胃癌FAT10表达与突变p53基因的相关性研究
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摘要
背景与目的
FAT10又被称为双泛素(diubiquitin)，属于泛素蛋白家族中的泛素样修饰蛋白(UBLs)，是一个分子量为18kDa的包含165个氨基酸残基的蛋白质，由两个泛素样结构域融合而成。FAT10在细胞周期的调控中起到十分重要作用，已被证明能与人纺锤体聚集检测点蛋白(mitotic arrest deficiency 2，MAD2)非共价结合，而此蛋白负责在有丝分裂时保持纺锤体的完整性，MAD2功能受到抑制则导致染色体不稳定，这是许多肿瘤发生的重要特征。已有发现肝癌、宫颈癌、卵巢癌、直肠癌、胰腺癌及小肠腺癌等FAT10表达明显上调，表明FAT10在肿瘤发生中起到一定作用。p53基因是一种肿瘤抑制基因，定位于人类17号染色体短臂，分为野生型p53(wt-p53)和突变型p53(mt-p53)两种，编码p53蛋白。人类一半以上恶性肿瘤都显示出p53基因突变或p53蛋白缺失。p53的突变在胃癌发生发展中也发挥着重要作用，许多研究均表明p53基因突变是胃癌发生发展的重要始动因素之一。最近研究发现FAT10启动子活性和表达在有野生型p53基因的细胞中比在p53基因缺失的受到更显著的抑制，表明p53基因存在使得FAT10基因表达下调。由于p53基因在体内结合于5′端的位点有一半共有系列定位于FAT10启动子，所以提出FAT10是p53基因的下游靶点，在p53基因缺陷细胞中FAT10基因表达的失调可能会导致肿瘤发生。胃癌是最常见的恶性肿瘤之一，中国男、女性胃癌世界调整死亡率居于首位。随着分子生物学技术的发展和应用，人们对胃癌发病机制的探讨已深入到基因水平，但至今未找到胃癌特异的分子病理学标志物。新近研究发现FAT10极有可能是胃癌发生的另一“元凶”。以往研究主要注重于FAT10的结构、表达产物的特征、诱导因子及其基本功能，但有关FAT10在胃癌组织中的表达、与临床病理学特征关系及其与p53基因突变的联系研究罕有报道。本研究采取胃癌、癌旁组织和正常组织，应用免疫组化及RT-PCR的方法检测FAT10及p53在人胃癌组织中的表达，试图探讨FAT10在人胃癌发生、发展及转移中的作用，以及突变p53基因在肿瘤中对其的促进作用。为肿瘤预防、早期诊断提供理论依据，并为基因治疗提供目标。
临床资料与方法
一、标本收集
1．病例资料：研究对象为胃癌患者共计62例，其中男性38例，女性24例，年龄21～86岁，平均59．62岁，所有标本均经病理证实。胃癌有淋巴结转移39例，无淋巴结转移23例；有远处转移15例，无远处转移47例；共62例。
2．收集方法：癌旁组织取距肿瘤旁2cm胃组织，正常胃组织取距肿瘤5cm以外胃组织。每个组织均为离体后半小时内取材，剔除坏死出血组织，编号标记并记录临床资料，将用于免疫组化的癌组织、癌旁组织及正常胃组织置于10％的甲醛液中保存，另一份癌组织、癌旁组织及正常胃组织立即置于液氮罐中并转入-80℃冰箱保存用于RT-PCR检测。
3．胃癌TNM分期按1997年国际抗癌联盟(UICC)公布的PTNM分期。
二、主要实验试剂及溶液配制
1．兔抗人FAT10多克隆抗体购自上海吉泰生物科技有限公司。
2．兔抗人p53单克隆抗体购自北京中杉金桥生物技术有限公司。
3．生物素标记的FAT10二抗与p53鼠抗兔二抗分别购自上海吉泰与武汉博士德。
4．免疫组化染色试剂盒购自福州迈新生物技术开发有限公司
5．DAB显色试剂盒、粘片剂APES购自福州迈新生物技术开发有限公司。
6．RNA快速抽提纯化(Trizol)试剂盒购自Gibco BRL公司。
7．Oligo dT及MMLV购自Promega公司
8．引物制备：上海生工生物工程有限公司合成，经Genebank检索为特异性引物：
内参照β-actin引物序列为：
上游引物：5′-TCA CCC ACA CCG TGC CCA TCT ACG A-3′
下游引物：5′-CAG CGG AAC CGC TCA TTG CCA ACG G-3′
FAT10特异性引物序列为：
上游引物：5′-AAT GCT TCC TGC CTC TGT GT-3′
下游引物：5′-GCC GTA ATC TGC CAT CAT CT-3′
突变的p53特异性引物序列为：
上游引物：5’—CCTATGGAAACTACTTCCTGAAAACAA—3’
下游引物：5’—ACAGCATCAAATCATCCATTGC—3’
三、主要仪器和设备
产品名称生产或经销公司国籍
石蜡包埋机Leica公司德国
病理石蜡切片机Leica公司德国
万能显微镜成像系统(Leica DMR) Leica公司德国
光学显微镜照相系统Olympus日本
GeneAmp 2400 PCR System：Perkin Elmer公司美国
SCR-300A稳流稳压电泳仪上海康华生化仪器制造厂中国
电泳槽上海康华生化仪器制造厂中国
数码凝胶成像系统BIO-RAD GDEQ杭州宝诚生物技术有限公司中国
紫外分析仪Beckman DU640 Beckman公司美国
ZF-1紫外分析仪上海康华生化仪器制造厂中国
DL-8R冷冻离心机上海市离心机械研究所中国
XW-80A旋涡混合器上海医科大学仪器厂中国
震荡器XW-80A上海医科大学仪器厂中国
低温冰箱Forma Scientific Inc美国
四、检测指标及方法
1．FAT10、p53蛋白免疫组化(二步法)测定：
免疫组化主要操作步骤如下：
1)10％的福尔马林固定的癌组织、癌旁组织及正常胃组织，石蜡包埋，切片。
2)切片常规脱蜡。
3)过氧化氢封闭内源性过氧化物酶。
4)抗原修复。
5)滴加正常兔血清封闭液。
6)滴加兔抗人FAT10多克隆抗体及一抗为兔抗人p53单克隆抗体工作液，以PBS溶液代替一抗作为阴性对照片。
7)加50ul生物素标记的FAT10二抗及生物素化p53鼠抗兔二抗。
8)DAB显色。
9)苏木素复染。
10)梯度酒精脱水。
11)光学显微镜下观察判定免疫组化结果。
2．FAT10、p53-mRNA RT-PCR测定：
1)RNA提取及分析：
(1)RNA提取：采用TRIZOL试剂一步提取法。
(2)RNA分析：
①普通琼脂糖凝胶电泳分析。
②分光光度仪分析。
2)逆转录cDNA的合成及电泳验证逆转录产物cDNA。
cDNA模板进行PCR或保存于-20℃冰箱备用．
3)PCR扩增反应：
PCR扩增体系：
cDNA样品2μl
内参上游引物0．5μl
内参下游引物0．5μl
10×Buffer 2．5μl
MgCl_2 2μl
Taq酶0．15μl
2mMdNTPs 2μl
ddH_2O 15．35μl
总体积25μl
(1)内参照β-actin基因PCR扩增反应条件：
PCR扩增产物为295bp。
(2)FAT10基因的PCR扩增反应条件：
PCR扩增产物为478bp。
(3)突变p53基因的PCR扩增反应条件：
PCR扩增产物为：492bp。
4)产物电泳分析。
五、统计学处理
1．免疫组化实验所得定性数据采用x~2检验或分类资料的关联性检验判定两者关系。用Spearman秩相关分析两者之间的关系。FAT10与肿瘤临床病理特征的关系用阳性／阴性计数进行定性数据的x~2检验进行统计。生存率制作Kaplan-Meier生存曲线，进行Log-rank生存曲线检验。对本组病例治疗后影响预后的因素进行Cox模型多因素分析。
2．RT-PCR条带灰度值结果用均数±标准差((?)±s)表示，样本间均数的比较采用t检验，用Pearson相关分析分析突变p53与FAT10表达的相关性。
3．所有数据采用SPSS12．0统计软件包处理，P<0．05表示差异有显著性；P<0.01表示差异非常显著。
结果
一、免疫组织化学检测FAT10及p53蛋白在胃癌组织、癌旁组织及正常胃组织中的表达
1．FAT10免疫组织化学染色：
FAT10的表达主要定位于细胞核，阳性物质呈颗粒状。FAT10在胃癌组织中的阳性表达率显著高于相应的癌旁组织及正常胃组织，其阳性率分别为51．61％、12．90％及6．45％，差异均有非常显著性(P<0．01)，而癌旁组织与正常胃组织比较，FAT10的阳性表达率差异无显著性(P>0．05)。
2．p53免疫组织化学染色：
p53的表达主要定位于胞核，阳性物质呈颗粒状，可弥散到胞浆中。p53在胃癌组织中的表达显著高于相应的癌旁组织及正常胃组织，其阳性率分别为45．16％、14．51％及9．68％，差异均有非常显著性(P<0．01)，而癌旁组织与正常胃组织比较，p53的阳性表达率差异无显著性(P>0．05)。
3．FAT10蛋白表达与胃癌临床病理特征的关系：
胃癌组织中FAT10的表达主要与胃癌的淋巴转移与否及TNM分期(Ⅲ+Ⅳ/Ⅰ+Ⅱ)密切相关(P均<0．05)。而与胃癌患者年龄、性别、肿瘤大小、发生部位、进展程度、分化程度及远处转移无显著相关性(P均>0．05)。39例淋巴转移患者胃癌组织FAT10阳性表达者转移淋巴也呈FAT10阳性表达。
4．FAT10与p53蛋白在胃癌组织中表达的相关性分析：
p53阳性胃癌中FAT10表达率为82．14％(23／28)，而p53阴性胃癌FAT10表达率仅26．47(9／34)，用双向无序分类资料的关联性检验说明FAT10与p53表达存在一定的相关性。Spearman秩相关分析得出胃癌组FAT10蛋白表达和突变型p53蛋白表达存在显著正相关性。
二、RT-PCR检测FAT10及突变p53-mRNA在胃癌组织、癌旁组织及正常胃组织中的表达
1．胃癌组织、癌旁组织以及正常胃组织中FAT10-mRNA表达：
胃癌组织、癌旁组织及正常胃组织中FAT10-mRNA条带灰度值分别为0．689±0．023、0．463±0．019及0．451±0．028，采用配对t检验，发现胃癌组织FAT10-mRNA条带灰度值显著高于相应的癌旁组织及正常胃组织(t值分别为3．12及4．64，P均<0．01)，癌旁组织与正常胃组织两者比较无显著性差异(t值为1．03，P>0．05)。
2．胃癌组织、癌旁组织以及正常胃组织中突变p53-mRNA表达：
胃癌组织、癌旁组织以及正常胃组织中突变p53-mRNA条带灰度值分别为0．471±0．021、0．398±0．017及0．421±0．019，采用配对t检验，发现胃癌组织突变p53-mRNA条带灰度值显著高于相应的癌旁组织及正常胃组织(t值分别为6．79及5．51，P均<0．01)，癌旁组织与正常胃组织两者比较无显著性差异(t值为1．22，P>0．05)。
3．FAT10-mRNA的表达与胃癌临床病理特征的关系：
