RNA干扰技术沉默STAT3基因对人肺腺癌A549细胞的生长抑制作用的研究
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摘要
目的:探讨RNA干扰技术沉默STAT3基因对人肺腺癌A549细胞的生长抑制作用。方法:①以STAT3 mRNA全基因序列作为模板,根据siRNA的设计原则,从基因编码区起始密码子下游寻找符合设计特征的靶序列–针对STAT3基因编码区989-1007与2144-2162核苷酸碱基序列,用BLAST软件进行同源分析证实为STAT3高度保守、与人类其它基因无同源性后,采用化学合成法合成四条寡核苷酸链。设计的寡核苷酸序列5'端和3'端分别含BglII和HindIII酶切位点,5'端开始为19个核苷酸的siRNA作用链序列,中间以9个核苷酸的TTCAAGAGA间隔形成发夹结构,最后为siRNA作用序列的反向重复序列,3'末端加有转录终止信号TTTTT。②取等量的正负链DNA合成片段,退火得到目的双链DNA(dsDNA)。含RNA聚合酶Ⅲ与U6启动子的真核表达质粒pSUPER经限制性内切酶BglII和HindIII双酶切,暴露BglII和HindIII酶切位点,可直接用于与制备好的目的基因连接。将纯化的双链DNA片段定向克隆到经BglII和HindIII酶双酶切的真核表达质粒pSUPER的多克隆位点,利用重组DNA技术构建含目的基因的重组质粒pSUPER–siRNA–STAT3。以常规方法制备感受态宿主E. coli DH5α,并将连接产物转化感受态细菌。将菌液涂布于含氨苄青霉素(终浓度60μg/ml)的固体LB培养板上,倒置于37℃温箱中孵育过夜。连接成功的重组体能在抗性培养板上生成单菌落,随机挑取生长良好的数个单菌落于含有适量氨苄青霉素的LB液体培养基3ml中,置于37℃摇床中250rpm过夜。以小量质粒抽提试剂提取扩增后的重组质粒,即得到目的表达载体pSUPER。经PCR初步筛选并进行DNA测序分析加以确定,证实能在体内转录针对STAT3基因转录体的发夹状siRNA的表达载体pSUPER–siRNA–STAT3构建成功。③采用脂质体介导方法,将重组质粒和脂质体按20:1的比例共转染肺腺癌A549细胞。同时设立空质粒对照组及空白对照组。分别于转染24小时、48小时、72小时收集细胞。
Objective: To develop an RNAi approach that specificallytargets the STAT3 gene sequence by short interfering RNA (siRNA), andto assess the inhibitory effect of pSUPER–siRNA–STAT3 on thegrowth of A549 cells. Methods: A siRNA targeting STAT3 genesequence were constructed based on pSUPER vector, the sequence weredigested with the restriction endonucleases BglII and HindIII, then it wascloned into pSUPER which contain RNA polymerase Ⅲ to constructpSUPER–siRNA–STAT3. The eukaryotic expression plasmid werecotransfected into A549 cells with either the RNAi plasmid pSUPER–siRNA–STAT3 or control plasmid to assess the inhibitory effect ofRNAi on STAT3 gene expression. At 24h,48h,72h post transfection, thelevel of STAT3 mRNA was detected by RT-PCR and cells viability byfluorescence microscope. Results: The siRNA eukaryotic expressionvector against STAT3 mRNA has been successfully conctructed, whichwas transfected into A549 cells. The IeveI of STAT3 mRNA in A549cells was inhibited by the specific siRNAs.The decrease of STAT3mRNA expression began to appear 24 hours after transfection.And themost apparent interfering efficiency was 85.32%and 75%,48h aftertransfection,which was markedly higher than that in the cells transfectedwith the controI siRNAs.Both siRNAs from different Ioci had interferingeffect on STAT3 mRNA expression,Compared with those controlcells ,the apoptotic cells were significantly higher in siRNA transfectedcells by fluorescence microscope. Conclusion: siRNA transcriptingplasmids pSUPER are successfully constructed.pSUPER–siRNA–STAT3 could significantly inhibit STAT3 expression,suppress the growthof A549 cells. The RNA interfering technique targeted on STAT3 mayProvide a new method in the gene therapy of lung cancer.
