组织微阵列技术研究肿瘤转移相关基因在鼻咽癌中的表达与临床意义

利用高通量组织微阵列结合免疫组化检测MT1-MMP、MT2-MMP、Ezrin、nm23-H1、E-cad和TIMP-2在鼻咽癌组织中的蛋白质表达,探讨肿瘤转移相关基因异常表达在鼻咽癌侵袭转移中的作用,筛选鼻咽癌转移相关分子标志物.结果发现,鼻咽癌组织存在MT1-MMP、Ezrin蛋白高表达(P<0.01)和nm23-H1、TIMP-2蛋白低表达(P<0.05).临床Ⅱ期、Ⅲ期和Ⅳ期鼻咽癌和淋巴结转移鼻咽癌中MT1-MMP、MT2-MMP和Ezrin蛋白阳性表达显著高于临床Ⅰ期鼻咽癌和无转移癌(P<0.05,P<0.01),但临床Ⅱ期、Ⅲ期和Ⅳ期鼻咽癌和淋巴结转移鼻咽癌中nm23-H1蛋白的阳性表达显著低于临床Ⅰ期鼻咽癌和无转移癌(P<0.05).鼻咽癌组织中MT1-MMP与MT2-MMP r=0.308,P<0.001),nm23-H1与E-cad(r=0.167,P<0.05)及TIMP-2(r=0.279,P=0.001),E-cad与TIMP-2(r=0.279,P=0.001)的蛋白质表达旱显著正相关.MT1-MMP与E-cad(r=-0.188,P<0.05)及TIMP-2(r=-0.233,P<0.05),Ezrin与E-cad(r=-0.204,P<0.05)的蛋白质表达呈显著性负相关.聚类分析显示,鼻咽癌MT1-MMP、MT2-MMP和Ezrin蛋白共同阳性表达显著高于慢性炎性鼻咽上皮(P<0.05),但nm23-H1、E-cad和TIMP-2蛋白在鼻咽癌组织中的共同阴性显著高于癌旁上皮和慢性炎性鼻咽上皮(P<0.05,P<0.01).多因素分析和有效性评估发现,MT1-MMP蛋白能较好地独立预测鼻咽癌淋巴结转移和临床进展.上述研究结果提示,多个肿瘤转移基因的蛋白质高表达,转移抑制基因的低表达和这些基因的蛋白质表达失平衡在鼻咽癌淋巴结转移和临床进展过程中起重要作用.MTI-MMP蛋白可作为预测鼻咽癌淋巴结转移的较好分子标志.     

MMP-2、Fhit、RECK、VEGF在喉癌组织中的表达及相关性分析

目的:探讨基质金属蛋白酶-2(MMP-2)、脆性组氨酸三联体基因(Fhit)、逆转录诱导蛋白基因(RECK)、血管内皮细胞生长因子(VEGF)在喉癌组织中的表达及其相关性。方法:选取2011年1月到2016年12月在陕西省人民医院接受治疗的喉癌患者80例,收集其手术中切除的喉癌组织和癌旁组织,另收集40例喉癌组织切除外缘的正常喉粘膜组织。比较喉癌组织、癌旁组织、正常喉粘膜组织中MMP-2、Fhit、RECK、VEGF的表达,分析喉癌组织中MMP-2、Fhit、RECK、VEGF的表达与临床病理特征的关系,并分析四个指标的相关性。结果:喉癌组织中MMP-2、VEGF表达明显高于癌旁组织和正常喉粘膜组织,Fhit、RECK表达明显低于癌旁组织和正常喉粘膜组织(P0.05)。喉癌组织中MMP-2的表达与淋巴结转移、临床分期、分化程度有关(P0.05);Fhit、RECK的表达与淋巴结转移、临床分期有关(P0.05);VEGF的表达与淋巴结转移有关(P0.05)。喉癌组织中MMP-2的表达水平与Fhit、RECK呈负相关(P0.05),与VEGF呈正相关(P0.05);Fhit与RECK呈正相关(P0.05),与VEGF呈负相关(P0.05);RECK与VEGF呈负相关(P0.05)。结论:在喉癌组织中MMP-2、Fhit、RECK、VEGF均存在异常表达;其相互影响,可能共同参与了喉癌的发生、发展。     

