微管末端结合蛋白1在Siha细胞自噬的表达和意义
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摘要
目的探讨宫颈癌Siha细胞自噬过程中微管末端结合蛋白1(EB1)基因的表达变化。方法 Hank平衡盐溶液(HBSS)和秋水仙素处理体外培养的宫颈癌Siha细胞,分别采用实时定量PCR和Western blotting检测EB1基因、LC3和p62的表达,荧光显微镜检测特异性绿色荧光蛋白(GFP)-LC3以证实自噬体形成。结果宫颈癌Siha细胞随HBSS作用时间延长,LC3 mRNA、EB1 mRNA、LC3-Ⅱ蛋白和EB1蛋白的表达均呈时间依赖性增加,差异具有统计学意义(P<0.05)。EB1 mRNA和LC3 mRNA表达呈正相关,具有统计学意义(P<0.05)。p62mRNA和蛋白在HBSS作用后的表达呈时间依赖性降低,差异具有统计学意义(P<0.05),EB1 mRNA和p62mRNA表达呈负相关,具有统计学意义(P<0.05)。GFP-LC3结果显示,在HBSS作用12 h后,Siha细胞胞质中GFP-LC3强度和数目明显增加,含有GFP信号的细胞数量也明显增加。秋水仙素处理后,LC3、p62和EB1 mRNA及蛋白均未发生显著改变,差异无统计学意义(P>0.05),但此时几乎检测不到含GFP-LC3荧光信号的细胞。结论EB1在饥饿状态Siha细胞中高表达,此时伴随LC3表达增加、p62表达降低以及自噬体形成增加,而用秋水仙素破坏EB1赖以作用的微管时,上述现象消失,充分表明EB1参与细胞自噬的可能。
Objective To explore the expressions and implications of end-binding protein 1( EB1) in autophagic cervical cancer Siha cells. Method Real-time quantitative PCR and Western blotting were introduced to detect the mRNA and protein expressions of EB1,LC3 and p62 in Siha cells treated with Hank 's balanced salt solution( HBSS) or colchicine in vitro. Specific green fluoresent protein( GFP)-LC3 was then be detected to confirm the autophagosome formation by fluorescence microscopy with FlowCellectTMGFP-LC3 reporter autophagy assay kit. Result The mRNA and protein levels of LC3 and EB1 in cervical cancer Siha cells were increased significantly when prolong the duration of HBSS treatment( P < 0. 05). EB1 mRNA was positively correlated with LC3 mRNA expression( P < 0. 05). The expressions of p62 mRNA and protein reduced dramatically after HBSS treated( P < 0. 05),and was negatively related with the EB1 mRNA( P < 0. 05). The number of cells with GFP-LC3 and intensity of GFP-LC3 per cells were increased when starvation for 12 hours. However,the mRNA and protein levels of LC3,p62 and EB1 were not significantly changed when the cells treated with colchicine( P > 0. 05),and we cannot detected the fluoresce signaling of GFP-LC3 in cells at this moment. Conclusion The expression of EB1 in starved cells is dramatically increased,and accompanied with the increased expression of LC3 and autophagosome,decreased expression of p62. However,when microtubules with which EB1 interaction were destructed by colchicine,the above phenomenon disappears. All these results fully indicate the probably roles of EB1 in autophagy.
Objective To explore the expressions and implications of end-binding protein 1( EB1) in autophagic cervical cancer Siha cells. Method Real-time quantitative PCR and Western blotting were introduced to detect the mRNA and protein expressions of EB1,LC3 and p62 in Siha cells treated with Hank 's balanced salt solution( HBSS) or colchicine in vitro. Specific green fluoresent protein( GFP)-LC3 was then be detected to confirm the autophagosome formation by fluorescence microscopy with FlowCellectTMGFP-LC3 reporter autophagy assay kit. Result The mRNA and protein levels of LC3 and EB1 in cervical cancer Siha cells were increased significantly when prolong the duration of HBSS treatment( P < 0. 05). EB1 mRNA was positively correlated with LC3 mRNA expression( P < 0. 05). The expressions of p62 mRNA and protein reduced dramatically after HBSS treated( P < 0. 05),and was negatively related with the EB1 mRNA( P < 0. 05). The number of cells with GFP-LC3 and intensity of GFP-LC3 per cells were increased when starvation for 12 hours. However,the mRNA and protein levels of LC3,p62 and EB1 were not significantly changed when the cells treated with colchicine( P > 0. 05),and we cannot detected the fluoresce signaling of GFP-LC3 in cells at this moment. Conclusion The expression of EB1 in starved cells is dramatically increased,and accompanied with the increased expression of LC3 and autophagosome,decreased expression of p62. However,when microtubules with which EB1 interaction were destructed by colchicine,the above phenomenon disappears. All these results fully indicate the probably roles of EB1 in autophagy.
