Coordinated expression of integrin subunits,matrix metalloproteinases (MMP), angiogenic genes and Ets transcription factors in advanced-stage ovarian carcinoma: a possible activation pathway?

The events that mediate tumor progression in ovarian carcinoma are poorly understood to date. This review summarizes our results studying metastasis-associated molecules in advanced-stage ovarian carcinomas, details the co-expression of mRNA of these genes, and discusses their prognostic role. Fifty-five primary and metastatic FIGO stage III-IV ovarian carcinomas were analyzed for the expression of alpha v and beta1 integrin subunits, the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP, the MMP inhibitor TIMP-2, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), PEA3 and Ets-1 using mRNA in situ hybridization. Tumor and adjacent stromal cell expression was scored. The association between integrin subunit expression and the expression of MMP, TIMP-2, angiogenic genes, PEA3 and Ets-1 was statistically analyzed. Alpha v integrin subunit mRNA expression in carcinoma cells showed significant association with that of MMP-2 and IL-8 in this cellular compartment, while the presence of beta1 integrin subunit mRNA showed similar association with that of PEA3, Ets-1, IL-8, bFGF and MMP-2. Expression of beta1 integrin subunit mRNA in stromal cells was associated with that of TIMP-2 and Ets-1 in this compartment. In addition, significant intercellular associations were found between alpha v integrin subunit mRNA expression in carcinoma cells and stromal cell expression of Ets-1, as well as between stromal cell expression of alpha v integrin subunit and labeling for IL-8 in carcinoma cells. The presence of beta1 integrin subunit mRNA in carcinoma cells showed a significant association with that of Ets-1, IL-8 and bFGF in stromal cells, while the presence of beta1 integrin subunit mRNA in stromal cells was associated with tumor PEA3 mRNA expression. To the best of our knowledge, this is the first evidence for coordinated autocrine and paracrine expression of members of these four families of metastasis-associated genes in human cancer. The results of this analysis support experimental data regarding cross-talk between carcinoma cells and peritumoral fibroblasts. They also suggest the existence of a putative activation sequence of metastatic genes, involving the beta1 (and possibly alpha v) integrin subunits, IL-8, PEA3, Ets-1 and MMP in ovarian carcinoma.     

EIF4E基因下调对胃癌细胞的侵袭转移能力的影响及机制研究

目的:探讨特异性下调真核起始因子EIF4E对胃癌细胞侵袭转移能力的影响及可能的作用机制。方法:采用siRNA技术下调EIF4E的表达,Western blot检测转染前后EIF4E和MMP9蛋白质表达,通过Traswell小室实验检测转染前后侵袭迁移能力的差异。结果:Western blot证实,与阴性对照相比,转染EIF4E siRNA组细胞EIF4E表达明显下调,同时与阴性对照相比,转染EIF4E siRNA组细胞MMP9蛋白表达同样明显下调,Traswell小室实验证实,下调EIF4E后,MKN28细胞的体外侵袭迁移能力显著降低。结论:在胃癌MKN28细胞中存在EIF4E基因对MMP9的调控作用,EIF4E基因可能通过调控MMP9的表达参与胃癌的侵袭迁移行为,EIF4E有望成为肿瘤基因治疗的新靶点。     

胃癌组织及细胞株细胞骨架蛋白Fascin-1表达临床意义分析

目的：分析胃癌组织及细胞株中Fascin-1蛋白表达的临床病理及预后意义。方法：从日本理化研究所获得胃癌细胞株MKN28、AGS、MKN45和KATO—Ⅲ。收集日本富山大学医学部1993—02—01～2006—10—01胃癌组织455例。利用实时PCR、蛋白质印迹法杂交或免疫组化检测胃癌细胞株（MKN28、AGS、MKN45和KATO-Ⅲ）和胃癌组织中Fascin-1mRNA和蛋白的表达,分析胃癌组织中Fascin-1蛋白表达与临床病理特征和预后的关系。结果：Fascin-1蛋白主要定位在胃癌细胞株的细胞质中,在MKN28和MKN45细胞株中呈强表达,在ACTS和KATO-Ⅲ细胞中弱表达。胃癌组织中Fascin-1mR—NA表达水平显著高于癌旁正常黏膜组织,t=5．267,P〈0．001;Fascin-1蛋白表达与胃癌肿块大小（rs=0．116,P〈0．001）、侵袭深度（n=0．177,P〈0．001）、淋巴管侵袭（rs=0．190,P=0．005）、静脉侵袭（^=0．131,P=0．003）、淋巴结转移（rs=0．141,P=0．013）和TNM分期（rs=0．174,P〈0．001）呈正相关。Cox风险比率回归模型显示,年龄（P〈0．001）、侵袭深度（P〈0．001）、淋巴管侵袭（P〈0．001）、淋巴结转移（P〈0．001）、TNM分期（P〈0．001）和Lauren分型（P=0．003）为独立的预后因素。结论：Fascin-1蛋白表达在胃癌细胞株的侵袭转移过程中发挥重要作用,Fascin-1蛋白表达上调参与胃癌发生和演进过程,可以作为胃癌恶性程度和预后判断的有效分子标志。     

