鼻咽癌组织中巨噬细胞移动抑制因子和MMP-2、MMP-9表达的关系

目的　研究巨噬细胞移动抑制因子 (macrophagemigrationinhibitoryfactor,MIF)和MMP 2、MMP 9在鼻咽癌组织中的表达水平及相互关系 ,探讨鼻咽癌细胞早期侵袭转移的机制。方法　收集 4 5例确诊的鼻咽原发癌活检组织标本 ,采用免疫组化LSAB法检测鼻咽癌组织中MIF和MMP 2、MMP 9的表达 ,并分析患者的临床参数的关系。结果　在 4 5例鼻咽原发癌组织中 ,MIF、MMP 2和MMP 9的阳性表达率分别为 77 8% (35 / 4 5 )、6 4 4 % (2 9/ 4 5 )和 71 1% (32 / 4 5 )。其中 ,癌细胞MIF和MMP 9的表达水平均显示与淋巴结转移有关 ,伴有淋巴结转移的癌组织中二者表达水平均高于无淋巴结转移的癌组织 (P值均 <0 0 5 )。MIF阳性组的癌细胞MMP 9的表达 (5 0 2 %± 33 5 % )明显高于MIF阴性病例 (11 7%± 2 2 7% ) ,两者差异有显著性 (P <0 0 1) ,且MIF的表达与MMP 9的表达亦呈正相关 (rs=0 .4 92 ,P <0 0 1) ,但癌细胞MMP 2的表达与MIF、MMP 9的表达以及是否有淋巴结转移则均未显示相关性。以Schmincke型生长方式分布的癌细胞MIF表达水平 (6 7 4 %±35 2 % )也高于以Regaud型方式分布的癌细胞 (32 9%± 2 9 7% ) ,差异有显著性 (P <0 0 1)。结论　鼻咽癌组织中癌细胞的MIF和MMP 9同步过表达 ,可能在鼻咽癌细胞的转移     

肝细胞生长因子/c-Met系统在鼻咽癌中的表达及意义

目的　探讨肝细胞生长因子(HGF)及其受体c- Met蛋白在鼻咽癌组织中的表达水平,研究HGF/c- Met系统对鼻咽癌细胞侵袭转移的影响。方法　收集1999—2003年期间45例确诊的鼻咽原发癌活检组织标本,采用免疫组织化学(LSAB)法检测鼻咽癌组织中HGF α亚单位和c- Met的表达,并与患者的病理及临床资料相联系。采用流式细胞术检测鼻咽癌细胞株CNE- 2在HGF刺激前后c- Met阳性癌细胞百分率的改变;蛋白印迹法和逆转录PCR法分别用于检测癌细胞株c -Met蛋白表达和mRNA表达的变化。结果　在45例鼻咽原发癌组织中,癌细胞c- Met的阳性表达率为91. 1% (41 /45),但仅有1例鼻咽癌细胞表达HGF( 2 .2%, 1 /45 )。HGF主要在鼻咽癌间质中的淋巴细胞表达。癌细胞c Met的表达水平与淋巴结转移有关(P=0 .024 ),且与淋巴细胞表达HGF呈正相关(rs=0 .450,P=0 .002)。癌细胞c Met表达量在间质淋巴细胞高表达HGF的病例中明显高于淋巴细胞低表达HGF的癌组织(P=0 .009)。但癌细胞c Met的表达与患者性别、年龄、病理组织学分型以及临床分期均未显示相关性。鼻咽癌细胞株CNE 2在HGF诱导24h后,c Met阳性的癌细胞比例即有明显增加,由(46 .6±9 .02)%增加至(85. 8±6. 05)% (P=0 .003 ),癌细胞c Met蛋白表达相对强度和mRNA表达水平均有显著提高,     

