细胞外基质蛋白-1(ECM_1)在人乳腺癌淋巴转移中的作用研究
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摘要
【目的】研究人乳腺癌中细胞外基质蛋白-1(ECM1)蛋白与mRNA的表达,以及ECM1蛋白的表达与微淋巴管密度(LMVD)的关系,揭示ECM1在人乳腺癌淋巴浸润和转移中的作用。
【方法】1.免疫组织化学技术半定量检测41例患者乳腺癌组织及其癌旁正常乳腺组织、腋窝淋巴结ECM1蛋白表达情况,以D2-40标记淋巴管,计数LMVD,分析ECM1蛋白的表达与LMVD的关系。2.实时荧光定量聚合酶链反应检测以上患者相应组织ECM1 mRNA的相对表达量。3.结合临床病理资料,探讨ECM1与人乳腺癌淋巴管生成的关系及其在乳腺癌淋巴浸润和转移中的作用。
【结果】1.乳腺癌组织LMVD(12.95±11.10个/400倍视野)高于正常乳腺组织(2.24±1.14个/400倍视野)及腋窝淋巴结(5.49±3.36个/400倍视野)(P<0.01);淋巴结转移阳性组的腋窝淋巴结LMVD(9.32±9.03个/400倍视野)高于阴性组腋窝淋巴结(2.18±1.50个/400倍视野)(P<0.01)。2.乳腺癌组织ECM1蛋白表达阳性率为75.6%(31/41),正常乳腺组织为9.8%(4/41),腋窝淋巴结为29.3%(12/41);乳腺癌组织ECM1蛋白表达阳性率高于相应的正常乳腺组织及腋窝淋巴结(χ2=39.91, P<0.01)。阳性组淋巴结ECM1蛋白表达阳性率63.2%(12/19)高于阴性组淋巴结(P<0.01)。乳腺癌组织ECM1 mRNA的相对表达量高于正常乳腺组织及腋窝淋巴结(P<0.01)。3. ECM1蛋白的表达与乳腺癌组织LMVD呈正相关(r=0.347, P<0.05)。4. ECM1蛋白的表达与患者绝经前后、病理类型、组织学分级、原发肿瘤大小及淋巴结转移情况无关(P>0.05)。
【结论】1.乳腺癌组织中ECM1蛋白及mRNA的表达高于相应的正常乳腺上皮组织和腋窝淋巴结。2. ECM1蛋白的表达与乳腺癌组织LMVD呈正相关,可能通过参与淋巴管生成,促进人乳腺癌淋巴浸润和转移。3.淋巴管生成在乳腺癌细胞淋巴管播散中发挥重要作用,新生淋巴管是乳腺癌淋巴转移的主要通路。
[Objective] To investigate the protein and mRNA expression of extracellular matrix protein-1 (ECM1) in human breast carcinoma and the correlation between ECM1 protein and lymphatic microvessel density (LMVD), and to reveal the roles of ECM1 in the lymphatic infiltration and metastasis of human breast carcinoma.
[Methods] 1. The protein expression of ECM1 was examined in the cancerous tissues, their normal counterparts and axillary lymph nodes in 41 cases of breast carcinoma by immunohistochemistry. Lymph vessels were labeled by using D2-40 and LMVD was calculated. The correlation between the expression of ECM1 protein and LMVD was analyzed.
2. The relative expression level of ECM1 mRNA was detected in the correspongding tissues of the above subjects by real-time fluorescent quantitative polymerase chain reaction.
3. The clinicopathological factors were taken into account to analyze the correlation between expression of ECM1 and lymphangiogenesis in breast carcinoma, and the roles of ECM1 in the lymphatic infiltration and metastasis of human breast carcinoma.
[Results] 1. LMVD in the carcinoma tissues (12.95±11.10 per 400x field of vision) was higher than that in the corresponding normal breast epithelial tissues (2.24±1.14 per 400x field of vision) and that in the axillary lymph nodes (5.49±3.36 per 400x field of vision) (P<0.01). LMVD in the axillary lymph nodes with lymph node metastases (9.32±9.03 per 400x field of vision) was higher than that without metastases (2.18±1.50 per 400x field of vision) (P<0.01).
