期刊存档
siRNA靶向沉默hTERT基因对人胰腺癌的凋亡抑制基因bcl-2和环氧合酶-2基因表达的影响
钟英强，黄花荣，付玉如，朱兆华钟英强、黄花荣、朱兆华，广州 中山大学附属第二医院消化内科，510120；付玉如，医学研究中心，510120
广东省自然科学基金(7001607)；广东省科技计划项目(2008B030301115)
摘 要:目的 应用siRNA靶向沉默人胰腺癌Capan-2细胞的hTERT基因表达，观察其对凋亡抑制基因(bcl-2)和环氧合酶-2(COX-2)基因表达的影响。方法 应用脂质体Lipofectamine 2000作为基因转染的载体，实时PCR进行扩增检测转染细胞的hTERT、bcl-2、COX-2基因的mRNA表达水平，应用Western blotting检测hTERT、bcl-2、COX-2蛋白表达水平。结果 在hTERT-siRNA完全沉默hTERT表达的基础上，bcl-2、COX-2基因mRNA表达的量在hTERT-siRNA转染后48 h均明显受到抑制(P<0.001)，抑制率分别为87.86%、100.00%，但在72 h后均恢复至与对照组相似的水平，而阴性对照组、Lipo对照组与空白对照组的各基因表达量差异均无统计学意义(P>0.05)。bcl-2、COX-2蛋白水平在转染48 h和72 h后被分别下调了58.54%和63.44%、50.06%和82.77%(P<0.01)，Lipo对照组和阴性对照组与空白对照组差异无统计学意义(P>0.05)。结论 siRNA靶向沉默hTERT基因表达，可在一定程度上抑制人胰腺癌Capan-2细胞的bcl-2、COX-2基因的表达。
关键词:胰腺肿瘤； 基因，bcl-2； 环氧化酶2； 端粒，末端转移酶； RNA干扰； 脂质体
Effects of silencing expression of hTERT gene by siRNA on the expression of bcl-2 and COX-2 genes of human pancreatic cancer cells in vitro
ZHONG Ying-qiang，HUANG Hua-rong，FU Yu-ru，ZHU Zhao-hua.Department of Gastroenterology，Second Affiliated Hospital，Sun Yat-sen University，Guangzhou 510120，China
Abstract:Objective To investiate effects of silencing expression of human telomerase reverse transcriptase (hTERT) gene by siRNA targeting to hTERT gene on the expressions of bcl-2，COX-2 genes of Capan-2 cells of human pancreatic cancer in vitro.Methods The media of transfection gene was Lipofectamine 2000.The expressions of hTERT mRNA，COX-2 mRNA and bcl-2 mRNA were tested by realtime PCR，and the control was GAPDH.The expressions of hTERT，COX-2 and bcl-2 proteins were tested by Western blotting，and the control was actin.Results Under the same experiment condition，the inhibited rate of expression of hTERT-mRNA by siRNA was 100%，and the expressions of COX-2，bcl-2 mRNA were inhibited remarkably by transfectation of hTERT-siRNA after 48 hours，and their inhibited rates were 100%,87.86% resprectively(P<0.001)，but they recovered the same level as the control after 72 hours.Meanwhile，the expressions of COX-2，bcl-2 proteins were downregulated 50.06% and 82.77%,58.54% and 63.44% after 48 hours and 72 hours resprectively (P<0.01).But no difference among the controls (P>0.05).Conclusions Targing silence to hTERT gene by siRNA，it can inhibit the expressions of COX-2，bcl-2 genes of Capan-2 cells of human pancreatic cancer in vitro in some degree.