鼻咽癌淋巴结转移组织中EB病毒BamH Ⅰ"f"变异分析

目的 探讨高发区原发鼻咽癌及相应的淋巴结转移癌组织中EB病毒BamH Ⅰ"f"变异情况.方法 原位杂交法检测了21对鼻咽原发癌及其相应的淋巴结转移癌组织、22例未伴淋巴结转移的鼻咽癌组织中EB病毒EBER的表达,进而采用PCR和限制性酶切分析技术检测了所有鼻咽癌病例及相应的淋巴结转移癌组织和50例鼻咽黏膜慢性炎组织中EB病毒BamH Ⅰ"f"变异情况.结果 所有鼻咽癌和相应的淋巴结转移癌组织均呈EBER阳性表达.21对鼻咽原发癌及其相应的淋巴结转移癌组织、22例未伴淋巴结转移的鼻咽癌组织和50例鼻咽黏膜慢性炎组织中EB病毒BamH Ⅰ"f"基因变异率分别为52.4%(11/21)、57.1%(12/21)、81.8%(18/22)和2.1%(1/47).统计学分析显示,鼻咽癌组织"f"变异率明显高于鼻咽黏膜慢性炎组织的变异率;伴淋巴结转移的鼻咽癌组织中"f"变异率并不比未伴淋巴结转移的鼻咽癌组织的变异率高.另外,表现为"f"变异的1例鼻咽黏膜慢性炎呈"F/f"型,且其组织学可见部分鼻咽上皮出现轻度的不典型增生.结论 高发区鼻咽癌组织中EB病毒BamH Ⅰ "f"变异率远高于同一地区鼻咽黏膜慢性炎组织中的变异率;鼻咽癌淋巴结转移组织中也有较高的BamH Ⅰ"f"变异率,且与其原发癌组织基本一致.     

上皮钙黏素/链接素黏附复合体在鼻咽癌组织中的表达及意义

目的探讨鼻咽癌组织中上皮钙黏素(E-CAD)、β-链接素(β-CAT)和α-链接素(α-CAT)的表达情况及与临床病理特征的关系。方法应用免疫组化SP法检测52例鼻咽非角化癌及其癌旁黏膜组织中E-CAD、β-CAT和α-CAT的表达。结果鼻咽癌组织中E-CAD、β-CAT及α-CAT的异常表达率分别为48.1%、32.7%及34.6%,而全部52例癌旁黏膜上皮中均正常表达E-CAD、β-CAT和α-CAT,癌组织的异常表达率显著高于癌旁组织(P<0.05)。鼻咽癌的E-CAD、β-CAT、α-CAT表达之间呈正相关(P<0.05)。α-CAT正常表达的鼻咽癌患者外周血中抗EB病毒病毒壳抗原IgA(VCA-IgA)滴度高于异常表达者(P<0.01)。E-CAD、β-CAT、α-CAT的表达与其他临床病理特征无相关性。结论鼻咽非角化癌中E-CAD/β-CAT/α-CAT黏附复合体明显异常表达,三者表达具有一致性。E-CAD、β-CAT、α-CAT表达下调与鼻咽癌临床分期和淋巴结转移缺乏必然联系。     

LMP1调节鼻咽上皮与鼻咽癌组织中p27与RPLP0蛋白的表达

目的: 观察p27和核糖体磷酸化蛋白大亚基P0(RPLP0)蛋白在潜伏膜蛋白1(LMP1)阳性鼻咽癌细胞中的表达,揭示蛋白质之间的相互作用规律及临床病理学意义。方法: 采用Western blotting分析p27和RPLP0蛋白表达与LMP1表达之间的关系,S-P免疫组织化学染色检测LMP1、p27和RPLP0蛋白在30例鼻咽上皮组织和60例鼻咽低分化鳞状细胞癌中表达,并分析其临床病理学意义。结果: (1)LMP1在鼻咽上皮和鼻咽低分化鳞状细胞癌中的阳性表达率分别为73.3%和90.0%;(2)与LMP1(-)组织比较,p27蛋白在LMP1(+)的鼻咽上皮和鼻咽低分化鳞状细胞癌中表达降低,而RPLP0蛋白表达与之相反;(3)p27蛋白和RPLP0蛋白表达与鼻咽癌患者年龄、LMP1蛋白的表达、淋巴结转移及TNM分期有关(P<0.05),患者年龄小、LMP1(-)、无淋巴结转移、TNM I期和II期时,p27蛋白表达阳性率较高,而RPLP0蛋白表达则反之(P<0.05)。结论: LMP1在鼻咽上皮和鼻咽低分化鳞状细胞癌中下调p27和上调RPLP0蛋白的表达;鼻咽癌患者年龄小、LMP1(-)、无淋巴结转移、TNM I期和II期时,p27蛋白表达阳性率较高,而RPLP0蛋白表达则与之相反。     

