Clinical significance of RRM1, CDX2 expression in different gastric mucosa lesions

目的探讨核苷酸还原酶亚单位M1（ribonucleotide reductase M1,RRM1）、尾型同源盒转录因子2（caudal-related homeobox transcription factor 2,CDX2）在不同胃黏膜组织中的表达,并分析其与胃癌发生发展及预后的关系。方法选取中国人民解放军第一七五医院病理科保存的组织蜡快240例,其中胃癌90例、高级别上皮内瘤变30例、低级别上皮内瘤变30例、肠上皮化生30例、浅表性胃炎30例、正常胃黏膜30例,采用S-P免疫组化方法检测6类不同胃黏膜组织RRM1、CDX2表达,进行临床病理相关性分析。结果正常胃黏膜、浅表性胃炎、肠上皮化生、低级别上皮内瘤变、高级别上皮内瘤变和胃癌中RRM1的阳性表达分别为40.0%、66.7%、33.3%、83.3%、83.3%、86.7%,胃癌、高级别上皮内瘤变、低级别上皮内瘤变组RRM1的阳性表达率均显著高于正常胃黏膜组与肠上皮化生组（P〈0.01）,亦高于浅表性胃炎组（P〈0.05）。CDX1的阳性表达分别为16.7%、0%、63.3%、33.3%、76.7%、61.1%,高级别上皮内瘤变、肠上皮化生、胃癌组的CDX2阳性表达率均显著高于浅表性胃炎组与正常胃黏膜组（P〈0.01）,高于低级别上皮内瘤变组（P〈0.05）。240例不同胃黏膜病变中,RRM1与CDX2之间表达呈正相关（rs=0.196,P=0.002）,90例胃癌中RRM1与CDX2之间呈正相关关系（rs=0.223,P=0.034）;RRM1、CDX2表达与肿瘤分化程度密切相关（P〈0.05）,CDX2表达与肿瘤浸润深度、淋巴结转移、TNM分期密切相关（P〈0.05）。结论 RRM1、CDX2表达均与胃癌的发生发展及预后有关,均可作为较为理想的肿瘤标志物,用于胃癌的早期诊断和转移的预警。Objective To explore the expression of ribonucleotide reductase M! （RRM1）and caudal- related homeobox transcription factor 2 （CDX2） in different gastric mucosa lesions and evaluate its relationship with the occurrence, development and prognosis of gastric cancer. Methods A total of 240 cases were selected from Department of Pathology of 175th Hospital of PLA, including 90 cases of gastric cancer,30 cases of highgrade intraepithelial neoplasia,30 cases of lowgrade intraepithelial neoplasia,30 cases of intestinal metaplasia, 30 cases of superficial gastritis, 30 cases of normal gastric mucosa. Expression of RRM1 and CDX2 was detected with immunohistochemistry SP method. Results The frequency of RRM1 expression were 86.7% ,83.3% and 83.3% in gastric cancer group, high - grade intraepithelial neoplasia group and low - grade intraepithelial neoplasia group, respectively. The frequencies were all higher than that in superficial gastritis group （66.7% ,P 〈0.05） and that in intestinal metaplasia group （33.3% ,P 〈0.01 ）. The frequency of RRM1 expression was higher in superficial gastritis group than that in intestinal metaplasia group（ P 〈 0.05 ）. The frequency of CDX2 expression were 76.7% ,63.3% and 61.1% in high - grade intraepithelial neoplasia group, intestinal metaplasia group and gastric cancer group, respectively. The frequencies were all higher than that in low - grade intraepithelial neoplasia group （33.3% ,P 〈 0.05 ） and that in superficial gastritis group （0%, P 〈 0.01 ）. In 240 cases of different gastric mucosa lesions, RRM1 expression was positively correlated with CDX2 expression（rs = 0. 196, P = 0. 002）. In 90 cases of gastric cancer, RRM1 expression was positively correlated with CDX2 （ r = 0. 223, P = 0. 034）. The expression of RRM1 and CDX2 were related with the differentiation degree （ P 〈 0.05 ）. CDX2 expression was related with the tumor invasion depth, TNM stages and the lymph node metastasis （P 〈 0.05）. Conclusion The expression of RRM1 and福建省教育厅B类科技基金资助项目（JB12319