Key words:Pancreatic neoplasms； Genes，bcl-2； Cyclooxygenase 2； Telomerase； RNA interference； Liposomes
参考文献:
[1] Chung HK，Cheong C，Song J，et al.Extratelomeric functions of telomerase.Curr Mol Med,2005,5(2)：233-241.[PubMed]
[2] Choi YH.Apoptosis of U937 human leukemic cells by sodium butyrate is associated with inhibition of telomerase activity.Int J Oncol,2006,29(5)：1207-1213.[PubMed]
[3] Rüegg C，Dormond O，Mariotti A.Endothelial cell integrins and COX-2：mediators and therapeutic targets of tumor angiogenesis.Biochim Biophys Acta,2004,1654(1)：51-67.[PubMed]
[4] 钟英强，魏菁，罗招凡，等.siRNA靶向沉默hTERT基因对人胰腺癌Capan-2细胞增殖、凋亡和周期的影响[J/CD].中华临床医师杂志：电子版，2008,2(10)：1155-1166.
[5] 钟英强，魏菁，傅玉如，等.阳离子脂质体Lipofectamine2000对人胰腺癌Capan-2细胞的毒性作用.南方医科大学学报，2008,28(11)：1981-1984.
[6] Verma UN，Surabhi RM，Schmaltieg A，et al.Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells.Clin Cancer Res,2003,9(4)：1291-1300.[PubMed]
[7] Li K，Lin SY，Brunicardi FC，et al.Use of RNA interference to target cyclin E-overexpressing hepatocellular carcinoma.Cancer Res,2003,63(13)：3593-3597.[PubMed]
[8] Mukhopadhyay D， Houchen CW， Kennedy S，et al.Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein，CUGBP2.Mol Cell,2003,11(1)：113-126.[PubMed]
[9] Vogler M，Walczak H，Stadel D，et al.Targeting XIAP bypasses Bcl-2-mediated resistance to TRAIL and cooperates with TRAIL to suppress pancreatic cancer growth in vitro and in vivo.Cancer Res,2008,68(19)：7956-7965.[PubMed]
[10] Grochola LF，Greither T，Taubert HW，et al.Prognostic relevance of hTERT mRNA expression in ductal adenocarcinoma of the pancreas.Neoplasia,2008,10(9)：973-976.[PubMed]
[11] Wack S，Rejiba S，Parmentier C，et al.Telomerase transcriptional targeting of inducible Bax/TRAIL gene therapy improves gemcitabine treatment of pancreatic cancer.Mol Ther,2008,16(2)：252-260.[PubMed]
[12] 钟英强，沈溪明，李海刚，等.hTERT在人胰腺癌组织中的表达及其COX-2、P-gp、Bcl-2蛋白表达表达和临床病理特征的关系[J/CD].中华临床医师杂志：电子版，2008,2(8)：877-885.
[13] Hashimoto Y，Murakami Y，Uemura K，et al.Detection of human telomerase reverse transcriptase (hTERT) expression in tissue and pancreatic juice from pancreatic cancer.Surgery,2008,143(1)：113-125.[PubMed]
[14] Kawahara R， Odo M， Kinoshita H， et al. Analysis of hTERT mRNA expression in biliary tract and pancreatic cancer.J Hepatobiliary Pancreat Surg,2007,14(2)：189-193.[PubMed]
[15] Park C，Jung JH，Kim ND，et al.Inhibition of cyclooxygenase-2 and telomerase activities in human leukemia cells by dideoxypetrosynol A，a polyacetylene from the marine sponge Petrosia sp.Int J Oncol,2007,30(1)：291-298.[PubMed]
[16] He H，Xia HH，Wang JD，et al.Inhibition of human telomerase reverse transcriptase by nonsteroidal antiinflammatory drugs in colon carcinoma.Cancer,2006,106(6)：1243-1249.[PubMed]
[17] 郭述良，罗永艾.RNA干扰及其在呼吸系统疾病研究中的应用.国外医学：内科学分册，2004,31(12)：507-510.
[18] 赵剑华，王秀琴，刘芝华，等.功能基因组学的研究内容与方法.生物化学与生物物理进展，2000,27(1)：6-8.