Maspin在鼻咽癌癌变和转移中的作用研究

目的：研究Maspin(mammary serine protease inhibitor)在鼻咽上皮癌变和鼻咽癌转移中的作用。方法：用免疫组织化学染色检测30例正常鼻咽黏膜上皮组织,24例鼻咽上皮鳞状化生组织、14例非典型增生鼻咽上皮、10例原位鼻咽癌和50例浸润性鼻咽癌(nasopharyngeal carcinoma,NPC)组织中表达水平变化;同时检测了189例不同转移潜能NPC组织中Maspin的表达,分析其表达水平与NPC临床病理特征和患者预后的关系。结果：Maspin在原位癌和浸润性NPC组织中的表达分别显著低于正常鼻咽黏膜上皮组织、鼻咽上皮鳞状化生组织(P<0.05)。在有转移NPC组织中的表达明显低于无转移NPC组织(P<0.001),在颈部淋巴结转移癌组织中的表达明显低于原发癌(P<0.05)。Maspin表达水平与NPC的淋巴结转移、远处转移、临床分期以及NPC患者的总生存期负相关,是一个NPC预后判断的独立因素。结论：Maspin可能是一个与NPC发生、发展和转移相关的抑制蛋白,Maspin低表达的NPC患者预后差。     

CDH1基因启动子甲基化在上皮性卵巢癌转移方面的研究

目的探讨CDH1基因启动子甲基化对上皮性卵巢癌转移的影响。方法采用免疫组织化学方法检测38例正常卵巢上皮和80例上皮性卵巢癌组织中E-钙黏附素（E-cadherin）表达;应用甲基化特异的PCR（MSP）检测上述组织中CDH1基因启动子区甲基化;应用5-氮-2＇-脱氧胞苷（5-aza-CdR）使SKOV3细胞去甲基化,观察SKOV3细胞体外侵袭性的改变,并通过RT-PCR检测CDH1基因的改变。结果E-cadherin在正常卵巢组织中表达明显高于上皮性卵巢癌（P〈0．05）。34例CDH1基因启动子区甲基化全部出现在卵巢癌组织中,有淋巴结转移组织中甲基化明显高于无淋巴结转移者（P〈0．05）,而CDH1基因启动子区有甲基化的卵巢癌组织中E-cadherin表达明显降低（P〈0．05）。经5-Aza-CdR处理后的SKOV3细胞体外侵袭性降低（P〈0．01）,CDH1基因的表达明显上调（P〈0．01）。结论E-cadherin表达降低与上皮性卵巢癌转移关系密切,CDH1基因启动子区甲基化可能是导致E-cadherin蛋白表达减低的重要原因之一,因此启动子区甲基化与上皮性卵巢癌转移有关。     

