乳腺癌细胞Id1 miRNA真核表达载体构建转染及对MMP9的影响
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摘要
[目的]:构建Id1 miRNA真核表达载体,并成功转染乳腺癌MDA-MB-231细胞株,筛选稳定表达的乳腺癌细胞株,观察对乳腺癌细胞的对Id 1、MMP 9表达的影响。
[方法]:
利用Invitrogen公司网上BLOCK-iT miR RNAi设计工具,设计针对Id 1基因(ID:3397)的两个转录版本mRNA(NM_002165.2和NM_181353.1)都有效的干扰序列。将干扰序列插入pcDNA6.2-GW/EmGFP-miR表达载体,构建Id1miRNA真核表达载体,通过脂质体转染法转染到乳腺癌MDA-MB-231细胞株,以杀稻瘟霉素筛选稳定表达的细胞株。经RT-PCR方法鉴定其对Id1、MMP9表达的影响。
[结果]:
成功构建Id1 miRNA真核表达载体,经电泳及测序鉴定正确,转染乳腺癌MDA-MB-231细胞系。经过2W杀稻瘟霉素筛选,获得稳定表达的细胞系。经RT-PCR方法鉴定,结果表明转染Id1 miRNA真核表达载体的乳腺癌MDA-MB-231细胞株其Id 1、MMP 9表达水平低于对照组,且Id 1、MMP 9表达水平正相关。未转染组与阴性对照转染组无明显差异。
[结论]:
本实验成功构建Id1 miRNA真核表达载体,并成功转染乳腺癌MDA-MB231细胞株,筛选出稳定表达Id1 miRNA的乳腺癌细胞株,证实干扰有效,并且Id1与mmp9表达相关,为将来进一步研究他们之间的生物学行为差异,或详细了解其分子作用机制奠定了基础。
[Purpose]: To construct the miRNA eukaryotic expression vector of Id1 and transfect the MDA-MB-231 breast cancer cell line,and finally select the stabilized expressed cells lines. To explore the relationship between Id 1 and MMP 9 expression in breast cancer line MDA-MB-231 .
[Method]: The miRNA array of Id1 gene was designed and insert into pcDNA6.2-GW/EmGFP-miR expression vector to construct the Id1 miRNA eukaryotic expression vector. The liposome infection protocol was used to transfect the MDA-MB-231 cell lines. Use Blasticidin to select stabilized expressed cell lines.the level of Id1 and MMP 9 was detected using RT-PCR methods.
[Result]: The eukaryotic expression vector of Id1 miRNA was successfully constructed After Identifying by electrophoresis and measuring array, the MDA-MB-231 breast cancer cell line was tranfected. We acquire the stabilized expressed cell lines through 2W Blasticidin selection. As a result of RT-PCR Identification, the level of Id1 and MMP 9 in Id1 miRNA transfeced cell line is lower than that in the control groups. While there is no difference between the not transfected and negative control transfected cell line.
[Conclusion]:The experiment successfully construct the eukaryotic expression vector of Id1 miRNA and successfully transfect the MDA-MB-231 breast cancer cell line and finally select the stabilized expressed cells lines.It prove that Id1and MMP9 are closely related.
