【作者】 向波；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 疾病基因组学， 2009， 博士

【摘要】 【C1ORF102基因的克隆、前期研究结果】近年来基因芯片技术被广泛应用于肿瘤研究。我室聂新民博士采用基因芯片筛查鼻咽癌细胞系HNE1与鼻咽原代培养上皮细胞之间的差异表达基因,从中筛选并克隆得到一个表达下调的新基因C1ORF102,最初命名为NOR1(oxidored-nitro domain containingprotein 1)。该基因定位于鼻咽癌高频等位基因杂合性丢失位点1p34.2,不仅在鼻咽癌组织中表达下调,而且检测到存在点突变。采用病例-对照的研究方法,熊炜等人证实C1ORF102(NOR1)基因编码区两个SNP位点之间存在连锁不平衡,且都与鼻咽癌发病相关。利用高通量的组织芯片进行原位杂交实验发现,C1ORF102(NOR1)基因mRNA在鼻咽癌中广泛下调。通过全基因组基因表达芯片筛查显微切割纯化的鼻咽癌组织和正常鼻咽上皮之间的差异表达基因,经SAM(Significance Analysis of Microarray)分析发现C1ORF102基因位列488个表达下调基因中的第6位,强烈提示C1ORF102基因在鼻咽癌发生过程中起到非常关键的作用。【C1ORF102编码蛋白的组织与亚细胞定位:鉴定其为鼻咽-气管组织相对特异性表达的线粒体蛋白】抗体是基因功能研究重要的工具。为了制备C1ORF102多克隆抗体,采用pET原核表达系统表达并纯化C1ORF102全长蛋白。将重组蛋白免疫新西兰兔,制备获得抗C1ORF102蛋白多克隆抗体。该抗体特异性好,效价高,可应用于Western blot(1:10000)、免疫组化(1:5000-10000)以及免疫荧光(1:500-1000)检测。应用该抗体研究发现,C10RF102在鼻咽组织、气管组织中高表达,在大脑和脊髓有微弱表达,而在心脏、肝脏、脾脏、胃、肾脏、肺、结肠、胸腺、骨骼肌、胰腺等组织中没有表达。采用RT-PCR检测也发现,C1ORF102基因mRNA在鼻咽、气管高表达,在脑组织存在微弱表达。免疫组化分析进一步证实,C1ORF102蛋白在鼻咽、气管、支气管上皮中呈强阳性表达,在鼻咽部腺体以及气管周边腺体有中等程度表达。而在心脏、肝脏、脾脏、胃、肾脏、肺、结肠、胸腺、骨骼肌、胰腺以及表皮等组织中未检测到阳性表达。有趣的是,我们发现,C1ORF102蛋白在中枢神经系统神经元中呈阳性,在胶质细胞中没有表达。该结果说明C1ORF102基因是一个新的组织特异性/选择性基因。在研究过程中还发现C1ORF102基因存在第二外显子选择性剪切。C1ORF102基因全长编码389aa,选择性剪切差异本编码379aa,不包括第二外显子。通过RT-PCR结合测序的方法分析了C1ORF102基因全长及剪切差异本在人胚胎组织及多种细胞系中的分布,结果表明,C1ORF102全长(389aa)仅见于脑组织,而脑组织同时也表达编码379aa的剪切本;鼻咽、气管组织仅表达379aa剪切本;在永生化成人正常鼻咽上皮细胞系NP69及鼻咽癌细胞系中表达的也是379aa差异本。前期研究发现在Hela细胞中瞬时转染C1ORF102表达质粒,C1ORF102蛋白表达于线粒体。免疫荧光检测表明NP69细胞中内源性C1ORF102蛋白与线粒体存在明显共定位特征。随后采用密度梯度离心方法分离NP69细胞线粒体和胞浆蛋白组分,Western blot检测发现内源性C1ORF102蛋白绝大部分定位于线粒体,且分子量较胞浆中的C1ORF102稍大,可能源于线粒体内存在某种翻译后修饰。上述结果表明天然C1ORF102蛋白是一个线粒体蛋白。另外我们发现外源性稳定表达的C1ORF102蛋白同样主要定位于线粒体。免疫电镜观察发现在5-8F细胞中瞬时转染myc-C1ORF102蛋白定位于线粒体。【C1ORF102在鼻咽癌中表达下调,转染C1ORF102抑制5-8F细胞生长与增殖,逆转5-8F细胞对缺氧的耐受能力】通过realtime RT-PCR、Western blot检测发现C1ORF102基因mRNA及蛋白在鼻咽癌细胞系(HNE1、HNE2、6-10B、5-8F)中表达下调,在5-8F细胞中检测不到C1ORF102蛋白表达。采用免疫组化检测发现C1ORF102蛋白在鼻咽癌组织中表达下调。为了研究C1ORF102基因是否是鼻咽癌抑瘤基因,基因转染建立稳定表达C1ORF102基因的5-8F细胞系,生长曲线、平板集落形成实验、软琼脂克隆形成实验显示C1ORF102具有抑制5-8F细胞生长和增殖的作用。流式细胞仪检测细胞周期发现,转染C1ORF102基因的5-8F细胞在G1期的比例增高,S期的比例下降,引起细胞周期G1-S期阻滞。Western blot检测发现转染C1ORF102基因引起cyclinD1、cyclinB1、c-Myc、CDK4和磷酸化Rb表达下调,上调p53表达。同时转染C1ORF102引起凋亡分子Bax、caspase3蛋白表达上调。更为重要的是,我们发现C1ORF102基因强烈抑制了5-8F细胞在化学缺氧状态下的生长与增殖能力。采用流式细胞计数及Hoechst 33258染色都证明C1ORF102基因显著促进了在缺氧状态下5-8F细胞的凋亡。将稳定转染的细胞接种裸鼠,发现转染C1ORF102基因可以明显抑制裸鼠移植瘤的生长,但不影响肿瘤细胞的分化水平。HE染色还发现在转染C1ORF102基因的5-8F细胞形成的移植瘤内,可见细胞坏死区域,该结果也提示C1ORF102可能具有逆转鼻咽癌细胞耐受缺氧的作用。【C1ORF102对鼻咽癌细胞系5-8F线粒体基因表达谱以及能量代谢和信号通路的影响】通过密度梯度离心分离线粒体和胞浆蛋白,应用Western blot检测发现,稳定转染的C1ORF102蛋白绝大部分定位于线粒体内,胞浆中有少量表达。应用基因芯片筛查C1ORF102对鼻咽癌细胞5-8F基因转录组的影响,发现共95个线粒体基因表达上调,这些上调的基因包括线粒体转录因子、线粒体翻译起始因子、线粒体核糖体蛋白、电子传递链组分蛋白、凋亡相关因子等。有8个线粒体靶向蛋白在转染C1ORF102基因后表达下调,其中包括丙酮酸脱氢酶激酶1(pyruvate dehydrogenase kinase 1,PDK1)。同时,我们还发现转染C1ORF102下调了葡萄糖转运受体GLUT3以及糖酵解关键酶6-磷酸果糖激酶(PFKL)的表达。有趣的是,PDK1、GLUT3、PFKL三者都是HIF1的靶基因。采用realtime RT-PCR检测了FXN、MRPS6、PDK1、GLUT3、PFKL mRNA表达水平,结果表明与芯片结果一致,另外还通过Western blot检测了PDK1、PFKL蛋白表达水平,证实转染C1ORF102引起PDK1、PFKL蛋白表达下调。随后我们考察了转染C1ORF102基因细胞的代谢特征,结果表明转染C1ORF102基因后5-8F细胞乳酸产量降低,而线粒体膜电位增高,与此同时,细胞内ATP含量明显增高,提示5-8F细胞糖酵解受抑制,能量代谢方式向线粒体有氧代谢转变。全基因组基因芯片筛查还发现转染C1ORF102基因引起多个信号通路分子表达水平改变。其中多个分子涉及MAPK信号通路。