Expression and clinical significance of RRM1,CDX2 in gastric cancer tissues

目的探讨RRM1、CDX2在胃癌组织中的表达,并分析其与胃癌侵袭与转移的关系。方法选取90例术前未行放化疗的胃癌石蜡标本及与其对应正常胃黏膜蜡标本30例,采用SP免疫组化方法检测RRM1和CDX2在两种组织中表达情况,分析二者表达与分化程度、浸润深度、淋巴结转移、TNM分期之间的关系。结果 （1）RRM1、CDX2在胃癌中的阳性表达率均显著高于正常胃黏膜组（86.7%vs 40.0%,P〈0.01;61.1%vs 16.7%,P〈0.01）。（2）90例胃癌中,RRM1与CDX2二者之间呈正相关关系（r_s=0.223,P=0.034）;RRM1、CDX2表达与分化程度密切相关（P〈0.05）,CDX2表达与浸润深度、淋巴结转移、TNM分期密切相关（P〈0.05）,RRM1表达与浸润深度、淋巴结转移、TNM分期无相关（P〉0.05）。结论 RRM1、CDX2可作为较为理想的肿瘤标志物,用于胃癌的诊断和预后的预警。Objective To explore the expression of RRM1 and CDX2 in gastric cancer tissues and its relationship with invasion and metastasis of gastric cancer. Methods Expression of RRM1 and CDX2 was detected in 90 cases of gastric cancer without preoperative chemoradiotherapy and 30 cases of normal gastric mucosa using immunohistochemistry SP method. The relationships of RRM1 and CDX2 expression with differentiation degree,tumor invasion depth,TNM stage and lymph node metastasis were analyzed. Results（1）The positive expression rates of RRM1 and CDX2 were both significantly higher in gastric cancer tissues than in those of normal gastric mucosa（86. 7% vs 40. 0%,P 〈 0. 01; 61. 1% vs 16. 7%,P 〈 0. 01）.（2） The expression of RRM1 in 90 cases of gastric cancer was positively correlated with CDX2（ r_s= 0. 223,P = 0. 034）. The expression of RRM1 and CDX2 was related with differentiation degree（ P〈 0. 05）. The expression of CDX2 was related with tumor invasion depth,TNM stage and lymph node metastasis（ P 〈 0. 05）. The expression of RRM1 was not related with tumor invasion depth,TNM stage,lymph node metastasis（ P 〉 0. 05）. Conclusion RRM1 and CDX2 can be used as ideal tumor markers in diagnosis of gastric cancer and prognosis.福建省教育厅B类科技基金资助项目（JB12319

Differentiation Induced by Ginsenoside Rg1 Combined Formula Involved the Alteration of Prohibitin Localization and Expression in Human Osteosarcoma MG-63 Cells

选择性抽提经人参皂苷Rg1组合(RCT)诱导处理前后的人成骨肉瘤MG-63细胞核基质,对prohibitin在核基质中的存在、分布及其与相关基因产物在RCT处理前后MG-63细胞中的共定位关系进行观察研究.蛋白质组学分析结果显示,prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在RCT处理后细胞核基质中表达下调;蛋白质印迹杂交确证了prohibitin在MG-63细胞核基质中的存在及其在RCT处理后下调变化;免疫荧光显微镜观察进一步证实prohibitin定位在核基质上,经RCT处理后出现分布位置与表达水平变化;激光共聚焦显微镜观察可见prohibitin与c-Fos、c-Myc、p53和Rb基因产物均存在共定位关系,并在RCT处理后共定位分布区域出现变化.本研究证实了prohibitin是一种新发现的核基质蛋白,其在核基质上的定位与表达在RCT诱导分化前后发生显著变化,并与相关癌基因、抑癌基因产物存在共定位关系.实验表明RCT处理引起的prohibitin的变化与人成骨肉瘤MG-63细胞的诱导分化与调控具有密切关系,为深入揭示RCT等中药有效成分诱导肿瘤细胞分化的机理提供了重要科学依据和深入探索的新方向.Prohibitin is a nuclear matrix protein and associates with various oncogene products.A combined formula with ginsenoside Rg1,cinnamic acid and tanshinoneⅡA(RCT)was used to treat human osteosarcoma MG-63 cells in this study.We found that the prohibitin expression in the nuclear matrix was of decreased following RCT treatment by Western blot.The colocalization of prohibitin with the products of oncogenes or tumor suppressor genes,including c-Fos,c-myc,p53 and Rb was evaluated by immunofluorescence staining and laser scanning confocal microscopy.We observed significant alternations of prohibiitin distribution and the degree of colocalization with the associated gene products after the cells exposed to RCT.The results suggested that prohibitin might be involved in the RCT-induced differentiation in MG-63 cells.Our findings may provide information to expand the applications of Traditional Chinese Medicine formulas in cancer-related studies.国家自然科学基金资助项目(No.30470877)~