[方法]:
利用Invitrogen公司网上BLOCK-iT miR RNAi设计工具,设计针对Id 1基因(ID:3397)的两个转录版本mRNA(NM_002165.2和NM_181353.1)都有效的干扰序列。将干扰序列插入pcDNA6.2-GW/EmGFP-miR表达载体,构建Id1miRNA真核表达载体,通过脂质体转染法转染到乳腺癌MDA-MB-231细胞株,以杀稻瘟霉素筛选稳定表达的细胞株。经RT-PCR方法鉴定其对Id1、MMP9表达的影响。
[结果]:
成功构建Id1 miRNA真核表达载体,经电泳及测序鉴定正确,转染乳腺癌MDA-MB-231细胞系。经过2W杀稻瘟霉素筛选,获得稳定表达的细胞系。经RT-PCR方法鉴定,结果表明转染Id1 miRNA真核表达载体的乳腺癌MDA-MB-231细胞株其Id 1、MMP 9表达水平低于对照组,且Id 1、MMP 9表达水平正相关。未转染组与阴性对照转染组无明显差异。
[结论]:
本实验成功构建Id1 miRNA真核表达载体,并成功转染乳腺癌MDA-MB231细胞株,筛选出稳定表达Id1 miRNA的乳腺癌细胞株,证实干扰有效,并且Id1与mmp9表达相关,为将来进一步研究他们之间的生物学行为差异,或详细了解其分子作用机制奠定了基础。
[Purpose]: To construct the miRNA eukaryotic expression vector of Id1 and transfect the MDA-MB-231 breast cancer cell line,and finally select the stabilized expressed cells lines. To explore the relationship between Id 1 and MMP 9 expression in breast cancer line MDA-MB-231 .
[Method]: The miRNA array of Id1 gene was designed and insert into pcDNA6.2-GW/EmGFP-miR expression vector to construct the Id1 miRNA eukaryotic expression vector. The liposome infection protocol was used to transfect the MDA-MB-231 cell lines. Use Blasticidin to select stabilized expressed cell lines.the level of Id1 and MMP 9 was detected using RT-PCR methods.
[Result]: The eukaryotic expression vector of Id1 miRNA was successfully constructed After Identifying by electrophoresis and measuring array, the MDA-MB-231 breast cancer cell line was tranfected. We acquire the stabilized expressed cell lines through 2W Blasticidin selection. As a result of RT-PCR Identification, the level of Id1 and MMP 9 in Id1 miRNA transfeced cell line is lower than that in the control groups. While there is no difference between the not transfected and negative control transfected cell line.
[Conclusion]:The experiment successfully construct the eukaryotic expression vector of Id1 miRNA and successfully transfect the MDA-MB-231 breast cancer cell line and finally select the stabilized expressed cells lines.It prove that Id1and MMP9 are closely related.
引文
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[1]Benezra R,Davis RL,Lockshon D,et a1.The protein Id:A negative regulator of helix-loop-helix DNA binding proteins[J].Cell,1990;61(1):49—59 [2 ]Wilson JW,Deed RW,1noue T,et a1.Expression of Id helix-loop-helix proteins in colorectal adenocarcinoma correlates with p53 expression and mitotic index [J].Cancer Res,2001;61(24):8803—88l0.
[3]Ouyang XS,Wang X,Lee DT,et a1.Over expression of ID-1 in prostate cancer l J.J Urof,2002;167(6):2598～2602.
[4]Maruyama H,Kleeff J,Wildi S,et a1.M-l and Id-2 are overexpressed in pancreatic cancer and in dysplastic lesions in chronic pancreatitis [J].Am J Pathol,l999;155(3):815—822.
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[17]Pagliuca A, Gallo P, De Luca P, Lania L. Class A helix-loop-helix proteins are positive regulators of several cyclin-dependent kinase inhibitors promoter activity and negatively affect cell growth. Cancer Res 2000;60:1376-1382
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[19]Schindl M, Schoppmann SF, Strobel T, Heinzl H, Leisser C, Horvat R, Birner P. Level of Id-1 protein expression correlates with poor differentiation, enhanced malignant potential, and more aggressive clinical behavior of epithelial ovarian tumors. Clin Cancer Res 2003;9:779-785 [20 ]Schindl M, Oberhuber G, Obermair A, Schoppmann SF, Karner B, Birner P. Overexpression of Id-1 protein is a marker for unfavorable prognosis in early-stage cervical cancer. Cancer Res 2001;61:5703-5706
[21]Ouyang XS, Wang X, Ling MT, Wong HL, Tsao SW, Wong YC. Id-1 stimulates serum independent prostate cancer cell proliferation through inactivation ofp16(INK4a)/pRB pathway. Carcinogenesis 2002;23:721-725
[22]Hasskarl J,Mtm4ger K.Id proteins-tumor markers or oncogenes[J].Cancer Biol Ther,2002,1(2):91-96.