哈族食管鳞癌中PLCE1基因启动子甲基化水平的实验研究

目的探讨PLCE1基因启动子甲基化与食管鳞癌发生发展的关系。方法分别用免疫组化(IHC)和甲基化特异PCR(MSP)方法检测51例新疆哈萨克族食管鳞癌及相应癌旁正常组织中PLCE1蛋白表达水平及其启动子甲基化水平,分析其与病理资料相关性;分别用Western blot及MSP检测食管鳞癌细胞在5-aza-dC作用前后PLCE1蛋白表达及启动子甲基化变化情况。结果新疆哈萨克族食管鳞癌组织中PLCE1蛋白表达高于癌旁正常组织,而其基因甲基化水平则较低(P 0.001),且蛋白高表达的癌组织中其甲基化水平低于蛋白低表达的癌组织(P=0.028),PLCE1甲基化水平与其蛋白表达水平具有明显的相关性(χ~2=4.791,P=0.028),并与患者的淋巴结及远处转移(χ~2=7.242,P=0.027)和TNM分期(χ~2=7.883,P=0.019)明显相关;在食管鳞癌细胞系中PLCE1甲基化程度同样与蛋白表达水平成反比,5-aza-dC处理可抑制TE-1和Kyse150的PLCE1甲基化,提高其蛋白表达。结论食管鳞癌组织中PLCE1甲基化低与其蛋白表达高、淋巴结和远处转移及TNM分期相关,5-aza-dC处理可抑制PLCE1甲基化程度,增高其蛋白表达。     

遗传性弥漫型胃癌家系上皮型钙黏素基因的分析

 摘要：目的 研究在遗传性弥漫型胃癌家系中是否存在上皮型钙黏素基因(CDH1)以及基因表达的异常。 方法 收集符合遗传性弥漫型胃癌(HDGC)诊断标准的1个家系中15例成员的外周血和组织标本。通过免疫组化和Western blot 的方法检测组织标本CDH1蛋白表达; 提取基因组DNA, 通过PCR扩增DNA直接测序检测CDH1基因16个外显子突变。用克隆测序法,鉴定CDH1基因启动子区CpG位点甲基化状况。 结果 先证者和另一胃癌患者(家系2号成员)的癌旁胃粘膜上皮细胞CDH1蛋白表达较正常胃粘膜减弱, 两者肿瘤组织的蛋白表达几乎为阴性。包括先证者在内的11例家系成员第14外显子mRNA水平2377位点存在一个C?T的单核苷酸多态性(single nucleotide polymorphism, SNP), 但未检测到16个外显子的胚系突变。相对于正常胃粘膜, 先证者和家系2号成员的胃癌组织均有CDH1基因启动子的高甲基化, 其瘤旁粘膜也有高甲基化。结论 此HDGC家系中,CDH1基因表达丢失可能和胃癌发生有关, CDH1基因外显子突变不是其肿瘤组织上皮型钙黏素蛋白表达丢失的直接原因, 基因启动子区的甲基化可能是导致其失活的原因之一。     

Calreticulin通过诱导细胞EMT促进鼻咽癌迁移和侵袭

目的:观察钙网蛋白(calreticulin,CRT)在鼻咽癌组织中的表达并分析其临床病理学意义,揭示其对鼻咽癌CNE2细胞上皮-间充质转化(EMT)的影响。方法:S-P免疫组化法检测CRT在52例鼻咽癌组织和57例鼻咽部良性病变组织中的表达情况,并分析其临床病理学意义;构建特异性干扰CRT基因的小干扰RNA干扰载体,瞬时转染鼻咽癌CNE2细胞,观察CRT低表达对CNE2细胞形态的影响;Transwell迁移和侵袭实验检测CRT低表达对CNE2细胞迁移和侵袭能力的影响;Western blot检测CRT低表达对上皮型钙黏蛋白(E-cadherin)、波形蛋白(vimentin)、转化生长因子-β(TGF-β)及基质金属蛋白酶9(MMP-9)等EMT相关蛋白表达的影响。结果:CRT在鼻咽癌组织中的阳性表达率为82.69%(43/52),在鼻咽部良性病变组织的阳性表达率为19.29%(11/57),CRT在鼻咽癌组织中表达显著增高(P0.05);CRT的阳性表达率与鼻咽癌分期及淋巴结转移呈正相关(P0.05)。敲降CRT呈低表达后,CNE2细胞由梭形演变为扁平和鹅卵石样,且细胞排列紧密,细胞的迁移侵袭能力减弱(P0.05)。敲降CRT呈低表达后,E-cadherin表达显著增高,vimentin、TGF-β以及MMP-9蛋白的表达降低(P0.05)。结论:Calreticulin在鼻咽癌组织中表达显著增高,且与鼻咽癌临床分期及淋巴结转移呈正相关。Calreticulin可诱导鼻咽癌CNE2细胞发生EMT,进而促进鼻咽癌的迁移和侵袭。     