沉默Snail基因对胃癌MKN-28细胞的生物学影响

目的:探讨转录因子Snail对胃癌MKN-28细胞的增殖、凋亡及侵袭的影响。方法:慢病毒沉默胃癌MKN-28细胞株的Snail基因,顺铂处理MKN-28细胞,CCK-8法检测各组细胞的增殖情况,透射电镜观察各组细胞的凋亡情况,Transwell小室观察各组细胞的侵袭能力,Real time PCR检测Snail mRNA、E-cadherin mRNA和Bcl-2 mRNA的表达水平。结果:细胞增殖情况,慢病毒沉默Snail基因组和顺铂处理组的细胞出现了增殖抑制,特别是shRNA-Snail+顺铂组的细胞增殖抑制率明显增高（P<0.05）。各组细胞的凋亡表现,不同刺激因素组细胞的凋亡程度明显高于单纯MKN-28细胞组。细胞侵袭实验显示,shRNA+Snail+顺铂组的细胞迁出的细胞数明显低于其他各组（P<0.05）。顺铂处理MKN-28细胞后,shRNA+Snail+顺铂组的细胞Snail mRNA和Bcl-2 mRNA的表达水平均低于单纯MKN-28细胞组（P<0.05）,E-cadherin mRNA的水平高于单纯MKN-28细胞组（P<0.05）。结论:慢病毒沉默Snail基因,影响胃癌MKN-28细胞的增殖、促进了细胞的凋亡,抑制MKN-28细胞的侵袭,增加了化疗药的敏感性,可为肿瘤的靶向治疗提供一定理论依据。     

Expression of E1AF, an ets-oncogene transcription factor, highly correlates with malignant phenotype of malignant melanoma through up-regulation of the membrane-type-1 matrix metalloproteinase gene

Matrix metalloproteinase (MMP) is closely involved in the degradation of extracellular matrix and confers invasive and metastatic potential to malignant tumors. MMP-2 is a type-IV collagenase secreted as a proenzyme that is activated on the surface of the tumor cell by membrane-type 1-MMP (MT1-MMP). MT1-MMP plays a critical role during tumor progression and metastasis. We investigated the expression levels of E1AF and MT1-MMP in malignant melanoma cell lines and specimens from patients in order to clarify the mechanisms responsible for the invasion and metastasis of malignant melanoma. High levels of E1AF and MT1-MMP mRNA expression were observed in melanoma cells by Northern blotting and real-time PCR. The expression level was highly correlated with an invasive potential determined by an in vitro invasion assay. The down-regulation of MT1-MMP was identified when E1AF was knocked down by RNA interference. These results suggest that E1AF plays a crucial role in the invasion and metastasis of malignant melanoma through up-regulating the MT1-MMP expression.     

实时荧光定量RT-PCR分析E1AF在直肠癌中的表达及其临床意义

 【摘要】　目的　检测E1AF在直肠癌组织及正常组织中的表达，并分析其与直肠癌侵袭转移的相关性。方法　应用实时荧光素酶反转录聚合酶链反应（RT-PCR）检测E1AF在86份直肠癌组织与正常组织中的表达。结果　在86份直肠癌标本中，55份E1AF mRNA表达上调。直肠癌组E1AF mRNA的表达与对照组差异有统计学意义。在直肠癌中，E1AF mRNA表达与组织分型、浸润深度、淋巴结和远处转移以及Duke分期明显相关。结论　E1AF与直肠癌的侵袭转移明显相关，可能是直肠癌侵袭转移的一个重要因子。     

