骨桥蛋白（OPN）和RAS基因在子宫内膜癌中的表达及其相关性研究
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摘要
目的
子宫内膜癌是女性生殖器常见的三大恶性肿瘤之一,近年来其发病率逐年上升,已趋于接近甚至超过宫颈癌,严重威胁着女性的健康,成为研究的重点之一但子宫内膜癌发生发展的分子机制及病理生理学机制仍不明确,因此,有必要进一步探索子宫内膜癌发生发展的机制,从而达到早期诊断、及时治疗、降低死亡率、延缓生命、提高生存质量的目的。骨桥蛋白(Osteopontin,OPN)是一类存在于细胞外基质中的酸性钙结合糖蛋白,通过与其受体整合素和CD44结合来促进细胞的趋化、黏附和迁移。近年来研究发现OPN的高表达和许多恶性肿瘤的发生发展有关。RAS基因是第一个被鉴定的人类癌基因,是在人类肿瘤中最常见的癌基因,近年来的研究表明RAS参与多种肿瘤的发生及发展。但有关OPN和RAS在子宫内膜癌中的相关性研究尚少见报道。
本研究旨在检测OPN与RAS在正常增生期子宫内膜、不典型增生子宫内膜、子宫内膜癌组织中的表达及特点,从而探讨二者与子宫内膜癌发生发展的关系,为子宫内膜癌病因学研究提供分子水平的依据,并有助于为子宫内膜癌的诊断治疗提供新思路。
方法
采用免疫组化(SABC)法检测45例子宫内膜癌、28例不典型增生子宫内膜组织和18例正常增生期子宫内膜组织中OPN和RAS的表达情况。每批染色均设已知阳性切片(肝癌)作阳性对照,用磷酸盐缓冲液代替一抗作阴性对照。实验数据以x±s表示,采用SPSS16.0统计软件分析,组间差异使用单因素方差分析(One-Way ANOVA)和独立样本Dunnett t检验,相关性分析采用Spearman相关分析。P<0.05为差异有统计学意义。
结果
1、OPN主要定位于细胞浆中,OPN在正常增生期子宫内膜组织、不典型增生子宫内膜组织、子宫内膜癌组织中表达水平呈明显上升趋势。进一步分析OPN蛋白的表达与子宫内膜癌某些临床病理参数之间的关系,经统计学检验,OPN的表达与组织病理学分级(G1、G2及G3间两两比较P均<0.05)、手术病理分期(P<0.05)和肌层浸润(P<0.05)呈明显正相关,与是否淋巴转移(P>0.05)无明显相关。
2、RAS主要定位于细胞浆中,RAS在正常增生期子宫内膜组织、不典型增生子宫内膜组织、子宫内膜癌组织中表达水平呈明显上升趋势。进一步分析RAS的表达与子宫内膜癌某些临床病理参数之间的关系,经统计学检验,RAS的表达与肌层浸润(P<0.05)和是否淋巴转移(P<0.05)呈明显正相关,与组织病理学分级(P>0.05)、手术病理分期(P>0.05)无明显相关。
3、OPN和RAS在子宫内膜癌中均呈高表达,分类变量相关性分析显示,OPN与RAS表达呈明显正相关(r=0.3,P<0.05)。
结论
1、子宫内膜癌组织中OPN和RAS的表达明显高于正常增生期子宫内膜组织,说明OPN和RAS可能参与子宫内膜癌的发生。
2、OPN和RAS的表达均与子宫内膜癌的肌层浸润呈明显正相关,提示二者参与了子宫内膜癌的局部浸润。
3、子宫内膜癌组织中OPN和RAS的表达存在显著相关性,联合检测两者的表达将有可能作为诊断子宫内膜癌的指标。
Endometrial carcinoma is a common neoplasm of the female genital tract, in contract to cervical cancer, it's incidence rate is stepping up constantly and is seriously threading women's health and has been one of the focus of the neoplasm research. It is molecular and pathophysiocal mechanisms are still unclear and lack of effective early detection. Recently, it has been found to contribute to tumorigenes is in several types of cancers. RAS genes are the first to be identified in human tumors, recent studies have shown that RAS participation in a variety of tumors and development. However the relationship between the two are rarely researched. In this study, we detected the expression of Osteopontin and RAS protein in endometrial carcinoma and normal control tissues, to evaluated the relationship between the expression of Osteopontin andRAS with endometrial carcinoma. So as to provide molecular evidence for endometrial carcinoma etiology and also to give a new way of diagnosis for endometrial carcinoma.