2. The positive rate of ECM1 protein expression in the breast carcinoma tissues, their normal counterparts and the axillary lymph nodes were 75.6%(31/41), 9.8%(4/41), 29.3%(12/41), respectively. The positive rate of ECM1 expression in the breast carcinoma tissues was higher than that in the corresponding epithelial tissues and axillary lymph nodes (χ2=39.91, P<0.01). The positive rate of ECM1 in positive lymph nodes (12/19, 63.2%) was higher than that in negative lymph nodes (P<0.01).
The relative expression of ECM1 mRNA in the breast carcinoma tissues was higher than that in the corresponding epithelial tissues and axillary lymph nodes (P<0.01).
3. The protein expression of ECM1 was positively correlated with LMVD in the breast carcinoma tissues (r=0.347, P<0.05).
4. The protein expression of ECM1 in the breast carcinoma tissues didn’t correlate with premenopause or postmenopause, pathological features, histological classfication, size of primary tumor or status of lymph node metastasis (P>0.05, respectively).
[Conclutions] 1. The expression of ECM1 protein and mRNA in the breast carcinoma tissues were both higher than those in the corresponding breast epithelial tissues and axillary lymph nodes.
2. The protein expression of ECM1 was positively correlated with LMVD in the breast carcinoma tissues. ECM1 might faciliate lymphangiogenesis, to promote lymphatic infiltration and metastasis of human breast carcinoma.
3. Lymphangiogenesis played a key role in the process of breast carcinoma cells disseminating to lymph vessels. The new-born lymph vessels were the main access for breast carcinoma cells to lymphatic metastasis.
【方法】1.免疫组织化学技术半定量检测41例患者乳腺癌组织及其癌旁正常乳腺组织、腋窝淋巴结ECM1蛋白表达情况,以D2-40标记淋巴管,计数LMVD,分析ECM1蛋白的表达与LMVD的关系。2.实时荧光定量聚合酶链反应检测以上患者相应组织ECM1 mRNA的相对表达量。3.结合临床病理资料,探讨ECM1与人乳腺癌淋巴管生成的关系及其在乳腺癌淋巴浸润和转移中的作用。
【结果】1.乳腺癌组织LMVD(12.95±11.10个/400倍视野)高于正常乳腺组织(2.24±1.14个/400倍视野)及腋窝淋巴结(5.49±3.36个/400倍视野)(P<0.01);淋巴结转移阳性组的腋窝淋巴结LMVD(9.32±9.03个/400倍视野)高于阴性组腋窝淋巴结(2.18±1.50个/400倍视野)(P<0.01)。2.乳腺癌组织ECM1蛋白表达阳性率为75.6%(31/41),正常乳腺组织为9.8%(4/41),腋窝淋巴结为29.3%(12/41);乳腺癌组织ECM1蛋白表达阳性率高于相应的正常乳腺组织及腋窝淋巴结(χ2=39.91, P<0.01)。阳性组淋巴结ECM1蛋白表达阳性率63.2%(12/19)高于阴性组淋巴结(P<0.01)。乳腺癌组织ECM1 mRNA的相对表达量高于正常乳腺组织及腋窝淋巴结(P<0.01)。3. ECM1蛋白的表达与乳腺癌组织LMVD呈正相关(r=0.347, P<0.05)。4. ECM1蛋白的表达与患者绝经前后、病理类型、组织学分级、原发肿瘤大小及淋巴结转移情况无关(P>0.05)。
【结论】1.乳腺癌组织中ECM1蛋白及mRNA的表达高于相应的正常乳腺上皮组织和腋窝淋巴结。2. ECM1蛋白的表达与乳腺癌组织LMVD呈正相关,可能通过参与淋巴管生成,促进人乳腺癌淋巴浸润和转移。3.淋巴管生成在乳腺癌细胞淋巴管播散中发挥重要作用,新生淋巴管是乳腺癌淋巴转移的主要通路。
[Objective] To investigate the protein and mRNA expression of extracellular matrix protein-1 (ECM1) in human breast carcinoma and the correlation between ECM1 protein and lymphatic microvessel density (LMVD), and to reveal the roles of ECM1 in the lymphatic infiltration and metastasis of human breast carcinoma.
[Methods] 1. The protein expression of ECM1 was examined in the cancerous tissues, their normal counterparts and axillary lymph nodes in 41 cases of breast carcinoma by immunohistochemistry. Lymph vessels were labeled by using D2-40 and LMVD was calculated. The correlation between the expression of ECM1 protein and LMVD was analyzed.
2. The relative expression level of ECM1 mRNA was detected in the correspongding tissues of the above subjects by real-time fluorescent quantitative polymerase chain reaction.