新疆卡波西肉瘤组织中MT1-MMP和ARHGDIB蛋白的表达

目的:探讨新疆卡波氏肉瘤组织中膜型1型基质金属蛋白酶(Menberane 1 Matrix metalloproteinases,MT1-MMP),Rho蛋白解离抑制因子β(Rho GDP dissociation inhibitor GDI beta,ARHGDIB)的表达及临床意义.方法:应用免疫组织化学Envision二步法检测新疆17例卡波氏肉瘤和17例健康人正常皮肤组织内MT1-MMP和ARHGDIB的表达水平.结果:免疫组化显示MT1-MMP和ARHGDIB在卡波氏肉瘤组织阳性表达率为82.4%和100%,在正常皮肤组织中阳性率为6.9%和17.7%,差异均有统计学意义(P＜0.01).结论:新疆卡波西肉瘤组织中MT1-MMP和ARHGDIB蛋白呈异常表达,有可能在KS的发展过程中发挥了重要作用.     

PTEN及COX-2在乳腺癌的表达、意义及相关性

目的:研究PTEN及COX-2在乳腺癌的表达、与临床病理特征的关系及二者表达的相关性.方法:采用免疫组化技术(S-P法)检测48例乳腺癌和12例乳腺纤维腺瘤中PTEN、COX-2的表达情况.结果:PTEN在乳腺癌中的高表达率52.08%(25/48)明显低于乳腺纤维腺瘤91.67%(11/12)(p<0.05);PTEN的表达水平与腋淋巴结转移呈负相关,与ER水平呈正相关.与乳腺纤维腺瘤组织16.67%(2/12)相比,COX-2在乳腺癌组织中的表达明显增强68.75%(33/48)(P<0.05),COX-2与肿瘤大小、临床分期、淋巴结转移及ER、PR水平有关.结论:PTEN和COX-2的异常表达与乳腺癌发生、发展密切相关;PTEN和COX-2在乳腺癌中的表达呈负相关,两者共同参与了乳腺癌发生和发展.     

RECK及MMP-9蛋白在皮肤鳞状细胞癌中的表达及临床病理意义（英文）

目的：观察RECK基因及基质金属蛋白酶-9（MMP-9）在皮肤鳞状细胞癌（CSCC）中的表达,探讨其与肿瘤浸润转移的关系。方法：应用免疫组化SP法检测40例CSCC组织、17例癌旁不典型增生组织及14例正常皮肤组织中RECK及MMP-9蛋白的表达。结果：①RECK在CSCC、癌旁不典型增生及正常皮肤中的表达率依次增高（30.0%、47.1%、85.7%）,组间比较差异有统计学意义（P＆lt;0.05）;MMP-9蛋白在CSCC、癌旁不典型增生及正常皮肤中的表达率依次降低（82.5%、76.4%、28.6%）,组间比较差异有统计学意义（P＆lt;0.05）②RECK及MMP-9与CSCC的组织学分级、淋巴结转移密切相关（P均＆lt;0.05）;与CSCC患者的性别、年龄无关（P均＆gt;0.05）③RECK及MMP-9在CSCC中的表达呈负相关（r=-0.475p＆lt;0.01）。结论：RECK和MMP-9与CSCC的浸润转移密切相关,RECK在CSCC中表达减少或缺失可能是通过上调MMP-9的表达从而促进CSCC的侵袭与转移。     

MT1-MMP在肿瘤转移中的研究进展

基质金属蛋白酶(matrix metallopeptidase, MMPs)家族是一类锌依赖性内肽酶,可降解大多数细胞外基质。MT1-MMP是MMPs家族的重要成员,定位在细胞膜上,并在多种肿瘤中高表达。MT1-MMP通过影响细胞外基质重塑、血管生成、脂质代谢、炎症反应等过程促进肿瘤转移。然而,其具体的调控作用机制仍未完全阐明。本文综述了目前MT1-MMP在各系统肿瘤中的最新研究进展,总结并探讨了其在肿瘤中的促转移调控机制,为其在肿瘤领域的深入研究和应用提供借鉴和参考。     