引文
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[22]Ligon LA,Shelly SS,Tokito M,et al.The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization[J].Mol Biol Cell,2003,14(4):1405-1417.
[23]Wang YH,Zhou XB,Zhu H,et al.Overexpression of EB1inhuman esophageal squamous cell carcinoma(ESCC)may promote cellular growth by activating beta-catenin/TCF pathway[J].Oncogene,2005,24(44):6637-6645.
[24]Kaplan KB,Burds AA,Swealow JR,et al.A role for the adenomatous polyposis coli protein in chromosome segregation[J].Nat Cell Bio,2001,3(4):429-432.
[2]Kchl R,Hu XW,Chan EY,et al.Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes[J].Traffic,2006,7(2):129-145.
[3]Bian ML.WHO(2006)practice guidelines for cervical cancer prevention[J].Chinese Journal of Practical Gynecology and Obostetrics,2007,23(7):557-560.(in Chinese)卞美璐.WHO(2006年)宫颈癌综合防治实践指南简介[J].中国实用妇科与产科杂志,2007,23(7):557-560.
[4]Chunrong H,Baocun S,Wei W,et al.Overexpression of microtubule-associated protein-1 light chain 3 is associated with melanoma metastasisand vasculogenic mimicry[J].Tohoku J Exp Med,2011,223(22):243-251.
[5]Duran A,Amanchy R,Linares JF,et al.p62 is a key regulator of nutrient sensing in the mTORC1 pathway[J].Mol Cell,2011,44(1):134-146.
[6]Kim DH,Sarbassov DD,Ali SM,et al.mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery[J].Cell,2002,110(2):163-175.
[7]Bjorkoy G,Lamark T,Brech A,et al.p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death[J].J Cell Biol,2005,171(4):603-614.
[8]Moscat J,Diaz-Meco MT.Feedback on fat:p62-mTORC1-autophagy connections[J].Cell,2011,147(4):724-727.
[9]Puissant A,Fenouille N,Auberger P.When autophagy meets cancer through p62/SQSTM1[J].Am J Cancer Res,2012,2(4):397-413.
[10]Wang Y,Singh R,Xiang Y,et al.Macroautophagy and chaperonemediated autophagy are required for hepatocyte resistance to oxidant stress[J].Hepatology,2010,52(1):266-277.
[11]Jahreiss L,Menzies FM,Rubinsztein DC.The itinerary of autophagosomes:from peripheral formationto kiss-and-run fusion with lysosomes[J].Traffic,2008,9(4):574-587.
[12]Aplin A,Jasionowski T,Tuttle DL,et al.Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles[J].J Cell Physiol,1992,152(3):458-466.
[13]Seglen PO,Berg TO,Blankson H,et al.Structural aspects of autophagy[J].Adv Exp Med Biol,1996,389:103-111.
[14]Monastyrska I,Rieter E,Klionsky DJ,et al.Multiple roles of the cytoskeleton in autophagy[J].Biol Rev Camb Philos Soc,2009,84(3):431-448.
[15]Orsi A,Polson HE,Tooze SA.Membrane trafficking events that partake in autophagy[J].Curr Opin Cell Biol,2010,22(2):150-156.
[16]Fass E,Shvets E,Degani I,et al.Microtubules support production of starvation-induced autophagosomes but not their targeting and fusion with lysosomes[J].J Biol Chem,2006,281(47):36303-36316.
[17]Kchl R,Hu XW,Chan EY,et al.Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes[J].Traffic,2006,7(2):129-145.
[18]Xie R,Nguyen S,McKeehan K,et al.Microtubule-associated protein 1S(MAP1S)bridges autophagic components with microtubules and mitochondria to affect autophagosomal biogenesis and degradation[J].J Biol Chem,2011,286(12):10367-10377.
[19]Jánosi IM,Chrétien D,Flyvbjerg H.Structural microtubule Cap:stability,catastrophe,rescue,and third state[J].Biophys J,2002,83(3):1317-1330.
[20]Jiang K,Akhmanova A.Microtubule tip-interacting proteins:a view from both ends[J].Curr Opin Cell Biol,2011,23(1):94-101.
[21]Tirnauer JS,Bierer BE.EB1 proteins regulate microtubule dynamics,cell polarity,and chromosome stability[J].J Cell Bio,2000,149(4)761-766.
[22]Ligon LA,Shelly SS,Tokito M,et al.The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization[J].Mol Biol Cell,2003,14(4):1405-1417.
[23]Wang YH,Zhou XB,Zhu H,et al.Overexpression of EB1inhuman esophageal squamous cell carcinoma(ESCC)may promote cellular growth by activating beta-catenin/TCF pathway[J].Oncogene,2005,24(44):6637-6645.
[24]Kaplan KB,Burds AA,Swealow JR,et al.A role for the adenomatous polyposis coli protein in chromosome segregation[J].Nat Cell Bio,2001,3(4):429-432.