胃癌组织中FAT10-mRNA的表达主要与胃癌的淋巴转移与否及TNM分期(Ⅲ+Ⅳ／Ⅰ+Ⅱ)密切相关(P均<0．05)。而与胃癌患者年龄、性别、肿瘤大小、发生部位、进展程度、分化程度及远处转移无显著相关性(P均>0．05)。FAT10-mRNA的表达与胃癌临床病理特征的关系结果与免疫组织化学基本一致。
4．FAT10与p53基因在胃癌组织中表达的相关性分析：
Spearman等级相关分析，胃癌组中FAT10基因表达和突变型p53基因表达存在正相关性。用Pearson相关分析其相关性，说明FAT10与p53基因呈显著正相关。
三、胃癌FAT10蛋白及FAT10-mRNA的表达与生存率的关系：
除4例患者失访外，其余病例均获随访，随访时间48～72个月。对本组患者术后累积生存率进行单因素分析，均经Log-rank生存曲线检验。
根据Log-rank生存曲线检验，FAT10蛋白表达阳性胃癌患者生存时间显著低于表达阴性者，P<0．05。
根据Log-rank生存曲线检验，FAT10-mRNA的表达阳性胃癌患者生存时间显著低于表达阴性者，P<0．05。
经Cox模型多因素分析，结果显示FAT10蛋白及mRNA的表达与肿瘤淋巴结转移、远处转移、临床TNM分期、p53蛋白及p53 mRNA同为影响胃癌预后的独立因素。
结论
1．胃癌组织FAT10蛋白表达及FAT10-mRNA相对表达量显著高于相应的癌旁组织及正常胃组织。胃癌组织中FAT10蛋白及mRNA的表达主要与胃癌的淋巴转移与否及TNM分期密切相关。而与胃癌患者年龄、性别、肿瘤大小、发生部位、进展程度、分化程度及远处转移无显著相关性。FAT10-mRNA的表达与胃癌临床病理特征的关系结果与免疫组织化学基本一致。
2．胃癌组织中p53蛋白表达及p53-mRNA相对表达量明显高于相应的癌旁组织及正常胃组织，而癌旁组织与正常胃组织两者比较无显著性差异。
3．胃癌组织中FAT10基因的表达与突变p53基因的表达均增强，蛋白水平与基因水平检测的结果一致。两者增高在统计学上均有统计学意义，并呈现明显的正相关。
4．FAT10蛋白及FAT10-mRNA的表达阳性胃癌患者生存时间均显著低于表达阴性者。经Cox模型多因素分析，结果显示FAT10蛋白及mRNA的表达与肿瘤淋巴结转移、远处转移、临床TNM分期、p53蛋白及p53 mRNA同为影响胃癌预后的独立因素。
Background
FAT10 is an 18KD protein comprising 165 amino-acid residues. It wasoriginally discovered through the identification of expressed genes covering theHLA-F genomic locus. A potential role of FAT10 in antigen presentation wassuggested by its expression in mature B cells and dendritic cells, and by its ability tobe generally and synergistically inducible with cytokines IFN_γand TNFα. FAT10belongs to the UBL family of proteins and contains two ubiquitin-like moietiesfused in tandem. It is 29% identical to ubiquitin at its N-terminus and 36% identicalat the C-terminus. It has the C-terminal Gly-Gly residues and there is a comservedLys residue in each moiety of FAT10 analogous to Lys 48 of ubiquitin, which mayserve as a potential site for polyubiquitination of FAT10. FAT10 has been reported tohave potential involvement in cell-cycle regulation and shown to bind noncovalently to the human spindle assembly checkpoint protein, MAD2, a protein responsible formaintaining spindle integrity suring mitosis. The inhibition of MAD2 function hasbeen associated with chromosomal instability, a characteristic of many tumoursoccurrence. It was reported that FAT10 expression in the nucleus of HCChepatocytes rather than the surrounding immune or non-HCC cells. FAT10overexpression was also found in other cancers of the gastrointestinal tract andgynecology. FAT10 may modulate tumorigenesis through its reported interactionwith the MAD2 spindle-assembly checkpoint protein.
Recently, It is reported that p53 is likely an upstream regulator of FAT10 andmay link FAT10 in the tumorigenesis process. As FAT10 was found to interact withMAD2, an hypothesis would be that the repression of FAT10 expression by p53may facilitate the interaction of MAD2 with cell division cycle 20 (CDC20)to induce mitotic arrest. P53 gene mutation may lead to the enhancement of FAT10gene expression during tumorigenesis. These results were observed in severaldifferent cancers. FAT10 overexpression may result in the deregulation of mitosis,cause genome instability and tumorigenesis.
Gastric cancer is one of the most common malignant tumor and a leading causeof mortality world-wide. With the development and appliance of molecule biologicaltechnology, research in the mechanism of gastric cancer has probed into the level ofgene. But, it hadn't been identified the specific marker of molecule pathology ingastric cancer yet. For a tumor to become established, compromises in the control oftumor related gene are necessary. The Ubiquitin-like modifier (UBL) which has generated much interest in the scientific community is implicated to play importantrole in gastric cancer. Previous study was concerned mainly about the struction ofthe FAT10, the character of its expression, its inducible factor and the function.There are no reports about the relationship between the expression of FAT10 proteinand mRNA and the characters of clinical pathology and correlation betweenFAT10 and mutant p53 in gastric cancer.
Objective
To investigate the expression of FAT10 and mutant p53 gene in the gastriccancerous tissue, latero-GC tissue and the normal gastric mucosa tissue from thelevel of cellular and molecular biology. To analyse the relationship of expression inFAT10 protein and gene and clinical pathological features of gastric cancer and thecorrelation between the FAT10 and mutant p53 expression in gastric cancer tissue.To explore the effects of FAT10 in the production , invasion and metastasis processof gastric cancer.