CXCR4 及VEGF 在肾母细胞瘤中的表达及其意义

目的:检测趋化因子受体4(CXCR4)及血管内皮生长因子(VEGF)在肾母细胞瘤中的表达,探讨其在肾母细胞瘤的发生、发展、浸润和转移中的作用。方法:选择临床及病理资料齐全的存档肾母细胞瘤标本30例,另取正常肾脏标本10例作对照。采用免疫组化(SABC)方法检测CXCR4及VEGF的表达。结果:CXCR4在肾母细胞瘤组织中高表达(66.67%),在正常肾脏组织中低表达(30.0%),两者相比有统计学意义(P<0.05),VEGF在肾母细胞瘤组织中高表达(73.33%),在正常肾脏组织中低表达(20.0%),两者相比同样有统计学意义(P<0.05),肾母细胞瘤组织中CXCR4与VEGF的表达两者之间呈正相关性(r=0.392,P<0.05)。结论:肾母细胞瘤组织中CXCR4和VEGF均高表达。CXCR4及VEGF可能成为预测肾母细胞瘤浸润转移的重要指标。     

VEGF-C和VEGF-D在胃癌组织中的表达与微血管生成的关系及临床意义

目的:观察胃癌患者血管内皮生长因子C和D(vascular endothelial growth factor-C and-D,VEGF-C,VEGF-D)在胃癌组织中的表达以及其与肿瘤微血管生成的关系.方法:采用免疫组化S-P法检测30例胃癌组织和30例正常胃组织中VEGF-C和VEGF-D蛋白的表达,检测微血管密度(microvessel density,MVD).结果:胃癌组织VEGF-C和VEGF-D表达明显高于正常胃组织(p<0.01),其中淋巴结转移组VEGF-C、VEGF-D的表达与淋巴结未转移组间差异显著(p<0.05).胃癌组织MVD值明显高于正常胃组织(p<0.01),淋巴结转移组MVD值明显高于未转移组(p<0.01).MVD与VEGF-C的蛋白在胃癌组织中的表达高度相关(r=0.735,p<0.05),与VEGF-D的蛋白在胃癌组织中的表达成正相关(r=0.623,p<0.05).结论:VEGF-C和VEGF-D的高表达与肿瘤微血管生成及淋巴道转移密切相关,可作为评估胃癌患者预后的重要参考指标.     

中华绒螯蟹第二次卵巢发育期间卵巢和肝胰腺中主要生化成分的变化

2005年3—5月,在室内养殖条件下对第一次产卵后的中华绒螯蟹进行连续采样,系统研究了其第二次卵巢发育期间的卵巢指数(GSI)、肝胰腺指数(HSI)、卵巢外部特征的变化,并测定了卵巢和肝胰腺中的主要生化成分(水分、蛋白、脂肪和碳水化合物)的变化。结果表明:1.中华绒螯蟹第二次卵巢发育过程中,卵巢指数(GSI)显著增加(p<0.05),肝胰腺指数(HSI)先增加后减少,GSI与发育天数呈显著正相关性(p<0.01),HSI和GSI的变化呈显著负相关性(p<0.05)。2.第二次卵巢发育过程中,卵巢中的水分含量显著下降,脂肪、蛋白质和碳水化合物的含量显著上升(p<0.05);肝胰腺中的水分含量显著增加(p<0.05),脂肪和碳水化合物的含量显著下降(p<0.01),蛋白质含量基本不变。3.第二次发育过程中,卵巢中的脂肪、蛋白质和碳水化合物的含量与GSI呈正相关(p<0.01),卵巢中的水分含量与GSI呈显著负相关(p<0.01);肝胰腺中的脂肪和碳水化合物的含量随着HSI的下降而显著下降,肝胰腺中的水分含量与HSI呈显著负相关(p<0.01);肝胰腺中的脂肪含量与GSI及卵巢中脂肪含量均呈显著负相关(p<0.01),这说明中华绒螯蟹第二次卵巢发育过程中肝胰腺中的部分脂肪转运到二次卵巢中去。     