[23]Lvden D,Young AZ,Zagzag D,et a1.Id1 and Id3 are required for neurogenesis,angiogenesis an d vascularization of tumour xenografts [J] Nature,1999;401(6754):670—677
[24]C. E. Caldon, A. Swarbrick, C.S.L. Lee, R. L. Sutherland, and E. A. Musgrove,The Helix-Loop-Helix Protein Id1 Requires Cyclin D1 to Promote the Proliferation of Mammary Epithelial Cell Acini,Cancer Res. April 15, 2008; 68(8): 3026– 3036.
[25]Swarbrick A, Akerfeldt MC, Lee CS, Sergio CM, Caldon CE, Hunter LJ, Sutherland RL, Musgrove EA.Regulation of cyclin expression and cell cycle progression in breast epithelial cells by the helix-loop-helix protein Id1.Oncogene. 2005 Jan 13;24(3):381-9
[26]Jankiewicz, Marcin; Groner, Bernd; Desrivieres, Sylvane,Mammalian Target of Rapamycin Regulates the Growth of Mammary Epithelial Cells through the Inhibitor of Deoxyribonucleic AcId Binding Id1 and Their Functional Differentiation through Id2. Molecular Endocrinology. 2006;20(10):2369-2381
[2]Ouyang XS,Wang X,Lee DT,et a1.Over expression of ID 1 in prostate cancer l J.J Urof,2002;167(6):2598～2602.
[3] Vandeputte DA,Troost D,Leenstra S,et a1.Expression and distribution of Id helix-loop-helix proteins in human astrocytic tumors.Glia,2002,38(4):329—338.
[4]Yokota Y,Moil S.Role of Id family proteins in growth contro.J Cell Physiol,2002,190(11:21—28.
[5]Swarbrick A, Akerfeldt MC, Lee cs, et al, Regulation of cyclin expression and cell cycle progression in breast epithelial cells by the helix-loop-helix protein ID1.oncogene ,2005 24(3):381-389
[6]Caldon CE, Swarbrick A, Lee CS, Sutherland RL, Musgrove EA. The helix-loop-helix protein Id1 requires cyclin D1 to promote the proliferation of mammary epithelial cell acini. Cancer Res. 2008 Apr 15;68(8):3026-36.
[7]Jang KS, Han HX, Paik SS, Brown PH, Kong G. Id-1 overexpression in invasive ductal carcinoma cells is significantly associated with intratumoral microvessel density in ER-negative/node-positive breast cancer. Cancer Lett, 2006, 244(2):203-10.
[8] Swarbrick A, Roy E, Allen T, Bishop JM. Id1 cooperates with oncogenic Ras to induce metastatic mammary carcinoma by subversion of the cellular senescence response. Proc Natl Acad Sci U S A , 2008 , 105(14):5402-7.
[9] Gupta GP, Perk J, Acharyya S, de Candia P, Mittal V, Todorova-Manova K, Gerald WL, Brogi E, Benezra R, MassaguéJ ID genes mediate tumor reinitiation during breast cancer lung metastasis. Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19506-11..
[10] Hasskarl J,Mtmtger K.Id proteins-tumor markers or oncogenes?.Cancer Biol Ther,2002,1(2):91-96.
[11] Wong vc,Wang X,Ling MT.Id-1 expression and cell survival. Apoptosis, 2004,9 (3):279-289.
[12] Visse R,Nagase H.M atrix meta11oproteinases and tissue Inhibitors of meta11oproteinases:structure,function,and biochemistry.Circ Res,2003,92:827—839.
[13] Chambers A F,Matrisian L M,et a1.Changing views of the role of matrix meta11oproteinases in metastasis.J Natl Cancer Inst,1997,89:1260—1270.