3. The clinicopathological factors were taken into account to analyze the correlation between expression of ECM1 and lymphangiogenesis in breast carcinoma, and the roles of ECM1 in the lymphatic infiltration and metastasis of human breast carcinoma.
[Results] 1. LMVD in the carcinoma tissues (12.95±11.10 per 400x field of vision) was higher than that in the corresponding normal breast epithelial tissues (2.24±1.14 per 400x field of vision) and that in the axillary lymph nodes (5.49±3.36 per 400x field of vision) (P<0.01). LMVD in the axillary lymph nodes with lymph node metastases (9.32±9.03 per 400x field of vision) was higher than that without metastases (2.18±1.50 per 400x field of vision) (P<0.01).
2. The positive rate of ECM1 protein expression in the breast carcinoma tissues, their normal counterparts and the axillary lymph nodes were 75.6%(31/41), 9.8%(4/41), 29.3%(12/41), respectively. The positive rate of ECM1 expression in the breast carcinoma tissues was higher than that in the corresponding epithelial tissues and axillary lymph nodes (χ2=39.91, P<0.01). The positive rate of ECM1 in positive lymph nodes (12/19, 63.2%) was higher than that in negative lymph nodes (P<0.01).
The relative expression of ECM1 mRNA in the breast carcinoma tissues was higher than that in the corresponding epithelial tissues and axillary lymph nodes (P<0.01).
3. The protein expression of ECM1 was positively correlated with LMVD in the breast carcinoma tissues (r=0.347, P<0.05).
4. The protein expression of ECM1 in the breast carcinoma tissues didn’t correlate with premenopause or postmenopause, pathological features, histological classfication, size of primary tumor or status of lymph node metastasis (P>0.05, respectively).
[Conclutions] 1. The expression of ECM1 protein and mRNA in the breast carcinoma tissues were both higher than those in the corresponding breast epithelial tissues and axillary lymph nodes.
2. The protein expression of ECM1 was positively correlated with LMVD in the breast carcinoma tissues. ECM1 might faciliate lymphangiogenesis, to promote lymphatic infiltration and metastasis of human breast carcinoma.
3. Lymphangiogenesis played a key role in the process of breast carcinoma cells disseminating to lymph vessels. The new-born lymph vessels were the main access for breast carcinoma cells to lymphatic metastasis.
引文
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[1] Mathieu E, Meheus L ,Raymackers J, et al. Characterization of the osteogenic stromal cell line MN7:identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis,Western blotting and microsequencing. Bone Miner Res,1994,9(6):903-913.
[2] Smits P, Ni J, Feng P, et al. The human extracellular matrix gene 1(ECM1):genomic structure, cDNA cloning,expression pattern and chromosomal localization. Genomics, 1997, 45(3): 487-495.
[3] Sercu S, Zhang M, Oyama N, et al. Interaction of extracellular matrix protein 1 with extracellular matrix components:ECM1 is a basement membrane protein of the skin. J Invest Dermato,2008,128(6):1397-1408.
[4] Sander C S, Sercu S, Ziemer M, et al. Expression of extracellular matrix protein 1(ECM1) in human skin is decreased by age and increased upon ultraviolet exposure. Br J Dermatol,2006,2(154):218-224.
[5] Smits P, Bhalerao J, Merregaert J. Molecular cloning and characterization of the mouse Ecm1 gene and its 5’regulatory sequences. Gene,1999,226(2):253-261.
[6] Horev L, Potikha T, Ayalon S, et al. A novel splice site mutation in ECM-1gene in a consanguineous family with lipoid proteinosis. Exp Dermatol,2005,14(12):891-897.
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[9] Han Z, Ni J, Smiths P, et al. Extracellular matrix protein 1(ECM1) has angiogenic properties and is expressed by breast tumor cells. FASEB J,2001,15(6):988-994.
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[15] Sercu S, Lambeir A M, Steenackers E, et al. ECM1 interacts with fibulin-3 and the beta 3 chain of laminin 332 through its serum albumin subdomain-like 2 domain. Matrix Biol,2009,28(3):160-169.
[16] Littlepage LE, Sternlicht MD, Rougier N, et al. Matrix metalloproteinases contribute distinct roles in neuroendocrine prostate carcinogenesis, metastasis, and angiogenesis progression. Cancer Res,2010,70(6):2224-2234.