Materials and Methods
Sixty-two samples which excisional specimen by operation confirmed asgastric cancer through pathological section in our hospital from March, 2003 to May,2004 were selected to study. The expression levels of FAT10 , FAT10-mRNA,mutant p53 and mutant p53-mRNA in the 62 specimens of gastric cancerous tissue,corresponding latero-GC tissue and normal mucosa tissue were determinedrespectively with the method of immunohistochemistry and RT-PCR. The featuresof expression of FAT10, FAT10-mRNA, mutant p53 and p53-mRNA in the sampleof gastric cancerous tissue, corresponding latero-GC tissue and normal mucosa tissue were analysed and the relationships between FAT10 overexpression with theinformation of age, sex, diameter of tumour, the location of tumor occurrence,invasion, differentiation, lymph nodes metastasis, distant metastasis and TNM stagesof the gastric cancer were observed. Kaplan-Meier survivial plot for patients withgastric cancer according to tumor expression of FAT10 and multivariate Coxproportional hazards model analysis of prognosis factors were studied.
Results
1、Immunohistochemistry
1) The rate of the FAT10's overexpression in the tissue of GC(51.61%) issiginificantly higher than that in latero-GC tissue and normal mucosa tissue(12.90%, 6.45%), the comparison between them:P<0.01.
2) The rate of the p53's positive expression in the tissue of GC(45.16%) issiginificantly higher than that in latero-GC tissue and normal mucosa tissue(14.51%,9.68%), the comparision between them:P<0.01. There are nosiginificant differences of the rate of the FAT10's and p53's positiveexpression between tissue in latero-GC and normal mucosa samples.
3) The rate of FAT10's overexpression in the tissue of GC with the lymphnodes metastasis (64.10%) is siginificantly higher than that without nodesmetastasis (30.43%), the comparision between them :P<0.05. And the rateof FAT10's overexpression in the TNM stageⅢ+Ⅳ(60.98%) issiginificantly higher than the TNM stageⅠ+Ⅱof GC (33.30%), thecomparision between them :P<0.05. Comparision the age,sex,diameter of tumour, the location of tumor occurrence,invasion,differentiation and distantmetastasis of the GC is no significant differences in the rate of FAT10'soverexpression.
2、RT-PCR analysis
4) The level of FAT10-mRNA expression in GC tissue is significantly higherthan that of latero-GC tissue and normal mucosa tissue (0.689±0.023,0.463±0.019 and 0.451±0.028), the comparision between them:P<0.01.
5) The level of mutant p53-mRNA expression in GC tissue is significantlyhigher than that latero-GC tissue and normal mucosa tissue (0.471±0.021, 0.398±0.017 and 0.421±0.019), the comparision betweenthem:P<0.01.
6) The level of FAT10-mRNA overexpression in the tissue of GC with thelymph nodes metastasis (0.656±0.016) is siginificantly higher than thatwithout nodes metastasis (0.531±0.026), the comparision betweenthem :P<0.01. And the level of FAT10's overexpression in the TNM stageⅢ+Ⅳ(0.667±0.023) is siginificantly higher than the TNM stageⅠ+ⅡGC(0.558±0.015), the comparision between them :P<0.05. Comparisionthe age, sex,diameter of tumour, the location of tumor occurrence,invasion,differentiation and distant metastasis of the GC is no significant differencesin the level of FAT10-mRNA overexpression.
3、Correlation Analysis
1) Immunohistochemistry shows: The rate of the positive expression of FAT10in the p53 positive samples is 82.14%(23/28). And 26.47%(9/34) of FAT10is overexpression in p53 negative expression group. Spearman orderprobabilities shows significant positive correlation with FAT10 and mutantp53(r=0.865, P<0.05).
2) RT-PCR shows: Pearson correlation shows significant positive correlationwith mutant p53 and FAT10(r=0.761, P<0.01).
4、Surviving rate Analysis shows: both overexpression of FAT10 protein and mRNAhave significant correlation with the life span of gastric cancer patients (P<0.05).Multivariate Cox proportional hazards model analysis of prognosis factorsshows:FAT10 protein and mRNA overexpression,lymph node and distantmetastases, TNM stage and p53 protein and mRNA overexpression are allsignificant in the multivariate analysis.
Conclusions
1. FAT10 overexpression plays important role in the process of the production,growth of gastric cancer. The level of FAT10's expression is related with thelymph nodes metastasis and TNM stages of the gastric cancer and isn't obviouslyrelated with the age,sex,diameter of tumour, the location of tumoroccurrence,invasion,differentiation and distant metastasis of the gastric cancer.
2. Mutant p53 overexpression plays important role in the process of the production,growth of gastric cancer.
3. FAT10 and mutant p53 are tightly related in the expression of gastric cancer.Mutant p53 intervenes the expression of FAT10. We suppose it's one pathwaysthat mutant p53 gene arouses FAT10 gene overexpression and accelerates thetumor's growth.
4. Both overexpression of FAT10 protein and mRNA have significant correlationwith the life span of gastric cancer patients and may be one of the most reliableindependent prognostic predictors.
FAT10又被称为双泛素(diubiquitin)，属于泛素蛋白家族中的泛素样修饰蛋白(UBLs)，是一个分子量为18kDa的包含165个氨基酸残基的蛋白质，由两个泛素样结构域融合而成。FAT10在细胞周期的调控中起到十分重要作用，已被证明能与人纺锤体聚集检测点蛋白(mitotic arrest deficiency 2，MAD2)非共价结合，而此蛋白负责在有丝分裂时保持纺锤体的完整性，MAD2功能受到抑制则导致染色体不稳定，这是许多肿瘤发生的重要特征。已有发现肝癌、宫颈癌、卵巢癌、直肠癌、胰腺癌及小肠腺癌等FAT10表达明显上调，表明FAT10在肿瘤发生中起到一定作用。p53基因是一种肿瘤抑制基因，定位于人类17号染色体短臂，分为野生型p53(wt-p53)和突变型p53(mt-p53)两种，编码p53蛋白。人类一半以上恶性肿瘤都显示出p53基因突变或p53蛋白缺失。p53的突变在胃癌发生发展中也发挥着重要作用，许多研究均表明p53基因突变是胃癌发生发展的重要始动因素之一。最近研究发现FAT10启动子活性和表达在有野生型p53基因的细胞中比在p53基因缺失的受到更显著的抑制，表明p53基因存在使得FAT10基因表达下调。由于p53基因在体内结合于5′端的位点有一半共有系列定位于FAT10启动子，所以提出FAT10是p53基因的下游靶点，在p53基因缺陷细胞中FAT10基因表达的失调可能会导致肿瘤发生。胃癌是最常见的恶性肿瘤之一，中国男、女性胃癌世界调整死亡率居于首位。随着分子生物学技术的发展和应用，人们对胃癌发病机制的探讨已深入到基因水平，但至今未找到胃癌特异的分子病理学标志物。新近研究发现FAT10极有可能是胃癌发生的另一“元凶”。以往研究主要注重于FAT10的结构、表达产物的特征、诱导因子及其基本功能，但有关FAT10在胃癌组织中的表达、与临床病理学特征关系及其与p53基因突变的联系研究罕有报道。本研究采取胃癌、癌旁组织和正常组织，应用免疫组化及RT-PCR的方法检测FAT10及p53在人胃癌组织中的表达，试图探讨FAT10在人胃癌发生、发展及转移中的作用，以及突变p53基因在肿瘤中对其的促进作用。为肿瘤预防、早期诊断提供理论依据，并为基因治疗提供目标。