[14] Papetti M,Herman I M , Mechanisms of normal and tumor-derived angiogenesis.Am J Physiol Cell Physiol,2002,282:947—970. [15 ]Ray J M ,Stetler—Stevenson W G.The role of matrix meta11oproteases and their inhibitors in tumour invasion,metastasis and angiogenesis.Eur Respir J,1994,7:2062—2072
[16] Kleiner D E , Stetler-Stevenson W G.Matrix metalloproteinases and m etastasis.Cancer Chemother Pharmacol,1999,43 (Supp1):S42—51.
[17] Sato H,Seiki M.v-Src activates the expression of 92 kDa type IV collagenase gene through the AP一1 site and the GT box homologous to retinoblastoma control elements.A mechanism regulating gene expression independent of that by inflammatory cytokines.J BioI Chem,1993,268:23460—23468.
[18] Olson M W,Bernardo M M,Pietila M,et a1.Characterization of the monomeric and dimeric forms of latent and active matrix etalloproteinase-9.J Biol Chem,2000,275:2661—2668.
[19]Chambers A F,Matrisian L M,et a1.Changing views of the role of matrix meta11oproteinases in metastasis.J Natl Cancer Inst,1997,89:1260—1270.
[20]Papetti M,Herman I M , Mechanisms of normal and tumor-derived angiogenesis.Am J Physiol Cell Physiol,2002,282:947—970.
[21]Kleiner D E , Stetler-Stevenson W G.Matrix metalloproteinases and m etastasis.Cancer Chemother Pharmacol,1999,43 (Supp1):S42—51.
[22]Brew K , Dinakarpandian D , Nagase H . Tissue inhibitors of metalloproteinases:evolution,structure and function.Biochim Biophys Acta,2000,1477:267—283.
[23] Baker A H,Edwards D R,Murphy G.Metalloproteinase inhibitors:biologicalactions and therapeutic opportunities.J CelI Sci,2002,ll5:3719—3727.
[24]Olson M W,Bernardo M M,Pietila M,et a1.Characterization of the monomeric and dimeric forms of latent and active matrix etalloproteinase-9.J Biol Chem,2000,275:2661—2668.
[25]Visse R , Nagase H.M atrix meta11oproteinases and tissue Inhibitors of meta11oproteinases:structure,function,and biochemistry.Circ Res,2003,92:827—839.
[25]Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bIdentate ribonuclease in the initiation step of RNA interference. Nature, 2001,409 (6818): 363–6.
[26]Kurihara Y, Watanabe Y. ArabIdopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc. Natl. Acad. Sci. U.S.A. 2004,101 (34): 12753–8..
[27]Gao FB. Posttranscriptional control of neuronal development by microRNA networks. Trends Neurosci.2008, 31 (1): 20–6..
[28]Stark A, Bushati N, Jan CH, et al. A single Hox locus in Drosophila produces functional microRNAs from opposite DNA strands. Genes Dev. 2008,22 (1): 8–13..
[29]李海明,施南峰.miRNA——一种新的调控基因表达的小分子RNA [J].中国癌症杂志,2006, 16(8):675~678..
[30]Han J, Lee Y, Yeom K-H, Nam J-W, Heo I, Rhee J-K, Sohn SY, Cho Y, Zhang B-T, Kim VN. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell .2006,125 (5): 887–901..
[31]Zeng Y, Yi R, Cullen BR. Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. Embo J. 2005,24 (1): 138–48.
[32]Preall JB, He Z, Gorra JM, Sontheimer EJ. Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila. Curr. Biol.2006, 16 (5): 530–5..
[33]Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cel.2005,l 123 (4): 631–40.
[34]Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson, J.M., Song, J.J.,Hammond, S.M., Joshua-Tor, L., and Hannon, G.J. Argonaute2 is the catalytic engine of mammalian RNAi. Science.2004 305, 1437-1441.
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