大鼠MIP-1α基因启动子荧光素酶报告质粒的构建及其IRF-8结合元件的初步鉴定
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摘要
目的:构建大鼠巨噬细胞炎性蛋白-1α(macrophage inflammatory protein-1α,MIP-1α)基因启动子(全长和截短)荧光素酶报告质粒,并观察在人胚肾细胞(HEK-293T)中过表达干扰素调节因子-8(interferon regulatory factor-8,IRF-8)对MIP-1α基因启动活性的影响,同时筛选其可能的IRF-8结合元件。方法:采用PCR技术,扩增出大鼠MIP-1α基因启动子序列,将MIP-1α基因启动子插入到荧光素酶报告基因载体pGL3-basic中,构建MIP-1α基因启动子全长荧光素酶报告质粒(pGL3-MIP-1α-FL)。将上述p GL3-MIP-1α-FL和本课题组已构建的大鼠IRF-8过表达质粒(pIRES2-IRF-8)共转染HEK-293T细胞,检测细胞内荧光素酶活性,确定IRF-8对MIP-1α基因的启动作用。同时,应用生物信息学软件预测MIP-1α基因启动子上IRF-8的结合元件,并据此构建3个MIP-1α基因启动子截短的荧光素酶报告质粒(pGL3-MIP-1α-1～3)。将上述MIP-1α基因启动子全长和各截短的荧光素酶报告质粒和IRF-8过表达质粒共转染HEK-293T细胞,再测定荧光素酶活性,初步确定IRF-8的结合元件。结果:菌液PCR及核酸测序证实,上述pGL3-MIP-1α-FL(-1 400～+94 nt)质粒构建成功。将pGL3-MIP-1α-FL和pIRES2-IRF-8共转染HEK-293T后发现,过表达IRF-8可显著增加MIP-1α基因启动子活性。应用生物信息学软件预测发现MIP-1α基因启动子上IRF-8的结合元件(-1 157～-1 144nt、-740～-734 nt、-683～-670 nt、-365～-359 nt、-249～-236 nt),并据此构建3个MIP-1α基因启动子截短的荧光素酶报告质粒,即pGL3-MIP-1α-1(-453～+94 nt)、p GL3-MIP-1α-2(-352～+94 nt)和pGL3-MIP-1α-3(-3～+94 nt)。将pGL3-MIP-1α-FL、pGL3-MIP-1α-1～3和pIRES2-IRF-8共转染HEK-293T后发现,p GL3-MIP-1α-3的启动活性显著低于pGL3-MIP-1α-FL、pGL3-MIP-1α-1和pGL3-MIP-1α-2。提示IRF-8可能结合在大鼠MIP-1α基因启动子的-352～-3 nt区域的IRF-8结合元件(-249～-236 nt)上。结论:本实验成功构建了大鼠MIP-1α基因启动子全长及截短荧光素酶报告质粒,并初步筛查出IRF-8在MIP-1α基因启动子上的可能结合元件,为后续研究奠定了基础。
Objective:To construct luciferase reporter plasmids of full-length and truncated promotors of rat macrophage inflammatory protein-1α(MIP-1α)gene and detect their activity in HEK-293 T cells in response to interferon regulatory factor-8(IRF-8)overexpression,screening the possible binding elements for IRF-8. Methods:Rat MIP-1α promoter was amplified by PCR and clonedinto the luciferase reporter plasmid(pGL3-basic). The recombinant plasmid(pGL3-MIP-1α-FL)and rat IRF-8 overexpression plasmid(pIRES2-IRF-8)were co-transfected into HEK-293 T cells and then the luciferase activity was detected to determine the role of IRF-8 in MIP-1α gene transcription. Meanwhile,the potential IRF-8 binding elements within MIP-1α promoter were predicted by usingbioinformatics software. Based on the predicted results,three luciferase reporter plasmids of truncated MIP-1α gene promotor(pGL3-MIP-1α-1～3)were constructed. The promoter luciferase reporter plasmids of pGL3-MIP-1α-FL or p GL3-MIP-1α-1～3 and the plasmidof pIRES2-IRF-8 were co-transfected into HEK-293 T cells. Then,the luciferase activity was detected to screen the IRF-8 binding elements. Results:It was verified that pGL3-MIP-1α-FL(-1 400～+94 nt)plasmid was constructed correctly by PCR analysis andnucleotide sequencing. The plasmids of pGL3-MIP-1α-FL and pIRES2-IRF-8 were co-transfected into HEK-293 T cells,and then theluciferase activity of MIP-1α gene promotor was markedly increased in response to IRF-8 overexpression. The potential IRF-8 bindingelements(-1 157～-1 144 nt,-740～-734 nt,-683～-670 nt,-365～-359 nt,and-249 ～-236 nt)within MIP-1α promoter werepredicted by using bioinformatics software. Based on the predicted results,we constructed three luciferase reporter plasmids oftruncated MIP-1α gene promotor,namely pGL3-MIP-1α-1(-453 ～ +94 