临床资料与方法
一、标本收集
1．病例资料：研究对象为胃癌患者共计62例，其中男性38例，女性24例，年龄21～86岁，平均59．62岁，所有标本均经病理证实。胃癌有淋巴结转移39例，无淋巴结转移23例；有远处转移15例，无远处转移47例；共62例。
2．收集方法：癌旁组织取距肿瘤旁2cm胃组织，正常胃组织取距肿瘤5cm以外胃组织。每个组织均为离体后半小时内取材，剔除坏死出血组织，编号标记并记录临床资料，将用于免疫组化的癌组织、癌旁组织及正常胃组织置于10％的甲醛液中保存，另一份癌组织、癌旁组织及正常胃组织立即置于液氮罐中并转入-80℃冰箱保存用于RT-PCR检测。
3．胃癌TNM分期按1997年国际抗癌联盟(UICC)公布的PTNM分期。
二、主要实验试剂及溶液配制
1．兔抗人FAT10多克隆抗体购自上海吉泰生物科技有限公司。
2．兔抗人p53单克隆抗体购自北京中杉金桥生物技术有限公司。
3．生物素标记的FAT10二抗与p53鼠抗兔二抗分别购自上海吉泰与武汉博士德。
4．免疫组化染色试剂盒购自福州迈新生物技术开发有限公司
5．DAB显色试剂盒、粘片剂APES购自福州迈新生物技术开发有限公司。
6．RNA快速抽提纯化(Trizol)试剂盒购自Gibco BRL公司。
7．Oligo dT及MMLV购自Promega公司
8．引物制备：上海生工生物工程有限公司合成，经Genebank检索为特异性引物：
内参照β-actin引物序列为：
上游引物：5′-TCA CCC ACA CCG TGC CCA TCT ACG A-3′
下游引物：5′-CAG CGG AAC CGC TCA TTG CCA ACG G-3′
FAT10特异性引物序列为：
上游引物：5′-AAT GCT TCC TGC CTC TGT GT-3′
下游引物：5′-GCC GTA ATC TGC CAT CAT CT-3′
突变的p53特异性引物序列为：
上游引物：5’—CCTATGGAAACTACTTCCTGAAAACAA—3’
下游引物：5’—ACAGCATCAAATCATCCATTGC—3’
三、主要仪器和设备
产品名称生产或经销公司国籍
石蜡包埋机Leica公司德国
病理石蜡切片机Leica公司德国
万能显微镜成像系统(Leica DMR) Leica公司德国
光学显微镜照相系统Olympus日本
GeneAmp 2400 PCR System：Perkin Elmer公司美国
SCR-300A稳流稳压电泳仪上海康华生化仪器制造厂中国
电泳槽上海康华生化仪器制造厂中国
数码凝胶成像系统BIO-RAD GDEQ杭州宝诚生物技术有限公司中国
紫外分析仪Beckman DU640 Beckman公司美国
ZF-1紫外分析仪上海康华生化仪器制造厂中国
DL-8R冷冻离心机上海市离心机械研究所中国
XW-80A旋涡混合器上海医科大学仪器厂中国
震荡器XW-80A上海医科大学仪器厂中国
低温冰箱Forma Scientific Inc美国
四、检测指标及方法
1．FAT10、p53蛋白免疫组化(二步法)测定：
免疫组化主要操作步骤如下：
1)10％的福尔马林固定的癌组织、癌旁组织及正常胃组织，石蜡包埋，切片。
2)切片常规脱蜡。
3)过氧化氢封闭内源性过氧化物酶。
4)抗原修复。
5)滴加正常兔血清封闭液。
6)滴加兔抗人FAT10多克隆抗体及一抗为兔抗人p53单克隆抗体工作液，以PBS溶液代替一抗作为阴性对照片。
7)加50ul生物素标记的FAT10二抗及生物素化p53鼠抗兔二抗。
8)DAB显色。
9)苏木素复染。
10)梯度酒精脱水。
11)光学显微镜下观察判定免疫组化结果。
2．FAT10、p53-mRNA RT-PCR测定：
1)RNA提取及分析：
(1)RNA提取：采用TRIZOL试剂一步提取法。
(2)RNA分析：
①普通琼脂糖凝胶电泳分析。
②分光光度仪分析。
2)逆转录cDNA的合成及电泳验证逆转录产物cDNA。
cDNA模板进行PCR或保存于-20℃冰箱备用．
3)PCR扩增反应：
PCR扩增体系：
cDNA样品2μl
内参上游引物0．5μl
内参下游引物0．5μl
10×Buffer 2．5μl
MgCl_2 2μl
Taq酶0．15μl
2mMdNTPs 2μl
ddH_2O 15．35μl
总体积25μl
(1)内参照β-actin基因PCR扩增反应条件：
PCR扩增产物为295bp。
(2)FAT10基因的PCR扩增反应条件：
PCR扩增产物为478bp。
(3)突变p53基因的PCR扩增反应条件：
PCR扩增产物为：492bp。
4)产物电泳分析。
五、统计学处理
1．免疫组化实验所得定性数据采用x~2检验或分类资料的关联性检验判定两者关系。用Spearman秩相关分析两者之间的关系。FAT10与肿瘤临床病理特征的关系用阳性／阴性计数进行定性数据的x~2检验进行统计。生存率制作Kaplan-Meier生存曲线，进行Log-rank生存曲线检验。对本组病例治疗后影响预后的因素进行Cox模型多因素分析。
2．RT-PCR条带灰度值结果用均数±标准差((?)±s)表示，样本间均数的比较采用t检验，用Pearson相关分析分析突变p53与FAT10表达的相关性。
3．所有数据采用SPSS12．0统计软件包处理，P<0．05表示差异有显著性；P<0.01表示差异非常显著。
结果
一、免疫组织化学检测FAT10及p53蛋白在胃癌组织、癌旁组织及正常胃组织中的表达
1．FAT10免疫组织化学染色：
FAT10的表达主要定位于细胞核，阳性物质呈颗粒状。FAT10在胃癌组织中的阳性表达率显著高于相应的癌旁组织及正常胃组织，其阳性率分别为51．61％、12．90％及6．45％，差异均有非常显著性(P<0．01)，而癌旁组织与正常胃组织比较，FAT10的阳性表达率差异无显著性(P>0．05)。
2．p53免疫组织化学染色：
p53的表达主要定位于胞核，阳性物质呈颗粒状，可弥散到胞浆中。p53在胃癌组织中的表达显著高于相应的癌旁组织及正常胃组织，其阳性率分别为45．16％、14．51％及9．68％，差异均有非常显著性(P<0．01)，而癌旁组织与正常胃组织比较，p53的阳性表达率差异无显著性(P>0．05)。
3．FAT10蛋白表达与胃癌临床病理特征的关系：
胃癌组织中FAT10的表达主要与胃癌的淋巴转移与否及TNM分期(Ⅲ+Ⅳ/Ⅰ+Ⅱ)密切相关(P均<0．05)。而与胃癌患者年龄、性别、肿瘤大小、发生部位、进展程度、分化程度及远处转移无显著相关性(P均>0．05)。39例淋巴转移患者胃癌组织FAT10阳性表达者转移淋巴也呈FAT10阳性表达。
4．FAT10与p53蛋白在胃癌组织中表达的相关性分析：
p53阳性胃癌中FAT10表达率为82．14％(23／28)，而p53阴性胃癌FAT10表达率仅26．47(9／34)，用双向无序分类资料的关联性检验说明FAT10与p53表达存在一定的相关性。Spearman秩相关分析得出胃癌组FAT10蛋白表达和突变型p53蛋白表达存在显著正相关性。
二、RT-PCR检测FAT10及突变p53-mRNA在胃癌组织、癌旁组织及正常胃组织中的表达
1．胃癌组织、癌旁组织以及正常胃组织中FAT10-mRNA表达：
胃癌组织、癌旁组织及正常胃组织中FAT10-mRNA条带灰度值分别为0．689±0．023、0．463±0．019及0．451±0．028，采用配对t检验，发现胃癌组织FAT10-mRNA条带灰度值显著高于相应的癌旁组织及正常胃组织(t值分别为3．12及4．64，P均<0．01)，癌旁组织与正常胃组织两者比较无显著性差异(t值为1．03，P>0．05)。
2．胃癌组织、癌旁组织以及正常胃组织中突变p53-mRNA表达：
胃癌组织、癌旁组织以及正常胃组织中突变p53-mRNA条带灰度值分别为0．471±0．021、0．398±0．017及0．421±0．019，采用配对t检验，发现胃癌组织突变p53-mRNA条带灰度值显著高于相应的癌旁组织及正常胃组织(t值分别为6．79及5．51，P均<0．01)，癌旁组织与正常胃组织两者比较无显著性差异(t值为1．22，P>0．05)。
3．FAT10-mRNA的表达与胃癌临床病理特征的关系：
胃癌组织中FAT10-mRNA的表达主要与胃癌的淋巴转移与否及TNM分期(Ⅲ+Ⅳ／Ⅰ+Ⅱ)密切相关(P均<0．05)。而与胃癌患者年龄、性别、肿瘤大小、发生部位、进展程度、分化程度及远处转移无显著相关性(P均>0．05)。FAT10-mRNA的表达与胃癌临床病理特征的关系结果与免疫组织化学基本一致。
4．FAT10与p53基因在胃癌组织中表达的相关性分析：
Spearman等级相关分析，胃癌组中FAT10基因表达和突变型p53基因表达存在正相关性。用Pearson相关分析其相关性，说明FAT10与p53基因呈显著正相关。
三、胃癌FAT10蛋白及FAT10-mRNA的表达与生存率的关系：
除4例患者失访外，其余病例均获随访，随访时间48～72个月。对本组患者术后累积生存率进行单因素分析，均经Log-rank生存曲线检验。
根据Log-rank生存曲线检验，FAT10蛋白表达阳性胃癌患者生存时间显著低于表达阴性者，P<0．05。
根据Log-rank生存曲线检验，FAT10-mRNA的表达阳性胃癌患者生存时间显著低于表达阴性者，P<0．05。
经Cox模型多因素分析，结果显示FAT10蛋白及mRNA的表达与肿瘤淋巴结转移、远处转移、临床TNM分期、p53蛋白及p53 mRNA同为影响胃癌预后的独立因素。
结论
1．胃癌组织FAT10蛋白表达及FAT10-mRNA相对表达量显著高于相应的癌旁组织及正常胃组织。胃癌组织中FAT10蛋白及mRNA的表达主要与胃癌的淋巴转移与否及TNM分期密切相关。而与胃癌患者年龄、性别、肿瘤大小、发生部位、进展程度、分化程度及远处转移无显著相关性。FAT10-mRNA的表达与胃癌临床病理特征的关系结果与免疫组织化学基本一致。
2．胃癌组织中p53蛋白表达及p53-mRNA相对表达量明显高于相应的癌旁组织及正常胃组织，而癌旁组织与正常胃组织两者比较无显著性差异。
3．胃癌组织中FAT10基因的表达与突变p53基因的表达均增强，蛋白水平与基因水平检测的结果一致。两者增高在统计学上均有统计学意义，并呈现明显的正相关。
4．FAT10蛋白及FAT10-mRNA的表达阳性胃癌患者生存时间均显著低于表达阴性者。经Cox模型多因素分析，结果显示FAT10蛋白及mRNA的表达与肿瘤淋巴结转移、远处转移、临床TNM分期、p53蛋白及p53 mRNA同为影响胃癌预后的独立因素。
Background
FAT10 is an 18KD protein comprising 165 amino-acid residues. It wasoriginally discovered through the identification of expressed genes covering theHLA-F genomic locus. A potential role of FAT10 in antigen presentation wassuggested by its expression in mature B cells and dendritic cells, and by its ability tobe generally and synergistically inducible with cytokines IFN_γand TNFα. FAT10belongs to the UBL family of proteins and contains two ubiquitin-like moietiesfused in tandem. It is 29% identical to ubiquitin at its N-terminus and 36% identicalat the C-terminus. It has the C-terminal Gly-Gly residues and there is a comservedLys residue in each moiety of FAT10 analogous to Lys 48 of ubiquitin, which mayserve as a potential site for polyubiquitination of FAT10. FAT10 has been reported tohave potential involvement in cell-cycle regulation and shown to bind noncovalently to the human spindle assembly checkpoint protein, MAD2, a protein responsible formaintaining spindle integrity suring mitosis. The inhibition of MAD2 function hasbeen associated with chromosomal instability, a characteristic of many tumoursoccurrence. It was reported that FAT10 expression in the nucleus of HCChepatocytes rather than the surrounding immune or non-HCC cells. FAT10overexpression was also found in other cancers of the gastrointestinal tract andgynecology. FAT10 may modulate tumorigenesis through its reported interactionwith the MAD2 spindle-assembly checkpoint protein.