为了进一步考察C1ORF102基因对MAPK信号通路的影响,通过Western blot检测MAPK信号通路关键分子的表达与活化。结果发现,转染C1ORF102并不影响NF-κB、total ERK1/2、total p38以及JNK2表达水平,但是抑制了p-ERK1/2、p- p38的表达水平,提示MAPK信号通路受抑制。基因芯片结果还发现Wnt/β-catenin信号通路分子Wnt5A、Wnt10A、FZD7、FZD5表达下调,采用realtimeRT-PCR方法检测Wnt5A、FZD7、FZD5 mRNA表达水平,证实三者在转染C1ORF102后表达显著下调。通过Western blot和免疫荧光检测β-catenin的表达和分布,结果表明:转染NOR1并不引起β-catenin表达水平改变,但是抑制了β-catenin从膜/胞浆向细胞核内转位,在转染C1ORF102的细胞内,定位于细胞核的β-catenin显著减少,表明Wnt/β-catenin信号通路受抑制。【采用酵母双杂交系统筛选C1ORF102交互作用蛋白,发现并证明C1ORF102通过OSCP蛋白与线粒体ATP合成酶发生交互作用】为了进一步明确C1ORF102基因的生物学功能,采用酵母双杂交技术筛选C1ORF102基因相互作用蛋白。以C1ORF102基因为诱饵,筛选人胎脑cDNA文库,得到15个阳性克隆,经测序发现代表7个不同的基因,分别为金属硫蛋白2A、神经元特异性的细胞间黏附分子(ICAM5)、线粒体ATP合成酶(FoF1-ATPase)亚基寡霉素敏感蛋白OSCP、微管相关蛋白MAP1S、补体C2、羧肽酶CPE和一个半胱氨酸富集的BMP调节因子CRIM2。因为C1ORF102是一个线粒体蛋白,因此我们推测C1ORF102与OSCP发生交互作用的可能性较大。首先采用特异性酵母双杂交证实C1ORF102与OSCP在酵母细胞内存在直接交互作用。免疫荧光共定位分析发现不论内源性还是外源性C1ORF102与OSCP在细胞内都存在明显共定位。进一步采用免疫共沉淀证实C1ORF102与OSCP蛋白在线粒体内存在交互作用。C1ORF102促进化学缺氧引起的凋亡作用部分依赖于线粒体ATP合成酶活性,当采用线粒体ATP合成酶抑制剂oligomycin阻断其酶活性后,C1ORF102促进化学缺氧引起的凋亡能力也随之下降。C1ORF102通过OSCP蛋白与ATP合成酶交互作用调节其酶活性,可能是转染C1ORF102基因后ATP含量增加的原因之一。更多还原

【Abstract】 【Background of C1ORF102】Recently,nonrandom loss of heterozygosity（LOH） at 1p34 in NPC has been detected by comparative genomic hybridization（CGH） and genome-wide analysis with microsatellite allelotyping,suggested that there is at least one tumor suppressor gene locates at this site. C1ORF102（formerly named as oxidored-nitro domain containing protein 1,NOR1） is a novel gene cloned by Dr.Xin-min Nie. C1ORF102 gene locates at chromosome 1p34.2 and down-regulated in NPC cell line HNE1 and NPC biopsies.By using case-control analysis stratege,two cSNPs（coding region single nucleotide polymorphism） of C1ORF102 were found to be associated with NPC.Fan.SQ et al. performed in site hybridization analysis on high through put NPC tissue microarray and found that C1ORF102 mRNA frequently down-expressed in NPC tissues.Further more,SAM analysis revealed that C1ORF102 gene locates at 6th among the 488 down-regulated genes in NPC tissues.All these data suggested C1ORF102 might be a good candidate tumor suppressor gene or related gene in nasopharyngeal tumorigenesis.However,to date,very few functional studies on C1ORF102 have been carried out,and the precise tissue and cell localization of C1ORF102 has not yet been resolved.【Prepared the polyclonal antibody specific for C1ORF102 and detected its expression pattern in the human fetus,identified it as a novel mitochondrial protein】Understanding the localization of gene product in humans is important with respect to characterization of gene function.In this study, His-C1ORF102 recombinant protein were expressed in E.coli. Polyclonal anti-C1ORF102 antibody was produced by immunizing New Zealand white rabbits with the purified His-C1ORF102 recombinant protein.The specificity of the antibody was identified by Western blot and showed that it have no cross-immunoreaction with other proteins. With the prepared C1ORF102 antibody,we detected the distribution of C1ORF102 in human fetus.In the Western blot assay,a high expression level of the