nt),pGL3-MIP-1α-2(-352 ～ +94 nt)and pGL3-MIP-1α-3(-3～ +94 nt). The plasmids of pGL3-MIP-1α-FL or pGL3-MIP-1α-1～3 and p IRES2-IRF-8 were co-transfected into HEK-293 T cells,andthe result displayed that the activity of pGL3-MIP-1α-3 was much lower than that in pGL3-MIP-1α-FL,pGL3-MIP-1α-1 and pGL3-MIP-1α-2,indicating that the region of rat MIP-1α promoter(-352 ～-3 nt)might contain an IRF-8 binding element(-249 ～-236 nt).Conclusion:The rat full-length and truncated rat MIP-1α promotor luciferase reporter plasmids were constructed successfully,and thepossible IRF-8 binding element was found,which could be beneficial to further studies.
Objective:To construct luciferase reporter plasmids of full-length and truncated promotors of rat macrophage inflammatory protein-1α(MIP-1α)gene and detect their activity in HEK-293 T cells in response to interferon regulatory factor-8(IRF-8)overexpression,screening the possible binding elements for IRF-8. Methods:Rat MIP-1α promoter was amplified by PCR and clonedinto the luciferase reporter plasmid(pGL3-basic). The recombinant plasmid(pGL3-MIP-1α-FL)and rat IRF-8 overexpression plasmid(pIRES2-IRF-8)were co-transfected into HEK-293 T cells and then the luciferase activity was detected to determine the role of IRF-8 in MIP-1α gene transcription. Meanwhile,the potential IRF-8 binding elements within MIP-1α promoter were predicted by usingbioinformatics software. Based on the predicted results,three luciferase reporter plasmids of truncated MIP-1α gene promotor(pGL3-MIP-1α-1～3)were constructed. The promoter luciferase reporter plasmids of pGL3-MIP-1α-FL or p GL3-MIP-1α-1～3 and the plasmidof pIRES2-IRF-8 were co-transfected into HEK-293 T cells. Then,the luciferase activity was detected to screen the IRF-8 binding elements. Results:It was verified that pGL3-MIP-1α-FL(-1 400～+94 nt)plasmid was constructed correctly by PCR analysis andnucleotide sequencing. The plasmids of pGL3-MIP-1α-FL and pIRES2-IRF-8 were co-transfected into HEK-293 T cells,and then theluciferase activity of MIP-1α gene promotor was markedly increased in response to IRF-8 overexpression. The potential IRF-8 bindingelements(-1 157～-1 144 nt,-740～-734 nt,-683～-670 nt,-365～-359 nt,and-249 ～-236 nt)within MIP-1α promoter werepredicted by using bioinformatics software. Based on the predicted results,we constructed three luciferase reporter plasmids oftruncated MIP-1α gene promotor,namely pGL3-MIP-1α-1(-453 ～ +94 nt),pGL3-MIP-1α-2(-352 ～ +94 nt)and pGL3-MIP-1α-3(-3～ +94 nt). The plasmids of pGL3-MIP-1α-FL or pGL3-MIP-1α-1～3 and p IRES2-IRF-8 were co-transfected into HEK-293 T cells,andthe result displayed that the activity of pGL3-MIP-1α-3 was much lower than that in pGL3-MIP-1α-FL,pGL3-MIP-1α-1 and pGL3-MIP-1α-2,indicating that the region of rat MIP-1α promoter(-352 ～-3 nt)might contain an IRF-8 binding element(-249 ～-236 nt).Conclusion:The rat full-length and truncated rat MIP-1α promotor luciferase reporter plasmids were constructed successfully,and thepossible IRF-8 binding element was found,which could be beneficial to further studies.