Methods
The SABC immunohistochemical method was used to detect the expressions of OPN and RAS proteins in 45 cases of endometrial cancer,28 cases of atypical endometrial hyperplasia and 18 cases of normal endometrial tissue. Every batch of staining are compared with HCC samples for positive-control, and replacing parmingen with control SPSS 16.0 software was employed to analyze all data, the data factors with Indepent sample Dunnett T-test and Anlysis of variance; And the relationship of the two factors was analyze by spearman rand correlation test.p<0.05 with significance.
Results
1. OPN was mainly stained in cytoplasm. The expressions of OPN in the tussues of endometrial carcinoma and atypical endometrial hyperplasia were significantly higher than that in normal endometrial tissues. The expressions of OPN correlated with the histopathological grading(G1 vs G2 Differentiated, P<0.05;G2 Differentiated vs G3 Differentiated, P<0.05), surgical pathological stage(P<0.05)and myometrial invasion(P<0.05), but no significant correlation with lymph node metastasis(P>0.05).
2. RAS was mainly stained in cytoplasm. The expressions of RAS in the tussues of endometrial carcinoma and atypical endometrial hyperplasia were significantly higher than that in normal endometrial tissues. The expressions of RAS correlated with myometrial invasion(P<0.05) and lymph node metastasis(P<0.05),but no significant correlation with histopathological grading(P>0.05) or surgical pathological stage(P>0.05).
3. There was significant positive correlation between OPN and RAS in endometrial carcinoma (r=0.3, P<0.05)
Conclusion
1. The expression of OPN and RAS were obviously stronger in endometrial carcinoma than in the normal control tissues, show that OPN and RAS may play a important role in the occurrounce of the endometrial carcinoma.
2. The expression of OPN and RAS had relationship with myometrial invasion and,show that the expression of OPN and RAS had relation with the maligmnt degree of endometrial carcinoma.
3. The expression of OPN and RAS had close correlation, indicated that OPN and RAS together play important role during the occurrence of endometrial carcinoma.
子宫内膜癌是女性生殖器常见的三大恶性肿瘤之一,近年来其发病率逐年上升,已趋于接近甚至超过宫颈癌,严重威胁着女性的健康,成为研究的重点之一但子宫内膜癌发生发展的分子机制及病理生理学机制仍不明确,因此,有必要进一步探索子宫内膜癌发生发展的机制,从而达到早期诊断、及时治疗、降低死亡率、延缓生命、提高生存质量的目的。骨桥蛋白(Osteopontin,OPN)是一类存在于细胞外基质中的酸性钙结合糖蛋白,通过与其受体整合素和CD44结合来促进细胞的趋化、黏附和迁移。近年来研究发现OPN的高表达和许多恶性肿瘤的发生发展有关。RAS基因是第一个被鉴定的人类癌基因,是在人类肿瘤中最常见的癌基因,近年来的研究表明RAS参与多种肿瘤的发生及发展。但有关OPN和RAS在子宫内膜癌中的相关性研究尚少见报道。
本研究旨在检测OPN与RAS在正常增生期子宫内膜、不典型增生子宫内膜、子宫内膜癌组织中的表达及特点,从而探讨二者与子宫内膜癌发生发展的关系,为子宫内膜癌病因学研究提供分子水平的依据,并有助于为子宫内膜癌的诊断治疗提供新思路。
方法
采用免疫组化(SABC)法检测45例子宫内膜癌、28例不典型增生子宫内膜组织和18例正常增生期子宫内膜组织中OPN和RAS的表达情况。每批染色均设已知阳性切片(肝癌)作阳性对照,用磷酸盐缓冲液代替一抗作阴性对照。实验数据以x±s表示,采用SPSS16.0统计软件分析,组间差异使用单因素方差分析(One-Way ANOVA)和独立样本Dunnett t检验,相关性分析采用Spearman相关分析。P<0.05为差异有统计学意义。
结果
1、OPN主要定位于细胞浆中,OPN在正常增生期子宫内膜组织、不典型增生子宫内膜组织、子宫内膜癌组织中表达水平呈明显上升趋势。进一步分析OPN蛋白的表达与子宫内膜癌某些临床病理参数之间的关系,经统计学检验,OPN的表达与组织病理学分级(G1、G2及G3间两两比较P均<0.05)、手术病理分期(P<0.05)和肌层浸润(P<0.05)呈明显正相关,与是否淋巴转移(P>0.05)无明显相关。
2、RAS主要定位于细胞浆中,RAS在正常增生期子宫内膜组织、不典型增生子宫内膜组织、子宫内膜癌组织中表达水平呈明显上升趋势。进一步分析RAS的表达与子宫内膜癌某些临床病理参数之间的关系,经统计学检验,RAS的表达与肌层浸润(P<0.05)和是否淋巴转移(P<0.05)呈明显正相关,与组织病理学分级(P>0.05)、手术病理分期(P>0.05)无明显相关。
3、OPN和RAS在子宫内膜癌中均呈高表达,分类变量相关性分析显示,OPN与RAS表达呈明显正相关(r=0.3,P<0.05)。
结论
1、子宫内膜癌组织中OPN和RAS的表达明显高于正常增生期子宫内膜组织,说明OPN和RAS可能参与子宫内膜癌的发生。
2、OPN和RAS的表达均与子宫内膜癌的肌层浸润呈明显正相关,提示二者参与了子宫内膜癌的局部浸润。
3、子宫内膜癌组织中OPN和RAS的表达存在显著相关性,联合检测两者的表达将有可能作为诊断子宫内膜癌的指标。