胃癌组织中Ets-1的表达及其与血管生成临床病理特征和预后的关系

目的 探讨胃癌组织、癌旁组织及淋巴结转移灶中Ets-1表达的临床意义,分析Ets-1表达与血管生成、临床病理特征及预后的关系.方法 应用免疫组化SP法,采用组织芯片技术,检测189例胃癌组织、54例癌旁组织、41例淋巴结转移灶及32例正常胃黏膜中Ets-1蛋白的表达.对胃癌患者通过上门或电话进行问卷随访.结果 胃癌组织、癌旁组织和正常胃黏膜Ets-1的阳性表达率分别为71.4%、29.6%和18.8%,3组间差异有统计学意义(P＜0.01).135例Ets-1表达阳性的胃癌组织微血管密度(MVD)为30.42±15.21,54例Ets-1表达阴性的胃癌组织MVD为25.73±11.50.两组差异有统计学意义(P=0.042).Ets-1蛋白表达与浸润深度、淋巴结转移有关(P＜0.01),与性别、年龄、肿瘤大小、分化程度、Lanren分型无关(P＞0.05).41例淋巴结转移灶和相对应的41例胃癌组织Ets-1表达阳性率分别为84.4%和58.5%,差异有统计学意义(P=0.007).单因素分析显示,Ets-1表达对胃癌患者生存期的影响有统计学意义(P＜0.05),Cox多元回归分析显示,Ets-1表达不是胃癌患者预后的独立影响因素(P＞0.05).结论 Ets-1在促进胃癌血管生成中发挥着重要作用,在胃癌的发生、发展中扮演了重要角色,Ets-1表达对胃癌患者的生存期有一定影响,胃癌淋巴结转移灶和原发灶肿瘤组织中Ets-1的表达不同,具有异质性.     

Upregulated EMMPRIN/CD147 might contribute to growth and angiogenesis of gastric carcinoma: a good marker for local invasion and prognosis

Tumour growth depends on angiogenesis, which is closely associated with vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Extracellular MMP inducer (EMMPRIN) was reported to involve in the progression of malignancies by regulating expression of VEGF and MMPs in stromal cells. To clarify the role of EMMPRIN in progression and angiogenesis of gastric carcinoma, expression of EMMPRIN, ki-67, MMP-2, MMP-9 and VEGF was examined on tissue microarray containing gastric carcinomas (n=234) and non-cancerous mucosa adjacent to carcinoma (n=85) by immunohistochemistry. Additionally, microvessel density (MVD) was assessed after labelling with anti-CD34 antibody. Extracellular MMP inducer expression was compared with clinicopathological parameters of tumours, including levels of ki-67, MMP-2, MMP-9 and vascular endothelial growth factor (VEGF), MVD as well as survival time of carcinoma patients. Gastric carcinoma cell lines (HGC-27, MKN28 and MKN45) were studied for EMMPRIN expression by immunohistochemistry and Western blot. Extracellular MMP inducer expression was gradually increased from normal mucosa to carcinomas through hyperplastic or metaplastic mucosa of the stomach (P<0.05). There was strong EMMPRIN expression in all gastric carcinoma cell lines despite different levels of glycosylation. Extracellular MMP inducer expression was positively correlated with tumour size, depth of invasion, lymphatic invasion, expression of ki-67, MMP-2, MMP-9 and VEGF of tumours (P<0.05), but not with lymph node metastasis, UICC staging or differentiation (P>0.05). Interestingly, there was a significantly positive relationship between EMMPRIN expression and MVD in gastric carcinomas (P<0.05). Survival analysis indicated EMMPRIN expression to be negatively linked to favourable prognosis (P<0.05), but not be independent factor for prognosis (P>0.05). Further analysis showed three independent prognostic factors, depth of invasion, lymphatic and venous invasion, to influence the relationship between EMMPRIN expression and prognosis. Upregulated expression of EMMPRIN possibly contributes to genesis, growth and local invasion of gastric carcinomas. Altered EMMPRIN expression might enhance growth, invasion and angiogenesis of gastric carcinoma via upregulating MMP expression of both stromal fibroblasts and gastric cancer cells and could be considered as an objective and effective marker to predict invasion and prognosis.     

人胃癌MMP-2、MMP-9及MMP-MT基因的过度表达—免疫组织化学和原位杂交研究

目的：探讨联和检测组织金属蛋白酶ＭＭＰ－２、ＭＭＰ－９和ＭＭＰ－ＭＴ对胃癌细胞侵袭的诊断价值。方法：以胃癌患者癌组织（４１例），癌旁组织（１７例）为研究对象，采用免疫组化（ＳＰ）和分子原位杂交法检测ＭＭＰ－２、ＭＭＰ－９和ＭＭＰ－ＭＴ的表达。结果：胃癌组ＭＭＰ－２、ＭＭＰ－９和ＭＭＰ－ＭＴ表达与侵袭程度成正相关（Ｐ＜０．０５），而且胃癌组ＭＭＰｓ表达显著高于癌旁组织（Ｐ＜０．０５）。结论：一致性检验显示ＭＭＰｓ的免疫组化和原位杂交检测结果的关系密切相关（Ｐ＜０．０５），是胃癌侵袭转移检测较好的分子标志，可用于胃癌的预后判断     

Expression of Ets-related transcription factors and matrix metalloproteinase genes in human breast cancer cells