Downregulation and abnormal expression of E-cadherin and beta-catenin in nasopharyngeal carcinoma: close association with advanced disease stage and lymph node metastasis

Nasopharyngeal carcinoma (NPC) is predominantly of the undifferentiated histological subtype. Histological differentiation is of limited prognostic significance in NPC. Recent studies have suggested that downregulation of the cadherin-catenin cell adhesion complex may play a crucial role in the initial stage of cancer invasion and metastasis and is associated with poor prognosis in human cancers. Expression of E-cadherin has not been reported previously in NPC, and its prognostic value in NPC is unknown. The purpose of this study was to examine the expression pattern of E-cadherin and its associated partner, beta-catenin, in NPC and their possible applications as prognostic markers to predict the clinical outcome of NPC. Expression of the E-cadherin and beta-catenin was examined by immunohistochemical methods in 74 cases of primary NPC and 17 of their corresponding lymph node metastases. Normal nasopharyngeal epithelium showed strong and homogeneous immunocytochemical staining of E-cadherin and beta-catenin at the cell membranes and intercellular junctions. In contrast, primary NPC showed variable and heterogeneous staining patterns of E-cadherin and beta-catenin. Loss of membranous E-cadherin expression was significantly associated with advanced stages of diseases (P<.001). Eighty percent to ninety percent of NPC in stages IV and V (Ho's staging), respectively, showed a reduced (<35%) membranous staining of E-cadherin compared with normal nasopharyngeal epithelium. Expression of beta-catenin also was downregulated in advanced NPC. Ninety percent to one hundred percent of NPC in stages IV and V (Ho's staging) expressed a reduction (<35%) of imnmunocytochemical staining of beta-catenin. The expression pattern of beta-catenin staining was strongly associated with the expression of E-cadherin (P<.001). Unlike E-cadherin, nuclear staining of beta-catenin expression was observed in some of the primary NPC and lymph node metastasis. Reduced expression of E-cadherin and beta-catenin expression was associated with a shorter survival of NPC patients (P<.001). In advanced NPC patients (stages IV and V), a significant difference in survival was observed in tumors with higher or lower levels of E-cadherin expression (P=.0224, log-rank test). These observations suggests that expression of E-cadherin and beta-catenin may have prognostic values in NPC patients.     

上皮性钙黏附蛋白和β-连环素的表达与非小细胞肺癌预后的关系

目的　探讨上皮性钙黏附蛋白 (E cad)及 β 连环素 (β cat)在非小细胞肺癌 (NSCLC)中的表达及其与分型、分化、转移和预后的关系。方法　免疫组织化学 (SP法 )和原位杂交法。结果　免疫组织化学 :10 1例NSCLC中E cad及 β cat的阳性率分别为 6 8 3%和 81 2 % ,异常表达率分别为6 1 4 %和 6 4 4 %。E cad和 β cat的异常表达率与组织类型无关 (P >0 0 5 ) ,而与分化程度有相关性 (P<0 0 5 ) ,在有淋巴结转移病例中E cad及 β cat的异常表达率明显高于无转移病例 (P <0 0 5 )。E cad和 β cat异常表达病例的平均生存时间和 5年生存率明显低于正常表达病例。原位杂交 :NSCLC中E cad和 β catmRNA的阳性率分别为 38 9%和 4 7 2 % ,与蛋白正常表达一致率分别为 78 6 %和 82 4 %。结论　检测β cat或E cad/ β cat的共同表达将是指导临床治疗及估计预后的有意义指标 ,可应用于临床预后的综合评估。     

Methylation status of T-lymphoma invasion and metastasis 1 promoter and its overexpression in colorectal cancer