Endometrial carcinoma is a common neoplasm of the female genital tract, in contract to cervical cancer, it's incidence rate is stepping up constantly and is seriously threading women's health and has been one of the focus of the neoplasm research. It is molecular and pathophysiocal mechanisms are still unclear and lack of effective early detection. Recently, it has been found to contribute to tumorigenes is in several types of cancers. RAS genes are the first to be identified in human tumors, recent studies have shown that RAS participation in a variety of tumors and development. However the relationship between the two are rarely researched. In this study, we detected the expression of Osteopontin and RAS protein in endometrial carcinoma and normal control tissues, to evaluated the relationship between the expression of Osteopontin andRAS with endometrial carcinoma. So as to provide molecular evidence for endometrial carcinoma etiology and also to give a new way of diagnosis for endometrial carcinoma.
Methods
The SABC immunohistochemical method was used to detect the expressions of OPN and RAS proteins in 45 cases of endometrial cancer,28 cases of atypical endometrial hyperplasia and 18 cases of normal endometrial tissue. Every batch of staining are compared with HCC samples for positive-control, and replacing parmingen with control SPSS 16.0 software was employed to analyze all data, the data factors with Indepent sample Dunnett T-test and Anlysis of variance; And the relationship of the two factors was analyze by spearman rand correlation test.p<0.05 with significance.
Results
1. OPN was mainly stained in cytoplasm. The expressions of OPN in the tussues of endometrial carcinoma and atypical endometrial hyperplasia were significantly higher than that in normal endometrial tissues. The expressions of OPN correlated with the histopathological grading(G1 vs G2 Differentiated, P<0.05;G2 Differentiated vs G3 Differentiated, P<0.05), surgical pathological stage(P<0.05)and myometrial invasion(P<0.05), but no significant correlation with lymph node metastasis(P>0.05).
2. RAS was mainly stained in cytoplasm. The expressions of RAS in the tussues of endometrial carcinoma and atypical endometrial hyperplasia were significantly higher than that in normal endometrial tissues. The expressions of RAS correlated with myometrial invasion(P<0.05) and lymph node metastasis(P<0.05),but no significant correlation with histopathological grading(P>0.05) or surgical pathological stage(P>0.05).
3. There was significant positive correlation between OPN and RAS in endometrial carcinoma (r=0.3, P<0.05)
Conclusion
1. The expression of OPN and RAS were obviously stronger in endometrial carcinoma than in the normal control tissues, show that OPN and RAS may play a important role in the occurrounce of the endometrial carcinoma.
2. The expression of OPN and RAS had relationship with myometrial invasion and,show that the expression of OPN and RAS had relation with the maligmnt degree of endometrial carcinoma.
3. The expression of OPN and RAS had close correlation, indicated that OPN and RAS together play important role during the occurrence of endometrial carcinoma.
引文
1 乐杰谢幸丰有吉等,《妇产科学第六版》,人民卫生出版社。
2 Tuck AB, Hota C, Wilson SM, et al. Osteopontin-induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways. Oncogene,2003,22(8):1198-1205.
3 刘思金,胡国法,刘思国,等,人类骨桥蛋白(h-OPN)在细胞增殖中的功能研究(J),高技术通讯,2003,2(4)：25-29。
4 Michihiro Hirana, Fumiyuki Takahashi, Kazuhisa Takahashi,et al. Osteopontin overproduced by tumor cells acts as a potent angiogenic factor contributing to tumor growth (J). Cancer Lett, 2003,198 (2):107-117.
5 Mi Z, Guo H, Russel MB, et al. RNA aptamer blockade of osteopontin inhibits growth and metastasis of MDA-MB231 breast cancer cells [J]. Mol Ther,2009,17(1):153-161.
6 侯志波,郑杰.骨桥蛋白下调对前列腺癌PC3细胞生物学行为的影响[J].肿瘤,2005,25(6)：542-546.
7 崔越宏,刘天舒,陈世耀,等.骨桥蛋白反义寡核苷酸抑制胃癌细胞MKN28粘附侵袭的实验研究[J].临床肿瘤学杂志,2008,13(7)：585-589.
