稳定干扰ITGB3基因可促进人乳腺癌BT549细胞的增殖
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摘要
目的 :探讨稳定干扰整合素β3(integrinβ3,ITGB3)基因对人乳腺癌BT549细胞增殖的影响。方法:采用实时荧光定量PCR法和蛋白质印迹法检测乳腺癌BT549、MCF-7和MDA-MB-453细胞中ITGB3 m RNA和蛋白的表达。将干扰ITGB3表达的LV-ITGB3-sh RNA慢病毒感染BT549细胞后,应用实时荧光定量PCR法和蛋白质印迹法检测BT549细胞中ITGB3 m RNA和蛋白的表达水平,MTT法和FCM法检测BT549细胞的增殖能力和细胞周期,蛋白质印迹法检测BT549细胞中c-Myc和cyclin D1蛋白的表达情况。结果:乳腺癌BT549细胞中ITGB3 m RNA和蛋白的表达水平高于MCF-7和MDA-MB-453细胞(P值均<0.05)。LV-ITGB3-sh RNA慢病毒感染后,BT549细胞中ITGB3 m RNA和蛋白的表达水平低于阴性对照组(LV-NCsh RNA感染BT549细胞)和空白对照组(BT549细胞未进行感染)(P值均<0.01),细胞增殖能力增强(P<0.01),S期细胞所占百分比上升(P<0.01),c-Myc和cyclin D1蛋白的表达水平上调(P值均<0.05)。结论:稳定干扰BT549细胞中ITGB3表达后,可能通过上调c-Myc和cyclin D1蛋白的表达而促进细胞增殖。
Objective:To investigate the effect of integrin β3(ITGB3) gene knockdown on the proliferation of human breast cancer BT549 cells.Ivethods:The expressions of ITGB3 mRNA and protein in breast cancer BT549,MCF-7 and MDA-MB-453 cells were detected by realtime fluorescent quantitative PCR and Western blotting.After BT549 cells infection with lentivirus containing LV-ITGB3-shRNA with ITGB3 knockdown,the expressions of ITGB3 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting,and the proliferation and cell cycle were measured by MTT and FCM assay,then the expressions of c- Myc and cyclin D1 proteins were determined by Western blotting.Results:The expression levels of ITGB3 mRNA and protein in BT549 cells were higher than those in MCF-7 and MDA-MB-453 cells(all P< 0.05).The expression levels of ITGB3 mRNA and protein in BT549 cells after infection with LV-ITGB3-shRNA lentivirus were lower than those in the negative control(BT549 cells infected with LV- NC- shRNA lentivirus) and the blank control(BT549 cells without any infection) groups(all P< 0.01),the proliferation ability was significantly enhanced(P < 0.01),the percentage of cells in S phase was increased(P < 0.01),and the expressions of c- Myc and cyclin D1 proteins were up- regulated(all P< 0.05).Conclusion:After BT549 cells with stable knockdown of ITGB3 expression,the cell proliferation can be promoted through up- regulation the expressions of c- Myc and cyclin D1 proteins.
Objective:To investigate the effect of integrin β3(ITGB3) gene knockdown on the proliferation of human breast cancer BT549 cells.Ivethods:The expressions of ITGB3 mRNA and protein in breast cancer BT549,MCF-7 and MDA-MB-453 cells were detected by realtime fluorescent quantitative PCR and Western blotting.After BT549 cells infection with lentivirus containing LV-ITGB3-shRNA with ITGB3 knockdown,the expressions of ITGB3 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting,and the proliferation and cell cycle were measured by MTT and FCM assay,then the expressions of c- Myc and cyclin D1 proteins were determined by Western blotting.Results:The expression levels of ITGB3 mRNA and protein in BT549 cells were higher than those in MCF-7 and MDA-MB-453 cells(all P< 0.05).The expression levels of ITGB3 mRNA and protein in BT549 cells after infection with LV-ITGB3-shRNA lentivirus were lower than those in the negative control(BT549 cells infected with LV- NC- shRNA lentivirus) and the blank control(BT549 cells without any infection) groups(all P< 0.01),the proliferation ability was significantly enhanced(P < 0.01),the percentage of cells in S phase was increased(P < 0.01),and the expressions of c- Myc and cyclin D1 proteins were up- regulated(all P< 0.05).Conclusion:After BT549 cells with stable knockdown of ITGB3 expression,the cell proliferation can be promoted through up- regulation the expressions of c- Myc and cyclin D1 proteins.
引文
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[23]Yoo NJ,Soung YH,Lee SH,et al.Mutational analysis of proapoptotic integrin beta 3 cytoplasmic domain in common human cancers[J].Tumori,2007,93(3):281-283.
[24]Desgrosellier JS Cheresh DA.Integrins in cancer:biological implications and therapeutic opportunities[J].Nat Rev Cancer,2010,10(1):9-22.
[25]Shao N,Lu Z,Zhang Y,et al.Interleukin-8 upregulates integrinβ3expression and promotes estrogen receptor-negative breast cancer cell invasion by activating the PI3K/Akt/NF-kappaB pathway[J].Cancer Lett,2015,364(2):165-172.