Objective: To develop an RNAi approach that specificallytargets the STAT3 gene sequence by short interfering RNA (siRNA), andto assess the inhibitory effect of pSUPER–siRNA–STAT3 on thegrowth of A549 cells. Methods: A siRNA targeting STAT3 genesequence were constructed based on pSUPER vector, the sequence weredigested with the restriction endonucleases BglII and HindIII, then it wascloned into pSUPER which contain RNA polymerase Ⅲ to constructpSUPER–siRNA–STAT3. The eukaryotic expression plasmid werecotransfected into A549 cells with either the RNAi plasmid pSUPER–siRNA–STAT3 or control plasmid to assess the inhibitory effect ofRNAi on STAT3 gene expression. At 24h,48h,72h post transfection, thelevel of STAT3 mRNA was detected by RT-PCR and cells viability byfluorescence microscope. Results: The siRNA eukaryotic expressionvector against STAT3 mRNA has been successfully conctructed, whichwas transfected into A549 cells. The IeveI of STAT3 mRNA in A549cells was inhibited by the specific siRNAs.The decrease of STAT3mRNA expression began to appear 24 hours after transfection.And themost apparent interfering efficiency was 85.32%and 75%,48h aftertransfection,which was markedly higher than that in the cells transfectedwith the controI siRNAs.Both siRNAs from different Ioci had interferingeffect on STAT3 mRNA expression,Compared with those controlcells ,the apoptotic cells were significantly higher in siRNA transfectedcells by fluorescence microscope. Conclusion: siRNA transcriptingplasmids pSUPER are successfully constructed.pSUPER–siRNA–STAT3 could significantly inhibit STAT3 expression,suppress the growthof A549 cells. The RNA interfering technique targeted on STAT3 mayProvide a new method in the gene therapy of lung cancer.
引文
1. 朱元珏,陈文斌. 呼吸病学[M]. 北京:人民卫生出版社,2003;1011
2. 蔡柏蔷,李龙芸. 协和呼吸病学[M]. 北京:中国协和医科大学出版社.,2005;915
3. Fu XY,Schindler C,Improta T, et a1.The proteins of ISGF-3,the interferon alpha-induced transcriptional activator define a gene family involved in signal transduction [J]. Proc Natl Acad Sci USA,1992;89(16) 7840～7843
4. Zhong Z, Wen Z, Darnel JE JR. Stat 3 and Stat 4: members of the family of signal transducers and activators of transcription [J] Proc Natl Acad Sci USA, 1994;91(1):4806～4810
5. YiZ,James T,Christin C ,et al .activation of Stat3 in v-Src transformed fibroblasts requires cooperation of Jak1 kinase activity [J].Biol Chem, 2000;275 (32):24935～24944
6. Chung CD,Liao J,Lin B,et al. Specific inhibition of STAT3 signal transduction by PIAS3 [J]. Science 1997;278:1803～1805
7. Kumisada K, Negoro S, Tone E, et al. signal transducers and activators of transcription in the heart transducers not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy [J]. Proc Nati Acad Sci USA .2000;97:315～319
8. Darnell JE, Reflections on STAT3,STAT5 and STAT6 as fat STATs [J]. Proc Natl Acad Sci USA. 2000;97:315～319
9. Bromberg J. Stat proteins and oncogenesis [J]. J Clin Invest 2002;109 (9):1139～1142