RNA干扰肝细胞肝癌中PECAM-1的表达对肿瘤细胞侵袭及增殖的影响

目的:探讨PECAM-1在肝细胞肝癌(Hepatocellular carcinoma,HCC)组织中的表达及意义。方法:选择2013年5月-2015年6月在我院接受治疗的HCC患者100例,收集肝癌患者HCC组织及癌旁组织,另选取100例正常肝脏组织作为对照组。应用免疫组织化学法检测PECAM-1在肝癌组织、癌旁组织以及正常肝脏组织中的阳性表达。利用小分子干扰RNA技术(si RNA)构建低表达的PECAM-1,并转染至肝癌细胞中抑制PECAM-1的表达。应用Transwell小室法检测肝癌细胞的侵袭能力,CCK-8法检测肝癌细胞的增殖能力。结果:PECAM-1在肝癌组织、癌旁组织及正常肝脏组织中呈不同程度阳性表达(P0.05);PECAM-1在肝癌组织及癌旁组织中的表达显著高于正常肝脏组织,差异具有统计学意义(P0.05);PECAM-1在肝癌组织中的表达显著高于癌旁组织,差异具有统计学意义(P0.05);转染si RNA PECAM-1后,肝癌细胞中PECAM-1 m RNA的表达水平明显下降,PECAM-1蛋白表达也明显降低,差异具有统计学意义(P0.05);转染si RNA PECAM-1后,肝癌细胞侵袭及增殖能力明显降低,差异具有统计学意义(P0.001)。结论:PECAM-1在肝癌患者血清中高表达,PECAM-1 si RNA能够抑制肝癌细胞的侵袭及增殖能力,提示PECAM-1可作为预测肝癌发生及发展的临床指标。     

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) interacts with membrane type 1 matrix metalloproteinase and CD13/aminopeptidase N and modulates their endocytic pathways

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is anchored to the cell surface via glycosylphosphatidylinositol. This molecule antagonizes the function of membrane type 1 matrix metalloproteinase (MT1-MMP) to promote proMMP-2 maturation. Here, we attempt to clarify the mechanism underlying RECK functions. First, we found that RECK forms a complex with MT1-MMP and inhibits its proteolytic activity. Notably, RECK increases the amount of MT1-MMP that associates with detergent-resistant membranes during sucrose gradient ultracentrifugation. Furthermore, perturbation of membrane cholesterol significantly affected the function of RECK in suppressing MT1-MMP function. These findings indicate that RECK possibly regulates MT1-MMP function by modulating its behavior on the cell surface as well as by enzymatic action; this prompted us to find another molecule whose behavior in detergent-resistant membranes is influenced by RECK. Subsequently, we found that RECK interacts with CD13/aminopeptidase N. Further, we found that RECK inhibits the proteolytic activity of CD13 in a cholesterol perturbation-sensitive manner. Finally, we examined whether RECK influences the behavior of MT1-MMP and CD13 during their internalization from the cell surface. In the absence of RECK, MT1-MMP and CD13 were internalized along with the markers of clathrin- or caveolae-dependent endocytosis. However, interestingly, in the presence of RECK these molecules were internalized preferentially with an endocytic marker that is neither clathrinnor caveolae-dependent, indicating that RECK modulates endocytic pathways of MT1-MMP and CD13. This modulation was correlated with the accelerated internalization and decay of MT1-MMP and CD13. This study unveils the novel function and target molecules of RECK.     

HPV16 oncoproteins induce MMPs/RECK-TIMP-2 imbalance in primary keratinocytes: possible implications in cervical carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.     