引文
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[12] Fosbrink M,Niculescu F,Rus V,et al. C5b-9-induced en-dothelial cell proliferation and migration are dependenton Akt inactivation of forkhead transcription factorFOXO1[J]. J Biol Chem,2006,281(28):19009-19018
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[14] Kunchithapautham K,Rohrer B. Sublytic membrane-at-tack-complex(MAC)activation alters regulated ratherthan constitutive vascular endothelial growth factor(VEGF)secretion in retinal pigment epithelium monolay-ers[J]. J Biol Chem,2011,286(27):23717-23724
[15] Liu J,Jha P,Lyzogubov VV,et al. Relationship betweencomplement membrane attack complex,chemokine(C-Cmotif)ligand 2(CCL2)and vascular endothelial growthfactor in mouse model of laser-induced choroidal neovas-cularization[J]. J Biol Chem,2011,286(23):20991-21001
[16] Sung JY,Park SY,Kim JH,et al. Interferon consensus se-quence-binding protein(ICSBP)promotes epithelial-to-mesenchymal transition(EMT)-like phenomena,cell-mo-tility,and invasion via TGF-beta signaling in U2OS cells[J]. Cell Death Dis,2014,5:e1224
[17] Sichien D,Scott CL,Martens L,et al. IRF8 transcriptionfactor controls survival and function of terminally differen-tiated conventional and plasmacytoid dendritic cells,re-spectively[J]. Immunity,2016,45(3):626-640
[18] Gupta M,Shin DM,Ramakrishna L,et al. IRF8 directsstress-induced autophagy in macrophages and promotesclearance of Listeria monocytogenes[J]. Nat Commun,2015,6:6379
[19] Liu J,Ma X. Interferon regulatory factor 8 regulates RAN-TES gene transcription in cooperation with interferon reg-ulatory factor-1,NF-kappaB,and PU.1[J]. J Biol Chem,2006,281(28):19188-19195
[20] Nardi V,Naveiras O,Azam M,et al. ICSBP-mediated im-mune protection against BCR-ABL-induced leukemia re-quires the CCL6 and CCL9 chemokines[J]. Blood,2009,113(16):3813-3820
[2] Al Hussain T,Hussein MH,Al Mana H,et al. Pathophysiology of IgA nephropathy[J]. Adv Anat Pathol,2017,24(1):56-62
[3] Zhang L,Han C,Ye F,et al. Plasma gelsolin induced glomerular fibrosis via the TGF-beta1/Smads signal transduction pathway in IgA nephropathy[J]. Int J Mol Sci,2017,18(2):390
[4] Tsai YL,Hua KF,Chen A,et al. NLRP3 inflammasome:Pathogenic role and potential therapeutic target for IgA nephropathy[J]. Sci Rep,2017,7:41123
[5] Wang PR,Kitamura H,Shimizu A,et al. Glomerular damage in experimental proliferative glomerulonephritis under glomerular capillary hypertension[J]. Kidney Blood Press Res,2015,40(2):188-199
[6] Rintala JM,Savikko J,Rintala SE,et al. Epidermal growth factor receptor inhibition with erlotinib ameliorates antiThy 1.1-induced experimental glomerulonephritis[J]. J Nephrol,2016,29(3):359-365
[7] Zhu G,Qiu W,Li Y,et al. Sublytic C5b-9 induces glomerular mesangial cell apoptosis through the cascade pathway of MEKK2-p38 MAPK-IRF-1-TRADD-Caspase 8 in rat Thy-1 nephritis[J]. J Immunol,2017,198(3):1104-1118