[17] Fujimoto N, Terlizzi J, Aho S, et al. Extracellular matrix protein 1 inhibits the activity of matrix metalloproteinase 9 through high-affinity protein/protein interactions. Exp Dermatol,2006,15 (4):300-307.
[18]侯彦强,仲人前,耿红莲,等.细胞外基质蛋白1在乳腺癌组织和细胞株中的表达及其意义.中国肿瘤生物治疗杂志,2008,15(4):384-387.
[19] McPherson L A, Woodfield G W, Weigel R J. AP2 transcription factors regulate expression of CRABPII in hormone responsive breast carcinoma. J Surg Res,2007,138(1):71-78.
[20] Kenny P A, Enver T, Ashworth A. Receptor and secreted targets of Wnt-1?β-catenin signalling in mouse mammary epithelial cells. BMC Cancer,2005,5:3.
[21] Du Q, Zhang X, Cardinal J, et al. Wnt/beta-catenin signaling regulates cytokine-induced human inducible nitric oxide synthase expression by inhibiting nuclear factor-kappaB activation in cancer cells. Cancer Res,2009,69(9):3764-4771.
[22] Lal G, Hashimi S, Smith B J, et al. Extracellular Matrix 1(ECM1) expression is a novel prognostic marker for poor long-term survival in breast cancer:a hospital-based cohort study in Iowa. Ann Surg Oncol,2009,16(8):2280-2287.
[23] Bergamaschi A, Tagliabue E, Srile T, et al. Extracellular matrix signature identifies breast cancer subgroups with different clinical outcome. J Pathol,2008,214(3):357-367.
[24] Kreunin P, Urquidi V, Lubman D M, et al. Identification of metastasis-associated proteins in a human tumor metastasis model using the mass-mapping technique. Proteomics,2004,4(9):2754-2765.
[25] Wang L, Yu J, Ni J, et al. Extracellular matrix protein 1(ECM1)is overexpressed in malignant epithelial tumors. Cancer Lett,2003,200:57-67.
[2] Billottet C, Jouanneau J. Tumor-stroma interactions[J]. Bull Cancer, 2008, 95(1):51-56.
[3] Guarino M, Rubino B, Ballabio G. The role of epithelial-mesenchymal transition in cancer pathology[J]. Pathology, 2007,39(3):305-318.
[4] Mathieu E, Meheus L, Raymackers J, et al. Characterization of the osteogenic stromal cell line MN7: identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis,Western blotting and microsequencing[J]. Bone Miner Res, 1994,9(6):903-913.
[5] Deckers MM,Smits P,Karperien M,et al.Recombinant human extracellular matrix protein 1 inhibits alkaline phosphatase activity and mineralization of mouse embryonic metatarsals in vitro[J].Bone, 2001,28(1):14-20.
[6] Chan I, Liu L, Hamada T, et al. The molecular basis of lipoproteinosis:mutations in extracellular matrix protein 1[J]. Exp Dermatol, 2007,16(11):881-890.
[7] Uchida T, Hayashi H, Inaoki M, et al. A failure of mucocutaneous lymphangiogenesis may underlie the clinical features of lipoid proteinosis[J]. Br J Dermatol, 2007,156(1):152-157.
[8] Zhenhu Li, Yuan Zhang, Zhiduo Liu, et al. ECM1 controls TH2 cell egress from lymph nodes through re-expression of S1P1[J]. Nature Immunology, 2011,12(1):178-185.
[9] Han Z, Ni J, Smiths P, et al. Extracellular matrix protein 1(ECM1) has angiogenic properties and is expressed by breast tumor cells[J]. FASEB J, 2001,15(6):988-994.
[10] Wang L, Yu J, Ni J, et al. Extracellular matrix protein 1(ECM1)is overexpressed in malignant epithelial tumors[J]. Cancer Lett, 2003,200:57-67.
[11] Chen H, Jia WD, Li JS, et al. Extracellular matrix protein 1, a novel prognostic factor, is associated with metastatic potential of hepatocellular carcinoma[J]. Med Oncol, 2010,12,3. [Epub ahead of print]
[12] McPherson LA, Woodfield GW, Weigel RJ. AP2 transcription factors regulate expression of CRABPII in hormone responsive breast carcinoma[J]. J Surg Res, 2007,138(1):71-78.
[13] Kenny PA, Enver T, Ashworth A. Receptor and secreted targets of Wnt-1?β-catenin signalling in mouse mammary epithelial cells[J]. BMC Cancer, 2005,5:3.
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