C1ORF102 protein was seen in the nasopharynx and trachea. Weak expression level of the C1ORF102 protein was seen in the homogenates from brain and spinal.A very weak expression level of the C1ORF102 protein was shown homogenate from human lung.Human heart,liver,spleen,stomach and kidney showed absent of C1ORF102 protein.Immunohistochemistry assays showed that intensive cytoplasma staining for C1ORF102 protein was seen in the mucosa of human nasopharynx,both columnar epithelium and stratified squamous epithelium have strong positive labeling.In trachea and small bronchie, intensive staining for C1ORF102 protein also were seen in epithelial cells,but lung alveoli showed negative staining.The submucosa matrices of these tissues were negative for staining.Tissue sections from the kidney,heart,liver,pancreas,stomach did not staining positive for C1ORF102.Tissue sections from skeletal muscle,smooth muscle, spleen,colon,thymus,adrenal gland also did not staining positive for C1ORF102.We further validated most of the positive expression by RT-PCR,and the results are consistent with the Western blot assay and immunochemistry observations.Interestely,neurons in tissue sections of central nervous system showed high expression level of C1ORF102 protein,but glial cell showed have no expression of C1ORF102.The data suggested C1ORF102 is a novel tissue specific/selective gene.In this study,beside the the fulllength C1ORF102 corresponding to a human cDNA（GenBank accession no.NM₁45047） which encoded 389-amino acid protein,a splice isoforms was also identified from human fetal brain cDNA library,which is lack of the second exon corresponding to previously cloned NOR1 and encoded a 379-amino acid protein.A human genome BLAST analysis using the NCBI Map Viewer program indicated that the human C1ORF102 gene is comprised of at least 11 exons,and the alternative use of exon 2 generates these splice variants.RT-PCR of mRNAs for C1ORF102 isoforms with 379 and 389 amino acids revealed that the former is the sole isoform presented in the nasopharynx,trachea and other tissues,whereas both isoforms are expressed in the brain.Further more,C1ORF102 isoforms with 379 amino acids is the sole isoform expressed in human nasopharygeal epithelial cell line NP69 and NPC cell lines（HNE1、HNE2、5-8F、6-10B）.However,immunofluoresence assay showed these two variants both locates in cytoplasma.The pilot study showed that the exogenous expressed GFP-NOR1 protein distributed in mitochondrion and cytoplasma.This result trigger us to suspect whether C1ORF102 is a mitochondrial targeted protein.To examine this hypothesis,we detected endogenous C1ORF102 in NP69 cell lines by immunofluoresence assay and probed mitochondrion with Mitotraker Red.The data showed that C1ORF102 protein co-localized with mitochondrion.We also isolated a mitochondrion-enriched fraction from human normal nasopharyngeal epithelial cell line NP69,and subjected it to immunoblot analysis with antibodies to C1ORF102.The result showed that most of endogenous expressed C1ORF102 protein was present in the