The expression levels of ets and MMP genes was examined in two breast cancer cell lines of differing invasive potential. The more invasive MDA-MB-231 cell line had higher levels of Ets-1, Ets-2, PEA3, ERM, Tel, Net, MMP-13 and -14 mRNA than MCF-7 cells. MMP-1, -3 and -16 mRNAs were expressed equally. TPA stimulated MMP-1, -9 and TIMP-1 mRNA expression in both cell lines. MMP-2 and MMP-7 mRNAs were not detected in either cell line. The Ets-1 protein was only detected in MDA-MB-231 cells and its level increased following TPA stimulation. TPA induced MMP-9 activity in MCF-7 cells and increased its activity in MDA-MB-231 cells, however, MMP-2 activity was not detected.     

Upregulation and differential expression of matrilysin (MMP-7) and metalloelastase (MMP-12) and their inhibitors TIMP-1 and TIMP-3 in Barrett's oesophageal adenocarcinoma

Oesophageal adenocarcinoma is believed to arise from metaplastic mucosa in the distal oesophagus, a condition also known as Barrett's oesophagus (BE). BE develops as a result of injury caused by refluxing gastric and duodenal contents and is associated with increased risk of malignant transformation. Matrix metalloproteinases (MMPs) have been implicated in all aspects of tumour progression; tumour growth, basement membrane degradation, invasion and metastatic spread. Using in situ hybridization, we investigated the expression patterns of collagenases-1 and -3, stromelysin-2, matrilysin, metalloelastase and TIMPs-1 and -3 in BE, adenocarcinoma and lymph-node metastases. Matrilysin was expressed abundantly in 12/15 tumours and in 4/6 lymph-node metastases and its expression correlated with the histological aggressiveness of tumour. Matrilysin and metalloelastase were upregulated already in BE. Stromelysin-2 and collagenase-3 expression was detected only in a few tumours. Collagenase-1 was expressed by cancer and stromal cells in 9/15 tumours. Tumour-infiltrating macrophages expressed metalloelastase in 13/15 cancers. TIMPs-1 and -3 were expressed in 12/15 and 11/15 tumours, respectively. Laminin-5 and tenascin were abundantly expressed at the invasive front of poorly differentiated tumours, but not in BE. Our results indicate that matrilysin is the principal MMP expressed by tumour cells in oesophageal adenocarcinoma, and further studies are needed to investigate whether matrilysin or tenascin-C could be used as a predictive marker for progression of BE to cancer.     

CD44s和CD44v6的mRNA在胃癌中的表达及其临床意义

目的　探讨CD4 4smRNA和CD4 4v6mRNA在胃癌中的表达及其临床意义。方法　应用巢式RT PCR检测 16例远隔癌灶部位“正常”胃黏膜及 5 8例胃癌新鲜组织中CD4 4smRNA和CD4 4v6mRNA表达水平。结果　CD4 4smRNA在胃癌组织和远隔癌灶部位“正常”胃黏膜中的表达无差异 ,和胃癌的各临床病理学指标无关 (P >0 .0 5 )。CD4 4v6mRNA在胃癌组织中的表达高于远隔癌灶部位“正常”胃黏膜 (P <0 .0 5 ) ,与Borrmann分型 (P <0 .0 5 )、脉管侵犯 (P <0 .0 1)、浆膜浸润 (P <0 .0 1)和淋巴结转移 (P <0 .0 1)有关。结论　CD4 4smRNA和胃癌的发生发展无关 ,CD4 4v6与胃癌的浸润及转移相关。     

胃癌组织中MMP-9和CD44V6表达的相互关系及其临床意义

目的　研究胃癌组织中MMP -9和CD 44V 6表达的相互关系及其临床意义。方法　采用免疫组化S -P方法 ,检测85例胃癌组织中CD44V 6和MMP -9的表达情况。结果　胃癌组织中CD44V 6阳性表达率为 47.0 6% (4 0 /85 ) ;MMP -9阳性表达率为 43 .5 3 % (3 7/85 )。胃癌组织中CD44V 6表达与MMP -9表达显著相关 (P <0 .0 5 ) ;CD44V 6和MMP -9均阳性表达与胃癌淋巴结转移、浸润深度、TNM分期及脉管浸润均呈显著正相关 (P <0 .0 5 ) ;CD 44V 6和MMP -9均阳性表达的胃癌患者术后 1、3、5年生存率分别为 5 6.2 5 %、2 8.13 %和 2 8.13 % ,其他患者术后 1、3、5年生存率分别为 89.2 5 %、71.19%和 5 9.3 3 % ,前者显著低于后者 (P均 <0 .0 5 )。结论　CD44V 6和MMP -9表达的与胃癌侵袭转移及预后显著相关