【作者】 刘敏；

【导师】 孟松树；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2016， 硕士

【摘要】 背景:细胞自噬(Autophagy)广泛发生于真核细胞中,是进化保守的自我保护、维持细胞内环境稳态的机制,是细胞死亡的一种重要方式。自噬发生时,通过内凹的膜结构不断延伸,形成内吞小体,内吞小体包裹衰老的细胞器及待分解的物质形成自噬小体,自噬小体不断成熟到达溶酶体,与溶酶体结合形成自噬溶酶体,在酸性溶酶体酶的作用下,降解自噬溶酶体内包裹的物质,使其分解代谢并循环利用,实现细胞内环境的稳态和细胞器的更新。在酵母菌中发现的一系列自噬相关基因(Autophagy associated gene,ATG)可以调控细胞自噬,目前已发现三十多个ATG基因参与自噬过程。微管相关蛋白1轻链3-β(Microtubules associated protein 1light chain 3-β,LC3)是哺乳动物中与酵母ATG8同源的基因,自噬发生时,LC3转变为脂型LC3ΙΙ,蛋白水平检测LC3ΙΙ的增多和LC3ΙΙ在自噬体的膜定位,可作为自噬发生的标志。自噬晚期,自噬小体成熟形成自噬溶酶体,降解胞内物质,自噬底物蛋白p62(SQSTM1)的降解,可以用来检测自噬流的发生。细胞自噬的过程主要受m TOR(mammalian target of rapamycin)、磷脂酰肌醇3-激酶(Phosphatidylinositol 3-kinase,PI3Ks)信号通路的调控。通过抑制m TOR和Ι型PI3K通路可以活化细胞自噬,而ΙΙΙ型PI3K信号通路与Ι型PI3K信号通路作用相反,活化ΙΙΙ型PI3K可以促进自噬的发生。同时AMPK、p53、Bcl2等信号分子也参与细胞自噬的调控。细胞自噬是生物体最基本的生物学现象,影响生物体生长发育等多个过程。细胞自噬的异常与肿瘤的发生发展相关。根据肿瘤类型、发展阶段的不同以及肿瘤细胞生长环境的不同,自噬在肿瘤的发生发展过程中起不同的作用,既可以促进肿瘤细胞的生长,也可以通过自噬抑制肿瘤的恶性扩增。基于自噬在肿瘤中作用的两面性,可以把自噬作为肿瘤治疗的一个新的靶点。Spred(Sprouty-related proteins with an EVH1 domain)蛋白家族是近年来发现的一类与Sprouty相关的膜蛋白。哺乳动物中包括Spred1、Spred2、Spred3三个家族成员。Spred蛋白通常含有3个结构域:EVH1(N-terminal Enabled/VASP homology 1 domain),KBD(central c-Kit binding domain)和SPR(C-terminal Sprouty-related domain)。研究表明Spred蛋白家族负调控ERK/MAPK信号转导通路,影响细胞的生长发育、增殖,血管淋巴管的生成,骨骼的形成和组织的运动能力等,并与肿瘤的发生发展、迁移侵袭有着密切的联系。Spred2是一个肿瘤抑制蛋白,抑制肿瘤细胞的增殖与分化。在一些肿瘤组织中,Spred1、Spred2均较正常组织表达量低,并且Spred的表达下降可能是肿瘤形成与恶化的重要标志。对Spred2的研究可能为肿瘤治疗提供新的治疗策略与靶点,但目前Spred2抑制细胞增殖的机制尚不明确。本研究发现在肿瘤细胞中,过表达Spred2促进自噬体的形成,加快自噬溶酶体的成熟,进而引起肿瘤细胞发生自噬依赖性细胞死亡。本研究证实,在人宫颈癌He La细胞中,稳定敲低Spred2后,可以显著的抑制自噬诱导剂雷帕霉素(Rapamycin,Rapa)诱导的自噬体成熟,重新过表达Spred2,可以回复该效应。我们进一步对Spred2引起肿瘤细胞自噬的分子机制进行了探讨。研究发现,Spred2通过其SPR结构域中的与LC3结合的基序LIR(LC3-interacting region)序列与LC3蛋白相互作用和细胞内共定位。LIR基序突变体显著减弱了Spred2调节的自噬体的成熟。此外,Spred2通过其SPR结构域与p62蛋白存在相互作用和细胞内的共定位。在人宫颈癌细胞He La细胞中,Spred2与p62和溶酶体的标志蛋白LAMP2存在三者的细胞内共定位,提示p62参与Spred2调节的促进自噬体的成熟。在证实了Spred2蛋白的分子作用机制基础上,我们进一步对Spred2蛋白引起的自噬是否与其介导的细胞死亡相关进行了研究。缺失Spred2的SPR结构域,或者将其LIR基序突变后,减弱了Spred2引起的肿瘤细胞死亡,表明LIR基序为Spred2促进肿瘤细胞死亡所必需。此外,自噬抑制剂氯喹(Choroquine,CQ)显著抑制了过表达Spred2引起的细胞死亡。而在人宫颈癌细胞He La和人肺癌细胞A549中,敲低自噬相关基因ATG5、LC3、p62,同样抑制了过表达Spred2引起的促进肿瘤死亡的效应。基于上述研究我们推断细胞自噬可能是Spred2蛋白促进肿瘤细胞死亡抑制肿瘤细胞增殖的一个新的机制。综上所述,本研究发现Spred2可以促进自噬体的成熟、自噬溶酶体的融合,引起肿瘤细胞自噬依赖性细胞死亡。方法:1.分子构建Spred2蛋白LIR基序的突变体2.细胞培养及转染3.腺病毒感染瞬时表达目的蛋白4.免疫共沉淀和体外GST-Pull-down检测蛋白的相互作用5.免疫荧光实验检测蛋白细胞内共定位6.细胞平板克隆形成、台盼蓝计数、流式细胞仪检测细胞增殖及凋亡结果:1.过表达Spred2抑制肿瘤细胞的增殖2.Spred2与LC3、p62存在相互作用和细胞内定位3.Spred2参与自噬过程,并促进自噬体的成熟4.过表达Spred2引起肿瘤细胞自噬依赖性的细胞死亡结论:1.肿瘤抑制蛋白Spred2促进自噬体的成熟;2.肿瘤抑制蛋白Spred2通过促进自噬溶酶体的融合,参与自噬引起肿瘤细胞自噬依赖性的细胞死亡。更多还原