mitochondrion fraction,as revealed by immunoblot with antibodies to mitochondrial ATP5αprotein.Interestely,molecular weight of C1ORF102 protein in mitochondrion is larger than the cytoplasmic counterpart,which may represent posttranslational modifications of C1ORF102 protein in mitochondrion.Most of exgenously expressed C1ORF102 protein in stable transfected 5-8F cells was also present in mitochondrian fraction.And this result was also confirmed by electrotranscopy.Thus,our results indicate that C1ORF102 is a novel nuclear encoded mitochondrial targed protein. 【C1ORF102 inhibited the growth and proliferation ability of NPC 5-8F cell line and induced tumor cells apoptosis under chemical hypoxia】We analyzed the expression of C1ORF102 in several human nasopharyngeal carcinoma cell lines using realtime RT-PCR and Western blot analyses.A high expression of C1ORF102 at the mRNA and protein level was detected in normal nasopharyngeal epithelial cell line NP69.However,all of the nasopharyngeal carcinoma cell lines showed downexpression of C1ORF102 at both mRNA and protein levels compared with NP69.5-8F cell line,a NPC cell line with high tumorigenic and metastatic potentiality,showed deficient of expression of C1ORF102.Immunohistochemistry assay also showed that C1ORF102 protein down-regulated in NPC biopsies.To investigate whether over-expression of C1ORF102 in cancer cells leads to suppression of cancer cells growth,C1ORF102 was stablely transfected into 5-8F cell line.Following stable transfection with C1ORF102, nasopharyngeal carcinoma cells showed significantly reduced growth rates when compared with mocked-transfected cells.Accordingly, population doubling times in cancer cells overexpressing C1ORF102 were significantly increased.Cell cycle analysis showed overexpression C1ORF102 induced cell cycle G1-S phase arrest,which accompanied by down-regulation of cell cycle regulators such as cyclin Dl,cyclin B,CDK4,c-Myc expression and Rb phosphorylation in C1ORF102 overexpressing cells.To further test whether C1ORF102 overexpression also affected anchorage-independent growth,soft agar assays were performed.A pronounced reduction in colony-forming capacity and colony size was observed in the case of C1ORF102 overexpressing cells. More importantly,proliferation assay showed over-expression C1ORF102 remarkblely impaired growth of 5-8F cells under CoCl₂ treatment.Further more,cells with C1ORF102 expression exhibit more sensitive to apoptosis induced by CoCl₂ treatment.Lastly,we tested whether tumor formation in nude mice would be similarly affected. Indeed,tumors derived from cancer cells with overexpression of C1ORF102 were significantly smaller than those derived from mock-transfected control cells.Thus,our data implied that C1ORF102 might impair the survival ability of NPC cells in hypoxia condition.