Recently, It is reported that p53 is likely an upstream regulator of FAT10 andmay link FAT10 in the tumorigenesis process. As FAT10 was found to interact withMAD2, an hypothesis would be that the repression of FAT10 expression by p53may facilitate the interaction of MAD2 with cell division cycle 20 (CDC20)to induce mitotic arrest. P53 gene mutation may lead to the enhancement of FAT10gene expression during tumorigenesis. These results were observed in severaldifferent cancers. FAT10 overexpression may result in the deregulation of mitosis,cause genome instability and tumorigenesis.
Gastric cancer is one of the most common malignant tumor and a leading causeof mortality world-wide. With the development and appliance of molecule biologicaltechnology, research in the mechanism of gastric cancer has probed into the level ofgene. But, it hadn't been identified the specific marker of molecule pathology ingastric cancer yet. For a tumor to become established, compromises in the control oftumor related gene are necessary. The Ubiquitin-like modifier (UBL) which has generated much interest in the scientific community is implicated to play importantrole in gastric cancer. Previous study was concerned mainly about the struction ofthe FAT10, the character of its expression, its inducible factor and the function.There are no reports about the relationship between the expression of FAT10 proteinand mRNA and the characters of clinical pathology and correlation betweenFAT10 and mutant p53 in gastric cancer.
Objective
To investigate the expression of FAT10 and mutant p53 gene in the gastriccancerous tissue, latero-GC tissue and the normal gastric mucosa tissue from thelevel of cellular and molecular biology. To analyse the relationship of expression inFAT10 protein and gene and clinical pathological features of gastric cancer and thecorrelation between the FAT10 and mutant p53 expression in gastric cancer tissue.To explore the effects of FAT10 in the production , invasion and metastasis processof gastric cancer.
Materials and Methods
Sixty-two samples which excisional specimen by operation confirmed asgastric cancer through pathological section in our hospital from March, 2003 to May,2004 were selected to study. The expression levels of FAT10 , FAT10-mRNA,mutant p53 and mutant p53-mRNA in the 62 specimens of gastric cancerous tissue,corresponding latero-GC tissue and normal mucosa tissue were determinedrespectively with the method of immunohistochemistry and RT-PCR. The featuresof expression of FAT10, FAT10-mRNA, mutant p53 and p53-mRNA in the sampleof gastric cancerous tissue, corresponding latero-GC tissue and normal mucosa tissue were analysed and the relationships between FAT10 overexpression with theinformation of age, sex, diameter of tumour, the location of tumor occurrence,invasion, differentiation, lymph nodes metastasis, distant metastasis and TNM stagesof the gastric cancer were observed. Kaplan-Meier survivial plot for patients withgastric cancer according to tumor expression of FAT10 and multivariate Coxproportional hazards model analysis of prognosis factors were studied.
Results
1、Immunohistochemistry
1) The rate of the FAT10's overexpression in the tissue of GC(51.61%) issiginificantly higher than that in latero-GC tissue and normal mucosa tissue(12.90%, 6.45%), the comparison between them:P<0.01.
2) The rate of the p53's positive expression in the tissue of GC(45.16%) issiginificantly higher than that in latero-GC tissue and normal mucosa tissue(14.51%,9.68%), the comparision between them:P<0.01. There are nosiginificant differences of the rate of the FAT10's and p53's positiveexpression between tissue in latero-GC and normal mucosa samples.
3) The rate of FAT10's overexpression in the tissue of GC with the lymphnodes metastasis (64.10%) is siginificantly higher than that without nodesmetastasis (30.43%), the comparision between them :P<0.05. And the rateof FAT10's overexpression in the TNM stageⅢ+Ⅳ(60.98%) issiginificantly higher than the TNM stageⅠ+Ⅱof GC (33.30%), thecomparision between them :P<0.05. Comparision the age,sex,diameter of tumour, the location of tumor occurrence,invasion,differentiation and distantmetastasis of the GC is no significant differences in the rate of FAT10'soverexpression.
2、RT-PCR analysis
4) The level of FAT10-mRNA expression in GC tissue is significantly higherthan that of latero-GC tissue and normal mucosa tissue (0.689±0.023,0.463±0.019 and 0.451±0.028), the comparision between them:P<0.01.
5) The level of mutant p53-mRNA expression in GC tissue is significantlyhigher than that latero-GC tissue and normal mucosa tissue (0.471±0.021, 0.398±0.017 and 0.421±0.019), the comparision betweenthem:P<0.01.
6) The level of FAT10-mRNA overexpression in the tissue of GC with thelymph nodes metastasis (0.656±0.016) is siginificantly higher than thatwithout nodes metastasis (0.531±0.026), the comparision betweenthem :P<0.01. And the level of FAT10's overexpression in the TNM stageⅢ+Ⅳ(0.667±0.023) is siginificantly higher than the TNM stageⅠ+ⅡGC(0.558±0.015), the comparision between them :P<0.05. Comparisionthe age, sex,diameter of tumour, the location of tumor occurrence,invasion,differentiation and distant metastasis of the GC is no significant differencesin the level of FAT10-mRNA overexpression.
3、Correlation Analysis
1) Immunohistochemistry shows: The rate of the positive expression of FAT10in the p53 positive samples is 82.14%(23/28). And 26.47%(9/34) of FAT10is overexpression in p53 negative expression group. Spearman orderprobabilities shows significant positive correlation with FAT10 and mutantp53(r=0.865, P<0.05).
2) RT-PCR shows: Pearson correlation shows significant positive correlationwith mutant p53 and FAT10(r=0.761, P<0.01).
4、Surviving rate Analysis shows: both overexpression of FAT10 protein and mRNAhave significant correlation with the life span of gastric cancer patients (P<0.05).Multivariate Cox proportional hazards model analysis of prognosis factorsshows:FAT10 protein and mRNA overexpression,lymph node and distantmetastases, TNM stage and p53 protein and mRNA overexpression are allsignificant in the multivariate analysis.
Conclusions
1. FAT10 overexpression plays important role in the process of the production,growth of gastric cancer. The level of FAT10's expression is related with thelymph nodes metastasis and TNM stages of the gastric cancer and isn't obviouslyrelated with the age,sex,diameter of tumour, the location of tumoroccurrence,invasion,differentiation and distant metastasis of the gastric cancer.