基质金属蛋白酶及其抑制剂在乳腺癌中的表达

目的:研究基质金属蛋白酶2(Matrix Metalloproteinase-2,MMP-2),基质金属蛋白酶7(MMP-7),基质金属蛋白酶9(MMP-9),膜型基质金属蛋白酶(Membrane Type-1 Matrix Metalloproteinase,MT1-MMP),金属蛋白酶组织抑制剂1(Tissue Inhibitor of Metalloproteinase,TIMP-1),金属蛋白酶组织抑制剂2(TIMP-2)在乳腺癌组织中mRNA的表达,及与临床病理变量之间的关联。方法:采用150例乳腺癌患者的组织样本。使用半定量逆转录-聚合酶链反应(RT-PCR)法来测定肿瘤组织和正常乳腺组织中MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2的mRNA表达。结果:MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2在乳腺癌中的mRNA表达显著高于正常组织。结论:MMP-2,MMP-7,MMP-9,和MTI-MMP的表达增加和临床病理参数之间的关联,可以用来预测乳腺癌的侵害行为。     

Spatial analysis of RECK,MT1-MMP,and TIMP-2 proteins during early Xenopus laevis development

Proper cell-cell and cell-ECM interactions are vital for cell migration and patterning of the vertebrate embryo. MMPs and their inhibitors, RECK and TIMPs, are all differentially expressed during embryogenesis to regulate such ECM remodeling and cell interactions. MT1-MMP, RECK, and TIMP-2 are unique amongst other ECM-regulating proteins as they act in the pericellular space. Past studies have shown that RECK and TIMP-2 interact with MT1-MMP on the cell surface, thereby influencing cell behaviour as well as the microenvironment immediately surrounding the cells. We investigated the localization of RECK, TIMP-2, and MT1-MMP proteins throughout early X. laevis development using immunohistochemistry. We found that during neural tube formation, axis elongation, and organogenesis, RECK, TIMP-2, and MT1-MMP proteins show highly similar localization patterns, particularly in the ectoderm and in the dorsal-ventral differentiation of the neural tube. Our data suggests they function together during patterning events in early Xenopus development.     

Clinical relevance of cyclooxygenase-2 and matrix metalloproteinases (MMP-2 and MT1-MMP) in human breast cancer tissue

Breast cancer (BC) is the most common neoplasm among women in most developed countries, including Egypt. Elevated levels of certain proteins in human BC are associated with unfavorable prognosis and progressive stages of the disease. The aim of our study was to evaluate the protein expression profile and prognostic significance of cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP-2), MMP-9 and membrane type 1-MMP (MT1-MMP) and their interaction in operable BC patients. The protein expression of COX-2, MMP-2 and MT1-MMP were evaluated by western blot technique, whereas enzymatic activity of MMP-2 and MMP-9 was determined by zymography in 47 breast cancer patients as well as normal adjacent tissues. Also, the correlation between these proteins and age, tumor size, LN stage, TNM stage, estrogen receptor, progesterone receptor, disease-free survival, and overall survival (OS) has been investigated. As compared to adjacent normal tissues, COX-2, MMP-2 and MT1-MMP were over-expressed in 43, 64, and 60 % of tumor tissues, respectively. In the same pattern, the activity of MMP-2 (62 %) and MMP-9 (45 %) was elevated in BC tissues. Multivariate analysis showed a positive correlation between the protein expression of COX-2, MMP-2, and MT1-MMP and the activity of MMP-2 and MMP-9 in BC patients. However, the enzymatic activity showed no correlation with clinicopathological features. This study confirms the preclinical evidence that COX-2 increased the expression of MT1-MMP, which in turn activates MMP-2. The lack of correlation with clinicopathological features, OS or disease-free survival ascertains the complexity of tumor progression and metastasis with many pro- and counter regulatory factors.     

The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.     

Higher expression and activity of metalloproteinases in human cervical carcinoma cell lines is associated with HPV presence.

Matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in 3 human invasive cervical carcinoma cell lines. Two HPV16-positive cell lines (SiHa and CaSki) and an HPV-negative cell line (C33A) were cultured either onto a type-I collagen gel, Matrigel, or plastic, to recreate their three-dimensional growth environment and evaluate the expression of these genes using quantitative real-time PCR. We also analyzed the gelatinolytic activity of MMP-2 and MMP-9 by zymography. We found that HPV (human papillomavirus)-positive cell lines express higher levels of MMP-2, MT1-MMP, and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with higher pro-MMP-2 activity. Furthermore, Matrigel substrate influences MMP-2 expression in both SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP-2, MT1-MMP, and TIMP-2, and that pro-MMP-2 activity is higher in HPV-positive than in HPV-negative cells.