT基因治疗大肠癌的研究[D].南宁:广西医科大学,2010.
[20]Ui-Tei K,Naito Y,Takahashi F,et al.Guidelines for the selection of highly effective siRNA sequences for mammalian and click RNA interference[J].Nucleic Acids Res,2004,32(3):936-948.
[21]Schwarz DS,Hutvágner G,Du T,et al.Asymmetry in the assembly of the RNAi enzyme complex[J].Cell,2003,115(2):199-208.