【C1ORF102 up-regulated mitochondrial gene expression and induced oxidative metabolism in NPC 5-8F cells】In order to identify genes modulated by C1ORF102 in NPC, high-density oligonucleotide microarray was performed on well-characterized C1ORF102-expressing 5-8F pooled clones and the mocked-transfected pooled clones.Change of mitochondrial targeted genes expression between transfected clones and control cells with a minimum of 1.6-fold were scored.Using these selection criteria,we identified 103 differentially expressed mitochondrial genes in the microarray analysis.Totally,95 mitochondrial targeted genes were upregulated following C1ORF102 overexpressing.These upregulated genes fall into several functional categories such as mitochondrial transcription（e.g.TFAM,TFB1M）,translation factors and protein synthesis（e.g.MTIF3,TSFM,MRPS6）,electron transport chain（e.g. ATP5D,ATP5I,NADH dehydrogenase）,mitochondrial translocase（e.g. TIMM8A,TIMM13） and apoptosis molecules（e.g.BAX,BID,BIK）. However,only eight genes in total were downregulated in C1ORF102 expressing cells,which included pyruvate dehydrogenase kinase, isozyme 1（PDK1）.Further more,several key molecules involved in glucose metabolism down-expressed in C1ORF102 transfected cells, which included glucose transporter 3（GLUT3） and phosphofructokinase, liver（PFKL）,a subunit of the key glycolytic enzyme.To examine the reliability of microarray data,we performed realtime RT-PCR analysis of the expression of frataxin,MRPS6,PDK1,PFKL and GLUT3.The fold change revealed by realtime RT-PCR well in accordance with microarray data.The inhibition of C1ORF102 on PDK1,PFKL was also accompanied by a remarkable decrease of PDK1,PFKL protein as demonstrated by immunoblot assay.Thus,our data suggested that over-expression C1ORF102 enhanced mitochondrial targeted genes expression level and possiblely regulates glucose metabolism.To investigate whether overexpression of C1ORF102 in cancer cells leads to metabolic alterations,we quantified lactate production,lastly ATP levels.We observed a 1.49-fold increase of cellular ATP content and less lactate production in C1ORF102 overexpressing cells than the mocked-transfected control cells.To evaluate whether mitochondrial membrane potentialΔΨ_m is increased in cells overexpressing C1ORF102,which is known to be the driving force for mitochondrial ATP synthesis,we stained the cells with the fluorescent rhodamine 123, a dye that accumulates in mitochondria in a potential-dependent manner. Rhodamine-stained cells overexpressing C1ORF102 showed a increased fluorescence of mitochondria when compared with control cells, suggested elevatedΔΨ_m in C1ORF102 expressing cells.To exclude that the elevation in mitochondrial capacity and in OXPHOS of C1ORF102 overexpressing cells could be explained by an increased number of mitochondria,we also stained the cells with the fluorescent Mitotraker Red,a mitochondrion-selective stains whose accumulation in mitochondria is directly proportional to mitochondria number. Mitotraker Red -stained cells overexpressing C1ORF102 showed similar fluorescence of mitochondria when compared with control cells.Thus, our primary data suggested C1ORF102 modulate energe metabolism of NPC 5-8F cells.Mitochondrial respiration defects in cancer cells could cause activation of important survival