T-lymphoma invasion and metastasis 1 has been implicated in tumor invasion and metastasis. However, the regulatory mechanisms underlying aberrant T-lymphoma invasion and metastasis 1 expression in human colorectal cancer have not been well defined. To investigate the relationship between methylation status and expression levels of T-lymphoma invasion and metastasis 1 gene, methylation-specific polymerase chain reaction, and immunohistochemistry staining were performed in 232 matched samples of human colorectal cancer tissue and normal colorectal mucosa. Results showed that T-lymphoma invasion and metastasis 1 protein was overexpressed in colorectal cancer, especially in metastatic cases (P < .001). The degree of T-lymphoma invasion and metastasis 1 promoter methylation was a little lower in cancer tissues than in matched normal mucosa (P < .05), and the expression level of T-lymphoma invasion and metastasis 1 was inversely related to the methylation status in cancer tissues (P < .001). Colon cancer cell lines HT29 and LS174T were treated with demethylating agent 5-aza-2'-deoxycytidine, resulting in promoter hypomethylation accompanied by reexpression of T-lymphoma invasion and metastasis 1 mRNA and protein. In contrast, colon cancer cell lines SW620 and LoVo were treated with hypermethylation agent S-adenosylmethionine, resulting in T-lymphoma invasion and metastasis 1 promoter hypermethylation, accompanied by suppression of T-lymphoma invasion and metastasis 1 expression and inhibition of cell growth, plate colony formation, and migration. The present study demonstrates that overexpression of T-lymphoma invasion and metastasis 1 is associated with hypomethylation status of T-lymphoma invasion and metastasis 1 promoter region in colorectal cancer tissues. It suggests that promotor hypomethylation of T-lymphoma invasion and metastasis 1 may play a role in the progression and metastasis of colorectal cancer. Pharmacologic reversal of T-lymphoma invasion and metastasis 1 promoter hypomethylation may inhibit cell proliferation and migration.     

Association of E-cadherin and beta-catenin immunoexpression with clinicopathologic features in primary ovarian carcinomas

Epithelial cadherin forms a complex with alpha-, beta-, and gamma-catenin proteins. Reduced expression of E-cadherin-catenins has been shown in human carcinomas and is associated with low histologic differentiation, increased risk of invasion, and metastatic disease. The immunoexpression pattern of E-cadherin and beta-catenin (reduced versus preserved phenotype) was evaluated in 104 primary ovarian carcinomas and related to clinicopathologic features of the tumors. The immunoexpression pattern of E-cadherin was associated with International Federation of Gynaecology and Obstetrics (FIGO) staging (P = 0.043), histologic subtype (P = 0.001), peritoneal metastasis (P = 0.006), and residual tumor (P = 0.036). The reduced phenotype of E-cadherin that was observed in 64% of the carcinomas (67/104) was associated with advanced stage tumors, serous carcinomas, presence of peritoneal metastasis, and residual tumor larger than 2 cm. The immunoexpression pattern of beta-catenin was associated with histologic subtype (P = 0.005), tumor differentiation (P = 0.025), and peritoneal metastasis (P = 0.041). The reduced phenotype of beta-catenin that was observed in 74% of the carcinomas (77/104) was associated with advanced stage tumors, poorly differentiated serous and clear cell carcinomas, presence of peritoneal metastasis, and residual tumor. The immunoexpression pattern of E-cadherin was correlated with beta-catenin (P = 0.001). The reduced phenotype for both E-cadherin and beta-catenin was associated with histologic subtype (P < 0.001) and peritoneal metastasis (P = 0.001). In conclusion, the immunohistochemical profile of E-cadherin and beta-catenin may be useful in identifying a particular subpopulation of ovarian cancer patients who are characterized by an adverse clinical outcome, because the reduced phenotype of these molecules was associated with poor tumor differentiation, peritoneal metastasis, and advanced FIGO stage tumors.     