10. Niu G ,Wright KL,Hung M, et al. Constitutive STAT3 activity up-regulates VEGF expression and tumor angiogenesis [J]. Oncogene, 2002;21(13):2000～2008
11. Riku Fagerlunds, Krister Melen, Leena Kinnunen, et al. Argine/Lysine-rich Nuclear Localization signals mediate interactions between dimeric Stats and importin a5. The journal of Biological Chemistry, 2002;277:3072～3078
12. Burke WM, Jin X, Lin HJ, et a1. Inhibition of constitutively active STAT3 suppresses growth of human ovarian and breast cancer cells [J] Oncogene, 2001;20(55):7925～7934
13. Leong PL, Andrews GA, Johnson DE, et a1. Targeted inhibition of STAT3 with a decoy o1igonucleotide abrogates head and neck cancer cell growth [J]. Proc Nat1 Acad Sci USA, 2003;100(7):4138～4143
14. 王鸿程,李艳萍,李万成等.肺腺癌细胞增殖分化及抗凋亡作用与STAT3 的调控效应[J].中国临床康复,2005;9(14):128
15. 王鸿程,张程,曹乃清等.非小细胞肺癌中 Bax 和 Cyclin D1 的表达及意义[J].四川医学,2004;25(9):978
16. 曹乃清,王鸿程,陈洁等.CyclinD1、MDR1 在非小细胞肺癌中的表达及临床意义[J].四川医学,2004;25(7):732
17. 张程,王鸿程,李万成等.非小细胞肺癌 BCL-2、PCNA 表达的临床研究[J].泸州医学院学报,2002;25(6):471
18. Zamore PD. RNA interference :listening to the sound of silence[J]. Nat Struct Biol, 2001;8(9):746
19. Persad S,Attwell S,Gray V,et a1.Inhibition of integrin–linked kinase(ILK)suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN–mutant prostate cancer ceHs[J].Proc Natl Acad Sci USA,2000;97(7):3207～32l2
20. DAmico M,Hulit J,Amanatunah DF,et a1.The integrin–linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP–responsive element–binding protein–dependent pathways[J].J Biol Chem,2000;275(42):32649～32657
21. Wu C,KeJighfley SY,Leung·Hagesteijn C.Integrin–linked protein kinase regulates fibroneetin matrix assembly,E-cadherin expression,and tumorigenicity[J].J Biol Chem,1998;273(1):528～536.
22. Zhang X,Li Y,Huang Q,et a1.Increased resistance of tumor cells to hyperthermia mediated by integrin–linked kinase[J].Clin Cancer Res,2003;9(3):1155～1160.
23. 戚晓平,方丽,陈群阳,等.原发性前列腺印戒细胞癌(附二例报告并文献复习)[J].中华泌尿外科杂志,2003;24(2):122～124.
24. 胥文春等。STAT3与VEGF在非小细胞肺癌中的表达及关系[J].重庆医科大学学报. 2003;28(4):460～466
25. Lipardi C, wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degr-adeed to generate new siRNAs[J] .Cell, 200l;107(3): 297～307
26. Sijen T, Fleenor J, Simmer F, et a1. On the role of RNA amplific-ation in dsRNA-triggered gene silencing[J]. Cell, 2001;107: 465～476
27. McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference[J].Nat Biotechnol, 2003;21: 639～644
2. 蔡柏蔷,李龙芸. 协和呼吸病学[M]. 北京:中国协和医科大学出版社.,2005;915
3. Fu XY,Schindler C,Improta T, et a1.The proteins of ISGF-3,the interferon alpha-induced transcriptional activator define a gene family involved in signal transduction [J]. Proc Natl Acad Sci USA,1992;89(16) 7840～7843
4. Zhong Z, Wen Z, Darnel JE JR. Stat 3 and Stat 4: members of the family of signal transducers and activators of transcription [J] Proc Natl Acad Sci USA, 1994;91(1):4806～4810
5. YiZ,James T,Christin C ,et al .activation of Stat3 in v-Src transformed fibroblasts requires cooperation of Jak1 kinase activity [J].Biol Chem, 2000;275 (32):24935～24944