2. Mutant p53 overexpression plays important role in the process of the production,growth of gastric cancer.
3. FAT10 and mutant p53 are tightly related in the expression of gastric cancer.Mutant p53 intervenes the expression of FAT10. We suppose it's one pathwaysthat mutant p53 gene arouses FAT10 gene overexpression and accelerates thetumor's growth.
4. Both overexpression of FAT10 protein and mRNA have significant correlationwith the life span of gastric cancer patients and may be one of the most reliableindependent prognostic predictors.
引文
[1] Fan W, Cai W, Parimoo S, et al. Identification of seven new haman MHC class I region genes around the HLA-F locus[J]. Immunogenetics, 1996, 44(2): 97-103.
[2] Bates EE, Ravel O, Dieu MC, et al.Identification and analysis of a novel member of the ubiquitin family expressed in dendritic cells and mature B cells. Eur J Immunol 1997,27:2471-2477.
[3] Jentsch S, Pyrowolakis G .Ubiquitin and its kin: how close are the family ties?Trends Cell Biol 2000,10:335-342.
[4] Yuan - Ching Liu, Julina Pan, Chunyu Zhang, et al. A MHC - encoded ubiquitin - like protein(FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2. Biochemistry, 1999,96:4313-4318.
[5] Shahri Raasi, Gunter Schmidtke, Marcus Groettrup. The Ubiquitin - like protein FAT10 Forms covalent conjugates and induces apoptosis. The Journal of Biological chemistry, 2001,276(38):35334-35343.
[6] Lee CG, Ren J, Cheong IS, et al. Expression of the FAT10 gene is highly upregulated in hepatocellular carcinoma and other gastrointestinal and gynecological cancers.Oncogene 2003,22:2592-2603.
[7] El-Deiry W S. Regulation of p53 downstream genes. Seminars in Cancer Biology, 1998,8:345-357.
[8] Tokino T,Nakamura Y.The role of p53-target genes in human cancer.Crit Revoncol Hematol,2000,33:1-6.
[9] Munna LA,William RT,Michail VC,et al.The p53 network[J].JBiochem,1998,278(1):1-4.
[10] 陈慧娟，饶周舟，陈汉春．p53对胃癌发生的影响．生命科学研究，2007，11(4)：35-38．
[11] Taguchi A,Ohmiya N,Itoh A,et al.Severity of atrophicgastritis related toantiparietal cell antibody and gastric carcinogenesis,including p53mutations[J].J Gastroenterol Hepatol,2006,21(3):545-551.
[12] Rugge M,Shiao Y H,Busatto G,et al.The p53 gene in patients under the ageof 40 with gastric cancer:mutationrates are low but are associated with acardiac location[J].Mol Pathol,2000,53(4):207-210.
[13] Shibata A,Parsonnet J,Longacre T A,et al.CagA status of Helicobacterpylori infection and p53 gene mutationsin gastric adenocarcinoma[J].Carcinogenesis,2002,13(3):419-424.
[14] Zhang DW,Jeang KT,Lee CG:p53 negatively regulates the expression ofFAT10,a gene upregulated in various cancers.Oncogene 2006,25:2318-2327.
[15] 孙秀娣，牧人，周有尚等．中国胃癌死亡率20年变化情况分析及其发展趋势预测．中华肿瘤杂志，2004，26(1)：4-9．
[16] Raasi S,G Schmidtke,R de Giuli,et al.A ubiquitin-like protein which issynergistically inducible by interferon-gamma and tumor necrosis factor-alpha. Eur. J. Immunol, 1999.29:4030-4036.
[17] Hipp MS, Kalveram B, Raasi S, et al. FAT10, a ubiquitin-independent signal for proteasomal degradation.Mol Cell Biol, 2005,25:3483-3491.
[18] Hipp MS, Raasi S, Groettrup M, et al. NEDD8 ultimate buster-1L interacts with the ubiquitin-like protein FAT10 and accelerates its degradation. J Biol Chem, 2004,279:16503-16510.
[19] Schmidtke G, Kalveram B, Weber E, et al. The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10. J Biol Chem, 2006,281:20045-20054.
[20] Santos Rosa; Caldas C.Chromatin modifier enzymes, the histone code and cancer, Europ. J. Cancer, 2005,41:2381-2402.
[21] Roomi MW; Gaal K; Yuan QX; French BA; Fu P; Bardag-Gorce F; French SW. Preneoplastic liver cell foci expansion induced by throacetamide toxicity in drug-primed mice, Exp. Mol. Pathol, 2006,81:8-14.
[22] Dedes, Jennifer; French, Barbara A; Oliva, Joan V; Li, Jun; French, Samuel W.Mallory body formation is associated with epigenetic phenotypic change in hepatocytes in vivo. Exp Mol Pathol, 2007,83(2): 160-8.
[23] Canaan,A. Canaan, X. Yu, C.J. Booth, et al. FAT10/diubiquitin-like protein-deficient mice exhibit minimal phenotypic differences. Mol Cell Biol, 2006, 26(13): 5180-5189.
[24] Joan Oliva, Fawzia Bardag-Gorce, Barbara A. French, et al.FatlO is an
epigenetic marker for liver preneoplasia in a drug-primed mouse model oftumorigenesis.Experimental and Molecular Pathology,2008,84(2):102-12.
[25] Tateishi K,Omata M,Tanaka K,et al.The NEDD8 system is essential for cellcycle progression and morphogenetic pathway in mice.J Cell Biol,2001,155(4):571-579.
[26] Kasai T,Kibler K,Jeang,Kuan-Teh,et al.Characterization of regions inhsMAD1 needed for binding hsMAD2.A polymorphic change in an hsMAD1leucine zipper affects MAD1-MAD2 interaction and spindle checkpointfunction.J Biol Chem,2002,277(34):31005-31013.
[27] Osima D,Kusima A.Detection p53 mutation in the plasma a DNA of patientswith ovarian cancer[J].International Journal of Gynecological Cancer,2004,14(3):459-464.
[28] Carazzola,Leandro Totti,da Rosa.Immmunohistochemical evaluation for p53and vascular endothelial growth factor(VEGF) is not prognostic for longtermsurvival in and stage esophageal adenocarcinoma[J].Disease of theEsophagus.2004,1(12):50-54.
[29] Allon C,Xiaofeng Y,Carmen JB,et al.FAT10/Diubiquitin-Like ProteinDeficientMice Exhibit Minimal Phenotypic Differences.Mol Cell Biol.2006,26(13):5180-5189.
[1] Fan W, Cai W, Parimoo S, et al. Identification of seven new haman MHC class I region genes around the HLA-F locus. Immunogenetics, 1996, 44(2): 97-103
[2] Bates EE, Ravel O, Dieu MC, Ho S, Guret C, et al. Identification and analysis of a novel member of the ubiquitin family expressed in dendritic cells and mature B cells. Eur J Immunol, 1997, 27:2471-2477.
[3] Jentsch S, Pyrowolakis G. U. Biquitin and its kin: how close are the family ties?Trends Cell Biol, 2000,10:335-342.
[4] Raasi, S, G. Schmidtke, R. de Giuli, and M. Groettrup. A ubiquitin-like protein which is synergistically inducible by interferon-gamma and tumor necrosis factor-alpha. Eur. J. Immunol, 1999,29: 4030-4036.
[5] Hipp MS, Kalveram B, Raasi S, Groettrup M, et al. FAT10, a ubiquitin-independent signal for proteasomal degradation. Mol Cell Biol ,2005, 25:3483-3491.
[6] Hipp MS, Raasi S, Groettrup M, Schmidtke G .NEDD8 ultimate buster-1L interacts with the ubiquitin-like protein FAT10 and accelerates its degradation. J Biol Chem, 2004, 279:16503-16510.
[7] Schmidtke G, Kalveram B, Weber E, et al. The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10. J Biol Chem ,2006,281:20045- 20054.
[8] Hanahan D, Weinberg RA .The hallmarks of cancer.Cell ,2000, 100:57-70.
[9] Momand J, Zambetti GP, Olson DC, et al. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell, 1992, 69:1237-1245.
[10] Zhang DW, Jeang KT, Lee CG . p53 negatively regulates the expression of FAT10, a gene upregulated in various cancers. Oncogene , 2006, 25:2318-2327.
[11] Jian Y, Lin Z, Paul M, et al. Identification and classification of p53-regulated genes. PNAS, 1999,96(25):14517-14522.
[12] El-Deiry W S. Regulation of p53 downstream genes. Seminars in Cancer Biology, 1998, 8: 345357.
[13] Tokino T, Nakamura Y. The role of p53-target genes in human cancer .Crit Rev oncol Hematol, 2000, 33:1-6.