Expression and Localization of Nucleophosmin During HMBA-induced Differentiation in Human Hepatocarcinoma SMMC-7721 Cells

To explore the existence and distribution of nucleophosmin in the nuclear matrix and its co-localization with the other related gene products following HMBA treatment in the human hepatocarcinoma SMMC-7721 cells,the nuclear matrix of SMMC-7721 cells was extracted pre/post HMBA induced differentiation.2D PAGE proteomics analyses showed that nucleophosmin existed in the fractions of nuclear matrix proteins and was down-regulated after HMBA treatment with further confirmation by Western blot analysis.The immunofluorescence observation revealed that nucleophosmin located in the nuclear matrix,HMBA treatment altered its expression level and distribution profile.The co-localization of nucleophosmin with cancer-related genes and the products of oncogenes or tumor repression genes,including c-fos,c-myc,p53 and Rb,using laser scanning confocal microscopy,were evaluated,and substantial differences were observed following HMBA treatment.The results implies that nucleophosmin,as a nuclear matrix protein,the level of its expression and the colocalization with cancer-related gene products may play an important role during the differentiation of SMMC-7721 cell.国家自然科学基金资助项目(No.30470877)~

Tachyplesin-Induced Differentiat ion of Human Hepatocarcinoma Cell Line SMMC-7721

背景与目的:海洋生物活性物质抗肿瘤活性研究是海洋生物活性物质开发与抗癌药物研究的一个重要领域。诱导肿瘤细胞分化则是肿瘤药物治疗的新策略。本文拟研究海洋生物活性物质鲎素对人肝癌SMMC-7721细胞分化的影响,以为鲎素抗肿瘤作用及其机理的深入研究提供实验依据。方法:从中国鲎血细胞酸抽提液提取鲎素,应用光镜和透射电镜观察3.0μg/ml鲎素处理前后人肝癌SMMC-7721细胞形态和超微结构的变化,应用细胞化学或免疫细胞化学方法观察鲎素处理前后细胞碱性磷酸酶活性与甲胎蛋白和增殖细胞核抗原表达的变化。结果:经3.0μg/ml鲎素处理的SMMC-7721细胞形态和超微结构发生恢复性变化,碱性磷酸酶活性减弱,甲胎蛋白和增殖细胞核抗原表达降低。结论:鲎素能有效改变肝癌细胞恶性形态和超微结构特征,改变肝癌细胞相关酶活性和抗原表达,对肝癌细胞具有一定的诱导分化作用。Background &Objective:The study on antitumor activities of marine bioactive substances is an important field in exploiting marin e bioactive substances and antitumo r drugs.And the induction of tumor cell differentiation is a new strategy for drug therapy of t umors.So the authors used tachyplesin,a marine bioactive substance,to investigate its effects on the differentiation of human hepatocarcinoma SMMC-7721cell s for further studying its antitumor activities and mechanisms.Methods:Tachyplesin,which was isolated fro m hemocytes of horseshoe crab(Tachypleus tridentatus),was used to treat human hepatocarcinoma SMMC-7721cells.Light microscopy a nd transmission electron microscop y were applied to examine the changes in morphology and ultrastructure of SMMC-7721cells,respectively.The activ ities of alkaline phosphatase or the expression of AFP and PCNA were assessed by cytochemis try or immunocytochemistry.Results:In the cells treated with 3.0μg /ml tachyplesin,the morphology an d ultrastructure returned to be norm al,the activity of alkaline phosphatase was decreased and the le vels of AFP and PCNA were downregulated.Conclusion:Tachyplesin might effectively reverse the malignant morphology an d ultrastructure,change the activity of enzyme and the levels of a ntigens associated with hepatocarcinoma cell,and induce th e differentiation of the human hepatocarcinoma SMMC-7721cells.教育部高等学校骨干教师资助计划项目;; 教育部重点实验室访问学者基金项

LOCALIZATION OF PROHIBITIN IN NUCLEAR MATRIX AND ITS EXPRESSIVE ALTERATION DURING THE DIFFERENTIATION OF HUMAN OSTEOSARCOMA MG-63 CELLS INDUCED BY HMBA