癌组织与癌栓上皮-间质转化相关标志物的对比研究

目的 通过对比上皮性原发癌、转移癌与其癌栓的组织形态学及E-cadherin和波形蛋白表达情况,从组织学水平探讨癌细胞在侵袭、转移过程中是否遵循上皮-间质转化(EMT)机制.方法 59例经病理诊断证实的原发性腺癌或原发性鳞状细胞癌及其淋巴结转移癌患者的68个石蜡组织标本,其中高分化癌组织13例,中分化癌11例,低分化癌30例,淋巴结转移癌14例,每例均有血管内或淋巴管内癌栓.挑选同一切面上同时具有癌组织(包含原发癌或淋巴结转移癌)及癌栓的蜡块进行HE染色观察形态及免疫组织化学(SP法)检测E-cadherin和波形蛋白表达.结果 癌组织与癌栓具有相似的形态学特征;54例原发性癌组织E-cadherin和波形蛋白阳性分别为50和23例,其癌栓阳性分别为51和22例,经统计学分析两者的E-cadherin和波形蛋白表达差异无统计学意义(P =0.804,P=0.842);14例淋巴结转移性癌组织E-cadherin和波形蛋白阳性分别为12和6例,其癌栓E-cadherin和波形蛋白阳性分别为12和7例,两者的E-cadherin和波形蛋白表达差异无统计学意义(P=0.084,P=0.878);癌组织和癌栓的E-cadherin和波形蛋白表达差异无统计学意义(P=0.410,P=0.824);癌栓部分癌细胞高表达E-cadherin而波形蛋白缺失,部分癌细胞高表达波形蛋白而E-cadherin缺失.结论 原发癌及其淋巴结转移癌、癌栓三者EMT特征无显著差异;癌栓细胞既有EMT细胞也有非EMT细胞,它们在肿瘤转移过程中的作用尚待进一步研究.     

Tumor-specific downregulation and methylation of the CDH13 (H-cadherin) and CDH1 (E-cadherin) genes correlate with aggressiveness of human pituitary adenomas.

The gene products of CDH13 and CDH1, H-cadherin and E-cadherin, respectively, play a key role in cell-cell adhesion. Inactivation of the cadherin-mediated cell adhesion system caused by aberrant methylation is a common finding in human cancers, indicating that the CDH13 and CDH1 function as tumor suppressor and invasion suppressor genes. In this study, we analyzed the expression of H-cadherin mRNA and E-cadherin protein in 5 normal pituitary tissues and 69 primary pituitary adenomas including all major types by quantitative real-time RT-PCR (qRT-PCR) and immunohistochemistry, respectively. Reduced expression of H-cadherin was detected in 54% (28/52) of pituitary tumors and was significantly associated with tumor aggressiveness (P<0.05). E-cadherin expression was lost in 30% (21 of 69) and significantly reduced in 32% (22 of 69) of tumors. E-cadherin expression was significantly lower in grade II, III, and IV than in grade I adenomas (P=0.015, P=0.029, and P=0.01, respectively). Using methylation-specific PCR (MSP), promoter hypermethylation of CDH13 and CDH1 was detected in 30 and 36% of 69 adenomas, respectively, but not in 5 normal pituitary tissues. Methylation of CDH13 was observed more frequently in invasive adenomas (42%) than in non-invasive adenomas (19%) (P<0.05) and methylation of CDH1 was more frequent in grade IV adenomas compared with grade I adenomas (P<0.05). Methylation of either CDH13 or CDH1 was identified in 35 cases (51%) and was more frequent in grade IV invasive adenomas than in grade I non-invasive adenomas (P<0.05 and P<0.05, respectively). Downregulation of expression was correlated with promoter hypermethylation in CDH13 and CDH1. In conclusion, the tumor-specific downregulation of expression and methylation of CDH13 and CDH1, alone or in combination, may be involved in the development and invasive growth of pituitary adenomas.     