[14] P Sacca, F Caballero, A Batlie, E Vazquez. Cell cycle arrest and modulation of HO-1 expression induced by acetyl salicylic acid in hepatocarcinogenesis. Int J Biochem Cell Biol, 2004, 36:1945-53.
[15] Xu H, Raafat el-Gewely M. P53-responsive genes and the potential for cancer diagnostics and therapeutics development. Biotechnol Annu Rev, 2001,7:131-164.
[16] Munna LA, William RT, Michail VC, et al. The p53 network. J Biochem, 1998, 278(1): 1-4.
[17] BertV, DavidL, Arnold JL. Surfing the p53 network. Nature, 2000, 408 (16): 307-310.
[18] Liu YC, Pan J, Zhang C, et al.A MHC-encoded ubiquitin-like protein (FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2. Proc Natl Acad Sci U S A , 1999, 96:4313-4318.
[19] Chuan-Bian Lim, Dongwei Zhang, Caroline GL Lee. FAT10, a gene up-regulated in various cancers, is cell-cycle regulated. Cell Division, 2006, 1:20.
[20] Ren J, Kan A, Leong SH, et al. Fat10 plays a role in the regulation of chromosomal stability. J Biol Chem ,2006, 281:11413 -111421.
[21] Li, Y., and Benezra, R. Identification of a Human Mitotic Checkpoint Gene: hsMAD2 .Science ,1996,274:246-248.
[22] Yu, H. Regulation of APC-Cdc20 by the spindle checkpoint. Curr. Opin. Cell Biol,2002,14:706-714.
[23] Shah, J. V., and Cleveland, D. W. Waiting for anaphase: Mad2 and the spindle assembly checkpoint. Cell ,2000,103: 997-1000.
[24] Musacchio, A., Hardwick, K. G. The spindle checkpoint: structural insights into dynamic signalling. Cell. Biol, 2002,3:731-741.
[25] Michel, L. S., Liberal, V., Chatterjee, A., et al. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature, 2001, 409:355-359.
[26] Loren S. Miche , Vasco Liberal , Anupam Chatterjee. MAD2 haplo- insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature, 2001,409:355-359.
[27] Eva Hernando, Zaher Nahle, Gloria Juan, Elena Diaz-Rodriguez. Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature, 2004,430:797-802.
[28] 28. Li GQ, Li H, Zhang HF.Mad2 and p53 expression profiles in colorectal cancer and its clinical significance. World J Gastroenterol, 2003,9(9): 1972-5.
[29] Kouji Tanaka, Junji Nishioka, Keiko Kato. Mitotic Checkpoint Protein hsMAD2 as a Marker Predicting Liver Metastasis of Human Gastric Cancers. Cancer Science, 2001,92 (9):952 - 958.
[30] Harith Rajagopalan, Martin A. Nowak, Bert Vogelstein, Christoph Lengauer. The significance of unstable chromosomes in colorectal cancer. Nature Reviews Cancer, 2003, 3(9):695-701.
[31] 31. N. Nishida, T. Nishimura, T. Ito, T. Komeda, et al. Chromosomal instability and human hepatocarcinogenesis. Histol Histopathol, 2003,18 (3): 897-909.
[32] 32.Habu, T., Kim, S. H., Weinstein, et al. Identification of a MAD2-binding protein, CMT2, and its role in mitosis. EMBO J, 2002, 21:6419-6428.
[33] 33. Nowak, M. A., Komarova, N. L., Sengupta, A., et al. The role of chromosomal instability in tumor initiation. Proc. Natl. Acad. Sci. U. S. A, 2002,99:16226-16231.
[34] 34. Lengauer, C., Kinzler, K. W., Vogelstein, B. Genetic instabilities in human cancers. Nature, 1998, 396:643-649.
[35] 35. Dyczkowski J, Vingron M .Comparative analysis of cell cycle regulated genes in eukaryotes. Genome Inform Ser Workshop Genome Inform ,2005, 16:125-131.
[36] 36. Zwicker J, Muller R .Cell cycle-regulated transcription in mammalian cells. Prog Cell Cycle Res ,1995, 1:91-99.
[37] 37. Santos-Rosa and Caldasa, H. Santos-Rosa and C. Caldasa. Chromatin modifier enzymes, the histone code and cancer. Eur J Cancer, 2005, 41(16):2381-402.
[38] 38.M.W. Roomi, K. Gaal, Q.X. Yuan, et al. Preneopl. astic liver foci expansion induced by throacetamide toxicity in drug-primed mice.Exp. Mol. Pathol, 2006, 81: 8-14.
[39] 39. Bardag-Gorce F, Dedes J, French BA, Oliva JV, Li J, French SW. French. Mailory body formation is associated with epigenetic phenotypic change in hepatocytes in vivo. Exp Mol Pathol, 2007, 83(2): 160-8.
[40] 40. A. Canaan, X. Yu, C.J. Booth, et al. FAT10/diubiquitin-like protein-deficient mice exhibit minimal phenotypic differences. Mol Cell Biol, 2006, 26(13): 5180-5189.
[41] 41. Joan Oliva, Fawzia Bardag-Gorce, Barbara A. French, et al.Fat10 is an epigenetic marker for liver preneoplasia in a drug-primed mouse model of tumorigenesis. Experimental and Molecular Pathology, 2008, 84(2):102-12.
[42] 42. Lee CG, Ren J, Cheong IS, Ban KH, et al. Expression of the FAT10 gene is highly upregulated in hepatocellular carcinoma and other gastrointestinal and gynecological cancers. Oncogene , 2003, 22:2592- 2603.
[2] Bates EE, Ravel O, Dieu MC, et al.Identification and analysis of a novel member of the ubiquitin family expressed in dendritic cells and mature B cells. Eur J Immunol 1997,27:2471-2477.
[3] Jentsch S, Pyrowolakis G .Ubiquitin and its kin: how close are the family ties?Trends Cell Biol 2000,10:335-342.
[4] Yuan - Ching Liu, Julina Pan, Chunyu Zhang, et al. A MHC - encoded ubiquitin - like protein(FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2. Biochemistry, 1999,96:4313-4318.
[5] Shahri Raasi, Gunter Schmidtke, Marcus Groettrup. The Ubiquitin - like protein FAT10 Forms covalent conjugates and induces apoptosis. The Journal of Biological chemistry, 2001,276(38):35334-35343.
[6] Lee CG, Ren J, Cheong IS, et al. Expression of the FAT10 gene is highly upregulated in hepatocellular carcinoma and other gastrointestinal and gynecological cancers.Oncogene 2003,22:2592-2603.
[7] El-Deiry W S. Regulation of p53 downstream genes. Seminars in Cancer Biology, 1998,8:345-357.
[8] Tokino T,Nakamura Y.The role of p53-target genes in human cancer.Crit Revoncol Hematol,2000,33:1-6.
[9] Munna LA,William RT,Michail VC,et al.The p53 network[J].JBiochem,1998,278(1):1-4.
[10] 陈慧娟，饶周舟，陈汉春．p53对胃癌发生的影响．生命科学研究，2007，11(4)：35-38．
[11] Taguchi A,Ohmiya N,Itoh A,et al.Severity of atrophicgastritis related toantiparietal cell antibody and gastric carcinogenesis,including p53mutations[J].J Gastroenterol Hepatol,2006,21(3):545-551.
[12] Rugge M,Shiao Y H,Busatto G,et al.The p53 gene in patients under the ageof 40 with gastric cancer:mutationrates are low but are associated with acardiac location[J].Mol Pathol,2000,53(4):207-210.
[13] Shibata A,Parsonnet J,Longacre T A,et al.CagA status of Helicobacterpylori infection and p53 gene mutationsin gastric adenocarcinoma[J].Carcinogenesis,2002,13(3):419-424.
[14] Zhang DW,Jeang KT,Lee CG:p53 negatively regulates the expression ofFAT10,a gene upregulated in various cancers.Oncogene 2006,25:2318-2327.
[15] 孙秀娣，牧人，周有尚等．中国胃癌死亡率20年变化情况分析及其发展趋势预测．中华肿瘤杂志，2004，26(1)：4-9．
[16] Raasi S,G Schmidtke,R de Giuli,et al.A ubiquitin-like protein which issynergistically inducible by interferon-gamma and tumor necrosis factor-alpha. Eur. J. Immunol, 1999.29:4030-4036.
[17] Hipp MS, Kalveram B, Raasi S, et al. FAT10, a ubiquitin-independent signal for proteasomal degradation.Mol Cell Biol, 2005,25:3483-3491.
[18] Hipp MS, Raasi S, Groettrup M, et al. NEDD8 ultimate buster-1L interacts with the ubiquitin-like protein FAT10 and accelerates its degradation. J Biol Chem, 2004,279:16503-16510.