本文以HMBA诱导处理前后的人成骨肉瘤MG-63细胞为对象,对prohibitin在核基质中存在、分布及其与相关基因产物在HMBA处理前后MG-63细胞中的共定位关系进行观察研究。蛋白印迹杂交结果确证prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在HMBA处理后细胞核基质中表达下调,免疫荧光显微镜观察显示prohibitin定位在核基质上,经HMBA处理后出现分布位置与表达水平的变化,激光共聚焦显微镜观察可见prohibitin与MG-63细胞中c-fos、c-myc、p53和rb基因产物均存在共定位关系,但在HMBA处理后细胞中其共定位分布区域出现变化。研究结果证实prohibitin是一种核基质蛋白,定位于核基质上,prohibitin在人成骨肉瘤MG-63细胞诱导分化过程中的表达分布及其与相关癌基因、抑癌基因产物的共定位现象值得进一步探索和研究。To explore the existence,distribution of prohibitin in nuclear matrix and the co-localization relationship between prohibitin and the products of some interrelated genes in the human osteosarcoma MG-63 cells before and after HMBA treatment, the nuclear matrix of MG-63 cells and the cells induced by HMBA were selectively extracted. It was confirmed by Western blot that prohibitin existed in the component of nuclear matrix protein of MG-63 cells and its expression was decreased by HMBA treatment. The immunofluorescence observation revealed that prohibitin located in the nuclear matrix, and its distribution regions and expression level had altered after HMBA treatment. The co-localization and its alternation of distributive area in the cells treated by HMBA were observed between prohibitin and the products of oncogenes or tumor repression genes including c-fos, c-myc, p53 and Rb by using laser scanning confocal microscopy.The results of this study demonstrated that prohibitin was a kind of nuclear matrix protein, and locacted in the nuclear matrix,and the distribution and expression of prohibitin and its relationship with associated genes play an important role during the differentiation of MG-63 cell.国家自然科学基金资助项目(No.30470877

The Localizational and Expressive Alteration of Nucleophosmin during the Differentiation of Human Osteosarcoma MG-63 cells Induced by the Combination of Ginsenoside Rg1

选择性抽提经中药有效成分人参皂甙Rg1组合(简称RCT)诱导处理前后的人成骨肉瘤MG-63细胞核基质,对nucleophosmin(NPM)在核基质中的存在、分布及其与相关基因产物在MG-63细胞中的共定位关系进行了观察研究。双向凝胶电泳和质谱鉴定结果显示NPM存在于MG-63细胞核基质蛋白组分中,在RCT处理后细胞核基质蛋白中表达下调。蛋白质印迹杂交结果证实了NPM在RCT处理前后的MG-63细胞核基质蛋白中的存在及其表达下调变化。免疫荧光显微镜观察显示NPM定位于MG-63细胞核基质上,经RCT处理后出现分布位置与表达水平的变化。免疫荧光-激光扫描共聚焦显微镜的观察结果显示NPM在MG-63细胞中与c-fos、c-myc、RB、p53等基因产物具有共定位关系,并在RCT处理后细胞核内其共定位区域发生了变化。研究结果证实了NPM存在于核基质上,是一种核基质蛋白,其在RCT诱导人成骨肉瘤MG-63分化过程中的表达与分布变化及其与相关癌基因、抑癌基因的关系显然对MG-63细胞分化具有重要影响,这为深入揭示中药有效成分诱导肿瘤细胞分化的机制提供了重要科学依据和深入探索的新方向。To explore the existence, distribution of nucleophosmin (NPM) in the nuclear matrix of the osteosarcoma MG-63 cells and the co-localizational relationship between NPM and the products of some interrelated genes, the MG-63 cells treated with or without the combination of ginsenoside Rg1, cinnamic acid and tanshinone II A (RCT) were selectively extracted and subjected to proteomic methodologies and laser scanning confocal microscopy (LSCM). 2-D PAGE, MS and Western blotting methods confirmed the existence of NPM and its decreased expression in the nuclear matrix proteins. LSCM observation showed that there were altered co-localization after RCT treatment between NPM and the product of genes c-fos, c-myc, RB and p53, respectively. Our study confirmed the existence of NPM in the nuclear matrix and the important role its expression and distribution played during the differentiation of MG-63 cells induced by effective ingredients from Chinese materia medica.国家自然科学基金资助项目(No.30470877)~