6. Chung CD,Liao J,Lin B,et al. Specific inhibition of STAT3 signal transduction by PIAS3 [J]. Science 1997;278:1803～1805
7. Kumisada K, Negoro S, Tone E, et al. signal transducers and activators of transcription in the heart transducers not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy [J]. Proc Nati Acad Sci USA .2000;97:315～319
8. Darnell JE, Reflections on STAT3,STAT5 and STAT6 as fat STATs [J]. Proc Natl Acad Sci USA. 2000;97:315～319
9. Bromberg J. Stat proteins and oncogenesis [J]. J Clin Invest 2002;109 (9):1139～1142
10. Niu G ,Wright KL,Hung M, et al. Constitutive STAT3 activity up-regulates VEGF expression and tumor angiogenesis [J]. Oncogene, 2002;21(13):2000～2008
11. Riku Fagerlunds, Krister Melen, Leena Kinnunen, et al. Argine/Lysine-rich Nuclear Localization signals mediate interactions between dimeric Stats and importin a5. The journal of Biological Chemistry, 2002;277:3072～3078
12. Burke WM, Jin X, Lin HJ, et a1. Inhibition of constitutively active STAT3 suppresses growth of human ovarian and breast cancer cells [J] Oncogene, 2001;20(55):7925～7934
13. Leong PL, Andrews GA, Johnson DE, et a1. Targeted inhibition of STAT3 with a decoy o1igonucleotide abrogates head and neck cancer cell growth [J]. Proc Nat1 Acad Sci USA, 2003;100(7):4138～4143
14. 王鸿程,李艳萍,李万成等.肺腺癌细胞增殖分化及抗凋亡作用与STAT3 的调控效应[J].中国临床康复,2005;9(14):128
15. 王鸿程,张程,曹乃清等.非小细胞肺癌中 Bax 和 Cyclin D1 的表达及意义[J].四川医学,2004;25(9):978
16. 曹乃清,王鸿程,陈洁等.CyclinD1、MDR1 在非小细胞肺癌中的表达及临床意义[J].四川医学,2004;25(7):732
17. 张程,王鸿程,李万成等.非小细胞肺癌 BCL-2、PCNA 表达的临床研究[J].泸州医学院学报,2002;25(6):471
18. Zamore PD. RNA interference :listening to the sound of silence[J]. Nat Struct Biol, 2001;8(9):746
19. Persad S,Attwell S,Gray V,et a1.Inhibition of integrin–linked kinase(ILK)suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN–mutant prostate cancer ceHs[J].Proc Natl Acad Sci USA,2000;97(7):3207～32l2
20. DAmico M,Hulit J,Amanatunah DF,et a1.The integrin–linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP–responsive element–binding protein–dependent pathways[J].J Biol Chem,2000;275(42):32649～32657
21. Wu C,KeJighfley SY,Leung·Hagesteijn C.Integrin–linked protein kinase regulates fibroneetin matrix assembly,E-cadherin expression,and tumorigenicity[J].J Biol Chem,1998;273(1):528～536.
22. Zhang X,Li Y,Huang Q,et a1.Increased resistance of tumor cells to hyperthermia mediated by integrin–linked kinase[J].Clin Cancer Res,2003;9(3):1155～1160.
23. 戚晓平,方丽,陈群阳,等.原发性前列腺印戒细胞癌(附二例报告并文献复习)[J].中华泌尿外科杂志,2003;24(2):122～124.
24. 胥文春等。STAT3与VEGF在非小细胞肺癌中的表达及关系[J].重庆医科大学学报. 2003;28(4):460～466
25. Lipardi C, wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degr-adeed to generate new siRNAs[J] .Cell, 200l;107(3): 297～307
26. Sijen T, Fleenor J, Simmer F, et a1. On the role of RNA amplific-ation in dsRNA-triggered gene silencing[J]. Cell, 2001;107: 465～476
27. McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference[J].Nat Biotechnol, 2003;21: 639～644