[19] Schmidtke G, Kalveram B, Weber E, et al. The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10. J Biol Chem, 2006,281:20045-20054.
[20] Santos Rosa; Caldas C.Chromatin modifier enzymes, the histone code and cancer, Europ. J. Cancer, 2005,41:2381-2402.
[21] Roomi MW; Gaal K; Yuan QX; French BA; Fu P; Bardag-Gorce F; French SW. Preneoplastic liver cell foci expansion induced by throacetamide toxicity in drug-primed mice, Exp. Mol. Pathol, 2006,81:8-14.
[22] Dedes, Jennifer; French, Barbara A; Oliva, Joan V; Li, Jun; French, Samuel W.Mallory body formation is associated with epigenetic phenotypic change in hepatocytes in vivo. Exp Mol Pathol, 2007,83(2): 160-8.
[23] Canaan,A. Canaan, X. Yu, C.J. Booth, et al. FAT10/diubiquitin-like protein-deficient mice exhibit minimal phenotypic differences. Mol Cell Biol, 2006, 26(13): 5180-5189.
[24] Joan Oliva, Fawzia Bardag-Gorce, Barbara A. French, et al.FatlO is an
epigenetic marker for liver preneoplasia in a drug-primed mouse model oftumorigenesis.Experimental and Molecular Pathology,2008,84(2):102-12.
[25] Tateishi K,Omata M,Tanaka K,et al.The NEDD8 system is essential for cellcycle progression and morphogenetic pathway in mice.J Cell Biol,2001,155(4):571-579.
[26] Kasai T,Kibler K,Jeang,Kuan-Teh,et al.Characterization of regions inhsMAD1 needed for binding hsMAD2.A polymorphic change in an hsMAD1leucine zipper affects MAD1-MAD2 interaction and spindle checkpointfunction.J Biol Chem,2002,277(34):31005-31013.
[27] Osima D,Kusima A.Detection p53 mutation in the plasma a DNA of patientswith ovarian cancer[J].International Journal of Gynecological Cancer,2004,14(3):459-464.
[28] Carazzola,Leandro Totti,da Rosa.Immmunohistochemical evaluation for p53and vascular endothelial growth factor(VEGF) is not prognostic for longtermsurvival in and stage esophageal adenocarcinoma[J].Disease of theEsophagus.2004,1(12):50-54.
[29] Allon C,Xiaofeng Y,Carmen JB,et al.FAT10/Diubiquitin-Like ProteinDeficientMice Exhibit Minimal Phenotypic Differences.Mol Cell Biol.2006,26(13):5180-5189.
[1] Fan W, Cai W, Parimoo S, et al. Identification of seven new haman MHC class I region genes around the HLA-F locus. Immunogenetics, 1996, 44(2): 97-103
[2] Bates EE, Ravel O, Dieu MC, Ho S, Guret C, et al. Identification and analysis of a novel member of the ubiquitin family expressed in dendritic cells and mature B cells. Eur J Immunol, 1997, 27:2471-2477.
[3] Jentsch S, Pyrowolakis G. U. Biquitin and its kin: how close are the family ties?Trends Cell Biol, 2000,10:335-342.
[4] Raasi, S, G. Schmidtke, R. de Giuli, and M. Groettrup. A ubiquitin-like protein which is synergistically inducible by interferon-gamma and tumor necrosis factor-alpha. Eur. J. Immunol, 1999,29: 4030-4036.
[5] Hipp MS, Kalveram B, Raasi S, Groettrup M, et al. FAT10, a ubiquitin-independent signal for proteasomal degradation. Mol Cell Biol ,2005, 25:3483-3491.
[6] Hipp MS, Raasi S, Groettrup M, Schmidtke G .NEDD8 ultimate buster-1L interacts with the ubiquitin-like protein FAT10 and accelerates its degradation. J Biol Chem, 2004, 279:16503-16510.
[7] Schmidtke G, Kalveram B, Weber E, et al. The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10. J Biol Chem ,2006,281:20045- 20054.
[8] Hanahan D, Weinberg RA .The hallmarks of cancer.Cell ,2000, 100:57-70.
[9] Momand J, Zambetti GP, Olson DC, et al. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell, 1992, 69:1237-1245.
[10] Zhang DW, Jeang KT, Lee CG . p53 negatively regulates the expression of FAT10, a gene upregulated in various cancers. Oncogene , 2006, 25:2318-2327.
[11] Jian Y, Lin Z, Paul M, et al. Identification and classification of p53-regulated genes. PNAS, 1999,96(25):14517-14522.
[12] El-Deiry W S. Regulation of p53 downstream genes. Seminars in Cancer Biology, 1998, 8: 345357.
[13] Tokino T, Nakamura Y. The role of p53-target genes in human cancer .Crit Rev oncol Hematol, 2000, 33:1-6.
[14] P Sacca, F Caballero, A Batlie, E Vazquez. Cell cycle arrest and modulation of HO-1 expression induced by acetyl salicylic acid in hepatocarcinogenesis. Int J Biochem Cell Biol, 2004, 36:1945-53.
[15] Xu H, Raafat el-Gewely M. P53-responsive genes and the potential for cancer diagnostics and therapeutics development. Biotechnol Annu Rev, 2001,7:131-164.
[16] Munna LA, William RT, Michail VC, et al. The p53 network. J Biochem, 1998, 278(1): 1-4.
[17] BertV, DavidL, Arnold JL. Surfing the p53 network. Nature, 2000, 408 (16): 307-310.
[18] Liu YC, Pan J, Zhang C, et al.A MHC-encoded ubiquitin-like protein (FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2. Proc Natl Acad Sci U S A , 1999, 96:4313-4318.
[19] Chuan-Bian Lim, Dongwei Zhang, Caroline GL Lee. FAT10, a gene up-regulated in various cancers, is cell-cycle regulated. Cell Division, 2006, 1:20.
[20] Ren J, Kan A, Leong SH, et al. Fat10 plays a role in the regulation of chromosomal stability. J Biol Chem ,2006, 281:11413 -111421.
[21] Li, Y., and Benezra, R. Identification of a Human Mitotic Checkpoint Gene: hsMAD2 .Science ,1996,274:246-248.
[22] Yu, H. Regulation of APC-Cdc20 by the spindle checkpoint. Curr. Opin. Cell Biol,2002,14:706-714.
[23] Shah, J. V., and Cleveland, D. W. Waiting for anaphase: Mad2 and the spindle assembly checkpoint. Cell ,2000,103: 997-1000.
[24] Musacchio, A., Hardwick, K. G. The spindle checkpoint: structural insights into dynamic signalling. Cell. Biol, 2002,3:731-741.
[25] Michel, L. S., Liberal, V., Chatterjee, A., et al. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature, 2001, 409:355-359.
[26] Loren S. Miche , Vasco Liberal , Anupam Chatterjee. MAD2 haplo- insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature, 2001,409:355-359.
[27] Eva Hernando, Zaher Nahle, Gloria Juan, Elena Diaz-Rodriguez. Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature, 2004,430:797-802.
[28] 28. Li GQ, Li H, Zhang HF.Mad2 and p53 expression profiles in colorectal cancer and its clinical significance. World J Gastroenterol, 2003,9(9): 1972-5.
[29] Kouji Tanaka, Junji Nishioka, Keiko Kato. Mitotic Checkpoint Protein hsMAD2 as a Marker Predicting Liver Metastasis of Human Gastric Cancers. Cancer Science, 2001,92 (9):952 - 958.
[30] Harith Rajagopalan, Martin A. Nowak, Bert Vogelstein, Christoph Lengauer. The significance of unstable chromosomes in colorectal cancer. Nature Reviews Cancer, 2003, 3(9):695-701.
[31] 31. N. Nishida, T. Nishimura, T. Ito, T. Komeda, et al. Chromosomal instability and human hepatocarcinogenesis. Histol Histopathol, 2003,18 (3): 897-909.
[32] 32.Habu, T., Kim, S. H., Weinstein, et al. Identification of a MAD2-binding protein, CMT2, and its role in mitosis. EMBO J, 2002, 21:6419-6428.
[33] 33. Nowak, M. A., Komarova, N. L., Sengupta, A., et al. The role of chromosomal instability in tumor initiation. Proc. Natl. Acad. Sci. U. S. A, 2002,99:16226-16231.
[34] 34. Lengauer, C., Kinzler, K. W., Vogelstein, B. Genetic instabilities in human cancers. Nature, 1998, 396:643-649.
[35] 35. Dyczkowski J, Vingron M .Comparative analysis of cell cycle regulated genes in eukaryotes. Genome Inform Ser Workshop Genome Inform ,2005, 16:125-131.
[36] 36. Zwicker J, Muller R .Cell cycle-regulated transcription in mammalian cells. Prog Cell Cycle Res ,1995, 1:91-99.
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