[26]Wang R,Li ZQ,Han X,et al.Integrinβ3and its ligand regulate the expression of uPA through p38 MAPK in breast cancer[J].APMIS,2010,118(12):909-917.
[2]Rossier O,Giannone G.The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking[J/OL].Exp Cell Res(2015-11-10)[2015-12-20].http://www.sciencedirect.com/science/article/pii/S0014482715301476.
[3]Plow EF,Haas TA,Zhang L,et al.Ligand binding to integrins[J].J Biol Chem,2000,275(29):21785-21788.
[4]Winograd-Katz SE,Fassler R,Geiger B,et al.The integrin adhesome:from genes and proteins to human disease[J].Nat Rev Mol Cell Biol,2014,15(4):273-288.
[5]Cabodi S,del Pilar Camacho-Leal M,Di Stefano P,et al.Integrin signalling adaptors:not only figurants in the cancer story[J].Nat Rev Cancer,2010,10(12):858-870.
[6]Kaur S,Kenny H,Jagadeeswaran S,et al.{beta}3-integrin expression on tumor cells inhibits tumor progression,reduces metastasis,and is associated with a favorable prognosis in patients with ovarian cancer[J].Am J Pathol,2009,175(5):2184-2196.
[7]Kim JH,Zheng LT,Lee WH,et al.Proapoptotic role of integrin beta3 in glioma cells[J].J Neurochem,2011,117(3):494-503.
[8]Liu M,Sakamaki T,Casimiro MC,et al.The canonical NF-kappaB pathway governs mammary tumorigenesis in transgenic mice and tumor stem cell expansion[J].Cancer Res,2010,70(24):10464-10473.
[9]Bouvard D,Pouwels J,De Franceschi N,et al.Integrin inactivators:balancing cellular functions in vitro and in vivo[J].Nat Rev Mol Cell Biol,2013,14(7):430-442.
[10]Kim C,Ye F,Hu X,et al.Talin activates integrins by altering the topology of theβtransmembrane domain[J].J Cell Biol,2012,197(5):605-611.
[11]Takagi J,Petre BM,Walz T,et al.Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling[J].Cell,2002,110(5):599-611.
[12]Shattil SJ,Kim C,Ginsberg MH.The final steps of integrin activation:the end game[J].Nat Rev Mol Cell Biol,2010,11(4):288-300.
[13]Manavski Y,Carmona G,Bennewitz K,et al.Brag2 differentially regulatesβ1-andβ3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis[J].Basic Res Cardiol,2014,109(2):404.
[14]Robinson SD,Hodivala-Dilke KM.The role ofβ3-integrins in tumor angiogenesis:context is everything[J].Curr Opin Cell Biol,2011,23(5):630-637.
[15]Ghevaert C,Salsmann A,Watkins NA,et al.A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the alphallbbeta3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia[J].Blood,2008,111(7):3407-3414.
[16]Zhao B,Han H,Chen J,et al.MicroRNA let-7c inhibits migration and invasion of human non-small cell lung cancer by targeting ITGB3 and MAP4K3[J].Cancer Lett,2014,342(1):43-51.
[17]Schneider JG,Amend SR,Weilbaecher KN.Integrins and bone metastasis:integrating tumor cell and stromal cell interactions[J].Bone,2011,48(1):54-65.
[18]Desgrosellier JS,Lesperance J,Seguin L,et al.lntegrinαvβ3 drives slug activation and sternness in the pregnant and neoplastic mammary gland[J].Dev Cell,2014,30(3):295-308.
[19]Miller PG,Al-Shahrour F,Hartwell KA,et al.In vivo RNAi screening identifies a leukemia-specific dependence on integrin beta 3 signaling[J].Cancer Cell,2013,24(1):45-58.
[20]Seguin L,Kato S,Franovic A,et al.An integrinβ_3-KRAS-RalB complex drives tumour sternness and resistance to EGFR inhibition[J].Nat Cell Biol,2014,16(5):457-468.
[21]Shin DH,Lee HJ,Min HY,et al.Combating resistance to anti-IGFR antibody by targeting the integrinβ3-Src pathway[J].J Natl Cancer Inst,2013,105(20):1558-1570.
[22]牟玲,陈婕,罗祎,等.高尔基体基质蛋白130、14-3-3ζ和整合素β3在卵巢上皮恶性肿瘤组织中的表达及其临床意义[J].肿瘤,2014,34(1):47-54.
[23]Yoo NJ,Soung YH,Lee SH,et al.Mutational analysis of proapoptotic integrin beta 3 cytoplasmic domain in common human cancers[J].Tumori,2007,93(3):281-283.
[24]Desgrosellier JS Cheresh DA.Integrins in cancer:biological implications and therapeutic opportunities[J].Nat Rev Cancer,2010,10(1):9-22.
[25]Shao N,Lu Z,Zhang Y,et al.Interleukin-8 upregulates integrinβ3expression and promotes estrogen receptor-negative breast cancer cell invasion by activating the PI3K/Akt/NF-kappaB pathway[J].Cancer Lett,2015,364(2):165-172.
[26]Wang R,Li ZQ,Han X,et al.Integrinβ3and its ligand regulate the expression of uPA through p38 MAPK in breast cancer[J].APMIS,2010,118(12):909-917.