鼻咽癌细胞中上皮细胞钙黏蛋白基因启动子甲基化的研究

目的 探讨鼻咽癌细胞中上皮细胞钙黏蛋白(E-cadherin)启动子甲基化对基因表达的影响,以及经去甲基化药物5-杂氮-2'-脱氧胞苷(5-Aza-dC)作用后肿瘤细胞增殖与侵袭能力的变化.方法采用逆转录(RT)-PCR、Western blot与免疫组织化学(polymer法)检测经5-Aza-dC处理前后HNE1和CNE2细胞中E-cadherin的表达水平,以甲基化特异性PCR(MSP)分析启动子甲基化状态,以四甲基偶氮唑盐(MTT)比色法与侵袭实验测定细胞增殖与侵袭能力的变化.结果 HNE1和CNE2细胞中E-cadherin表达水平减弱,其启动子区域存在部分甲基化现象;经5-Aza-dC作用后可显著上调鼻咽癌细胞E-cadherin的表达水平,降低基因启动子甲基化程度,同时显著抑制肿瘤细胞的增殖能力与侵袭能力.经20 μmol/L 5-Aza-dC作用后,HNE1与CNE2细胞的增殖能力与未处理组相比分别降低27.6%和34.3%,P＜0.05;HNE1与CNE2细胞经5-Aza-dC药物处理后,通过滤膜迁移进至侵袭小室下腔的数量与未处理组相比分别减少37.2%和29.7%,P＜0.05.结论基因启动子区域高甲基化状态是导致E-cadherin表达水平下调的机制之一,应用去甲基化药物可恢复E-cadherin表达并抑制肿瘤细胞的恶性生物学特征.     

Establishment and validation of real-time polymerase chain reaction method for CDH1 promoter methylation

Aberrant methylation of the promoter region has emerged as the major mechanism for silencing tumor suppressor genes. However, for some genes, such as E-cadherin (CDH1), methylation and protein expression demonstrate considerable heterogeneity, making correlations difficult. We compared methylation and protein expression status of CDH1 in 56 primary breast carcinomas using semiquantitative assays. Aberrant CDH1 methylation was studied by methylation-specific polymerase chain reaction (MSP) and semiquantitative real-time MSP assays. The Cdh1 expression was investigated by immunostaining on archival formalin-fixed sections from 34 primary carcinomas and their accompanying normal epithelium and preinvasive and metastatic lesions. Membrane-specific Cdh1 expression in the neoplastic cells was quantified by image analysis using an automated cellular imaging system and a continuous score. Aberrant promoter methylation of the CDH1 was present in 24 of 56 (43%) breast carcinomas by MSP assay. There was excellent concordance between the standard MSP assay and the real-time assay (91%, P < 0.0001). The concordance between loss of Cdh1 expression and CDH1 methylation by standard MSP was 71% (P = 0.02). Furthermore, there was a strong correlation between the semiquantitative assays for methylation and protein expression (r = 0.47, P = 0.005). We conclude that promoter methylation of CDH1 significantly correlated with the Cdh1 expression level, demonstrating that epigenetic silencing is a valid pathway for silencing of tumor suppressor genes in primary breast carcinomas.     

人肝细胞癌金属蛋白酶组织抑制因子-3的表达及与其基因启动子甲基化的关系

目的 探讨肝细胞癌的发生和门脉浸润机制。方法 20例肝细胞癌患者,每例在手术后分别取原发瘤、门脉瘤栓及远离肝癌之肝组织。用Western印迹法检测金属蛋白酶组织抑制因子(TIMP)的蛋白表达,用逆转录-聚合酶链反应(RT-PCR)检测TIMP-3 mRNA的表达,用甲基化特异性PCR检测TIMP-3基因启动子的甲基化。结果 远离肝癌之肝组织中均可见TIMP-3蛋白和mRNA的表达,原发瘤和门脉瘤栓组织TIMP-3蛋白和mRNA表达明显降低,其中分别有5例和6例完全丢失。远离肝癌之肝组织均未发现TIMP-3启动子甲基化。原发瘤中有7例、门脉瘤栓组织中有9例出现甲基化。所有有甲基化的肝癌组织,包括原发瘤和门脉瘤栓,有13例TIMP-3 mRNA和蛋白表达完全丢失,6例表达降低。TIMP-3启动子甲基化和肝细胞癌组织学分级无关(P>0.05)。结论 肝细胞癌的发生和门脉浸润与TIMP-3基因和蛋白缺失或降低相关、而TIMP-3启动子甲基化是其基因和蛋白缺失或降低的原因。