[8] Zhang J,Li Y,Shan K,et al. Sublytic C5b-9 induces IL-6and TGF-beta1 production by glomerular mesangial cells in rat Thy-1 nephritis through p300-mediated C/EBPbeta acetylation[J]. Faseb J,2014,28(3):1511-1525
[9] Ikezumi Y,Kawachi H,Toyabe S,et al. An anti-CD5monoclonal antibody ameliorates proteinuria and glomerular lesions in rat mesangioproliferative glomerulonephritis[J]. Kidney Int,2000,58(1):100-114
[10] Qin D,Morita H,Inui K,et al. Aldosterone mediates glo-merular inflammation in experimental mesangial prolifera-tive glomerulonephritis[J]. J Nephrol,2013,26(1):199-206
[11] Tan LX,Toops KA,Lakkaraju A. Protective responses to sublytic complement in the retinal pigment epithelium[J].Proc Natl Acad Sci USA,2016,113(31):8789-8794
[12] Fosbrink M,Niculescu F,Rus V,et al. C5b-9-induced en-dothelial cell proliferation and migration are dependenton Akt inactivation of forkhead transcription factorFOXO1[J]. J Biol Chem,2006,281(28):19009-19018
[13] He F,Zhou M,Yu T,et al. Sublytic C5b-9 triggers glomer-ular mesangial cell apoptosis in rat Thy-1 nephritis viaGadd45 activation mediated by Egr-1 and p300-depen-dent ATF3 acetylation[J]. J Mol Cell Biol,2016,8(6):477-491
[14] Kunchithapautham K,Rohrer B. Sublytic membrane-at-tack-complex(MAC)activation alters regulated ratherthan constitutive vascular endothelial growth factor(VEGF)secretion in retinal pigment epithelium monolay-ers[J]. J Biol Chem,2011,286(27):23717-23724
[15] Liu J,Jha P,Lyzogubov VV,et al. Relationship betweencomplement membrane attack complex,chemokine(C-Cmotif)ligand 2(CCL2)and vascular endothelial growthfactor in mouse model of laser-induced choroidal neovas-cularization[J]. J Biol Chem,2011,286(23):20991-21001
[16] Sung JY,Park SY,Kim JH,et al. Interferon consensus se-quence-binding protein(ICSBP)promotes epithelial-to-mesenchymal transition(EMT)-like phenomena,cell-mo-tility,and invasion via TGF-beta signaling in U2OS cells[J]. Cell Death Dis,2014,5:e1224
[17] Sichien D,Scott CL,Martens L,et al. IRF8 transcriptionfactor controls survival and function of terminally differen-tiated conventional and plasmacytoid dendritic cells,re-spectively[J]. Immunity,2016,45(3):626-640
[18] Gupta M,Shin DM,Ramakrishna L,et al. IRF8 directsstress-induced autophagy in macrophages and promotesclearance of Listeria monocytogenes[J]. Nat Commun,2015,6:6379
[19] Liu J,Ma X. Interferon regulatory factor 8 regulates RAN-TES gene transcription in cooperation with interferon reg-ulatory factor-1,NF-kappaB,and PU.1[J]. J Biol Chem,2006,281(28):19188-19195
[20] Nardi V,Naveiras O,Azam M,et al. ICSBP-mediated im-mune protection against BCR-ABL-induced leukemia re-quires the CCL6 and CCL9 chemokines[J]. Blood,2009,113(16):3813-3820
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