【Abstract】 Background:Autophagy is an evolutionarily conserved self-protective mechanism and a common process in eukaryotes maintaining cellular homeostasis.It is an important manner of cell death.The initiation of autophagy includes the formation of the phagophore,the initial sequestering compartment,which expands into an autophagosome.These autophagosomes mature by fusing with the endocytic compartments(e.g.early and late endosomes,multivesicular bodies)and then fusing with the lysosomal compartment to form autolysosomes,in which the cargo is degraded by acidic lysosomal hydrolases.The process is tightly regulated by a set of core autophagy-related(ATG)genes.More than 30 Atg genes have been identified in yeast so far.During autophagy,the microtubule-associated protein 1 light chain 3(LC3),which is the mammalian homologue of yeast ATG8,is converted to lipidated LC3 II and then associates with the autophagic membrane.The accumulation of LC3 II and its localization to the autophagosome(punctate dot formation)are generally used as markers for autophagy.During late autophagy,autophagosome matured by fusing with the lysosomal compartment to form autolysosomes,further to degrade its contents.The degradation of the substrate protein of autophagy p62(also known as SQSTM1)can be used as a hallmark of autophagy flux.Autophagy is mainly regulated by m TOR,class PI3 Kand classΙΙΙ PI3 K singaling pathways.Basically,autophagy is activated by the inhibition of m TOR and class Ι PI3 K,while the class ΙΙΙ PI3 K pathway has the opposing effect.Signaling molecules such as AMPK,p53 and Bcl-2 are also involved in autophagy.Autophagy is an important biology process playing significant roles during cell growth and differentiation,the dysfunction of autophagy always leads to various diseases including tumorigenesis.It can play both anti-and pro-tumorigenic roles depending on tumor type,stage,and cellular context.Based on the dual character of autophagy,autophagy could be used as a new therapeutic target for cancer.Sprouty-related EVH1 domain(Spreds)proteins are a new Sprouty-related membrane protein family,containing Spred1,Spred2,Spred3 three members in mammals.Several researches have shown that Spreds play a critical role in cell growth and differentiation,influence angiogenesis and lymphangion genesis also skeletogeny and histokinesis.Spreds are closely associated with tumorigenesis and EMT.Accumulating evidence indicates that expression of Spred1 and/or Spred2 was frequently reduced in hepatocellular carcinoma and prostate cancer,while decreased Spred levels were associated with increased tumor invasion and metastasis and may be a marker protein of tumorigenesis and aggressiveness.In this regard,to investigate the molecular mechanism of Spred may provide a new therapeutic target in tumor therapy.Tumor suppressor Spred2 functions as a negative regulator of ERK/MAPK signaling pathway,inducing cell death and differentiation.However,the underlying mechanism remains to be elucidated.In the current study we show that Spred2 modulates autophagosome maturation in tumor cells and induces autophagy-dependent cell death.We demonstrate that Spred2 knockdown induces accumulation of lapidated LC3,whereas Spred2 overexpression increases autophagosome formation and autophagosome-lysosome fusion.Mechanistically,Spred2 co-localizes and interacts with LC3 via the LC3-interacting region(LIR)motifs in its SPR domain.Mutations in the LIR motifs impair Spred2-mediated autophagosome maturation.In addition,Spred2 interacts and co-localizes with p62/SQSTM1 through its SPR domain,indicating that p62 may be involved in Spred2-mediated autophagosome maturation.Functionally,mutations in the LIR motifs impaired Spred2-mediated tumor cell death,indicating that the functional LIR is required for Spred2 to trigger cell death.Additionally,silencing the expression of autophagy-related genes ATG5,LC3 or p62 in He La cells reduced Spred2-mediated cell death.Pharmacological inhibition of autophagy with lysosomal inhibitors chloroquine gave similar results,suggesting that autophagy is required for Spred2-induced cell death.Collectively,these data indicate that Spred2 induces tumor cell death in an autophagy-dependent way.Method: 1.Molecular subcloning and PCR-based site-directed mutagenesis of LIR motif in Spred2 2.Cell culture and transfection 3.Adenovirus infecte cell induce the expression of aim proteins 4.Immunecoprecipitation and GST-Pull-down in vitro to test the interaction between proteins 5.Immunofluorescence assay explore the co-localization 6.Colony formation assay,Trypan blue exclusion assay and Flow cytometry analysis of apoptosisResults: 1.Overexpress Spred2 induce tumor cell growth 2.Spred2 interact and co-localization with LC3 and p62 3.Spred2 is involved in autophagy and promotes autophagosome maturation 4.Spred2 induces autophagy-dependent cell death Conclusion: 1.Tumor suppressor Spred2 promotes autophagosome maturation 2.Tumor suppressor Spred2 promotes autophagosome maturation induce autophagy-dependent cell death in tumor cell更多还原