8 Coppla D. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin Cancer Res.2004, Janl; 10(1):184-190.
9 Rudland PS, Platt-Higgins A, El-Tanani M, et al. Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res,2002,62:3417-3427.
10 Robillard PY, Chaouat G, Le Bouteiller P, et al. Current debates on immunology of preeclampsia. Report of the sixth international workshop of Reunion Island (Indian Ocean, December 2008). Gynecol Obstet Fertil,2009,37(6):570-578
11 Zhang J, Takahashi K, Takahashi F, et al. Differential osteopontin expression in lung cancer. Cancer Letters,2001,171:215-222.
12 Denhardt DT, Commentary re:Q-T,et al. identification of osteopontin as a prognostic marker for head and nece squamousce carcinomas. Clin Cancer Res,2003,9:31-32.
13 Denhardt DT, Mistretta D, Chambers AF,et al. Transcriptional regulation of osteopontin and the metastatic phenotype:evidence for a ras-activated enhance the human OPN promoter. ClinExpmetastasis,2003,20:77-84.
14何太平,严卫红,梁念慈.Ras信号转导通路[J].国外医学·临床生物化学与检验学分册,2004,25：74-76。
15 Futakawa N, Kimura M, Yamagata S, et al. Significance of K-RAS mutation and CEA level in pancreatic juice in the diagnosis of pancreatic cancer[J].J Hepatobiliary Pancreat Surg, 2000,7(1):63.
16 Yoo J, Robinson RA. H-RAS gene mutationin salivary gland mucoepidermoid carcinomas [J]. Cancer,2000,88 (3):518.
17 Denhardt DT, Mistretta D, Chambers AF,et al. Transcriptional regulation of osteopontin in and the metastatic phenotype:evidence for a RAS-activated enhanced the human OPN promoter. linExpmetastasis2003,20:77-84.
18 Wang Y, Chen B, Shen D, Xue S. Osteopontin protects against cardiac ischemia-reperfusion injury through late preconditioning. Heart Vessels,2009,24(2):116-123
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2 Tuck AB, Hota C, Wilson SM, et al. Osteopontin-induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways. Oncogene,2003,22(8):1198-1205.
3 刘思金,胡国法,刘思国,等,人类骨桥蛋白(h-OPN)在细胞增殖中的功能研究(J),高技术通讯,2003,2(4)：25-29。
4 Michihiro Hirana, Fumiyuki Takahashi, Kazuhisa Takahashi,et al. Osteopontin overproduced by tumor cells acts as a potent angiogenic factor contributing to tumor growth (J). Cancer Lett, 2003,198 (2):107-117.
5 Mi Z, Guo H, Russel MB, et al. RNA aptamer blockade of osteopontin inhibits growth and metastasis of MDA-MB231 breast cancer cells [J]. Mol Ther,2009,17(1):153-161.
6 侯志波,郑杰.骨桥蛋白下调对前列腺癌PC3细胞生物学行为的影响[J].肿瘤,2005,25(6)：542-546.
7 崔越宏,刘天舒,陈世耀,等.骨桥蛋白反义寡核苷酸抑制胃癌细胞MKN28粘附侵袭的实验研究[J].临床肿瘤学杂志,2008,13(7)：585-589.
8 Coppla D. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin Cancer Res.2004, Janl; 10(1):184-190.
9 Rudland PS, Platt-Higgins A, El-Tanani M, et al. Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res,2002,62:3417-3427.
10 Robillard PY, Chaouat G, Le Bouteiller P, et al. Current debates on immunology of preeclampsia. Report of the sixth international workshop of Reunion Island (Indian Ocean, December 2008). Gynecol Obstet Fertil,2009,37(6):570-578
11 Zhang J, Takahashi K, Takahashi F, et al. Differential osteopontin expression in lung cancer. Cancer Letters,2001,171:215-222.
12 Denhardt DT, Commentary re:Q-T,et al. identification of osteopontin as a prognostic marker for head and nece squamousce carcinomas. Clin Cancer Res,2003,9:31-32.
13 Denhardt DT, Mistretta D, Chambers AF,et al. Transcriptional regulation of osteopontin and the metastatic phenotype:evidence for a ras-activated enhance the human OPN promoter. ClinExpmetastasis,2003,20:77-84.
14何太平,严卫红,梁念慈.Ras信号转导通路[J].国外医学·临床生物化学与检验学分册,2004,25：74-76。
15 Futakawa N, Kimura M, Yamagata S, et al. Significance of K-RAS mutation and CEA level in pancreatic juice in the diagnosis of pancreatic cancer[J].J Hepatobiliary Pancreat Surg, 2000,7(1):63.
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