pathway.To examine whether C1ORF102 expression affects important cell signal transduction involved in tumorigenesis,we analyzed the expression and activation status of mitogen-activated protein kinase（MAPK） pathway.Although the levels of NF-κB,ERK1/2,p38,JNK2 expression in C1ORF102 overexpressing 5-8F cells were similar to that in mocked-transfected control cells,the levels of phosphorylated ERK1/2 and phosphorylated p38 were greatly reduced after C1ORF102 over-expression.Thus, overexpression C1ORF102 modulated the MAPK signaling pathway, which may in turn inhibit the proliferation of NPC 5-8F cells.Because of microarray data revealed that Wnt5A and two receptor FZD7,FZD5 down-regulated in C1ORF102 overexpressing cells,we examined Wnt5A,FZD7,FZD5 expression and distribution ofβ-catenin in cells. Realtime RT-PCR analysis indicates significant down-regulation of Wnt5A,FZD7,FZD5 in C1ORF102 overexpressing cells which in accordance with array data.Although totalβ-catenin protein unaltered between C10RF102 overexpressing cells and mocked-transfected cells, subfraction and immunofluoresence analysis showed that over-expression C1ORF102 in 5-8F cells effectively attenuatedβ-catenin translocation from membrane/cytoplasma to nucleis.Thus,our data demonstrated that C1ORF102 negatively modulate Wnt/β-catenin signaling pathway via down-regulate the expression level of Wnt5A and it’s receptor FZD7 and FZD5.【Screened the protein-protein interactors of C1ORF102 by yeast two-hybrid system,identified C1ORF102 as a novel interact partner of mitochondrial FoFl-ATPase via binding to OSCP protein】To establish a clearer picture of what the functions might be,and to identify cellular proteins that might bind to C1ORF102,we used a yeast two-hybrid approach to screen a human brain cDNA library for C1ORF102 binding proteins.With fulllength C1ORF102 as the bait,we screened a human fetal brain cDNA library and identified 10 positive clones,one of which encoded the fulllength of the oligomycin sensitivity conferring protein（OSCP）,which is a subunit of mitochondrial ATP synthase.The interaction between C1ORF102 and ATP50 was confirmed by specific yeast two-hybrid assay.To examine whether C1ORF102 binding to OSCP in mammalian cells,we transiently transfected 5-8F cells with expression vectors for Myc-tagged C1ORF102 and subjected lysates of the isolated mitochondria in transfected cells to co-immunoprecipitation and immunoblot analysis. Myc-C1ORF102 coprecipitated with endogenous OSCP from the mitochondria lysates.Immunofluorescence analysis also revealed that endogenous C1ORF102 protein co-localized with OSCP in human normal nasopharyngeal epithelium cell line NP69 and Hela cells.Thus, the data inticates that C1ORF102 interact with FoFl-ATPase by binding to OSCP.Further more,the effect of C1ORF102 on CoCl₂ induced apoptosis is partially dependent on FoFl-ATPase activity,as the FoFl-ATPase inhibitor,oligomycin strongly antagonized CoCl₂ induced apoptosis in C1ORF102 expressing cells,which implied that C1ORF102 might modulate FoFl-ATPase enzyme activity.更多还原