人肺腺癌转移相关分子的定量蛋白质组学研究
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摘要
肺癌是目前全球发病率和死亡率较高的恶性肿瘤之一,肺癌按组织学分类一般分为鳞癌、腺癌、大细胞癌和小细胞癌四型。近年来,人肺腺癌(lung adenocarcinoma,AdC)的发病率逐步上升,且病人的死亡率高、预后较差。肺腺癌死亡率高和预后差的主要原因是癌细胞易转移。临床上,对肺腺癌进行早期发现或控制癌细胞转移是降低其死亡率的有效手段。癌细胞转移是一个极其复杂的过程,涉及多因素、多阶段和多基因的参与,众多的正或负相关基因起着促进或抑制癌细胞转移的作用。然而,这些基因大多都是通过翻译成蛋白质来行使其促进或抑制癌细胞转移的功能。因此,直接筛选人肺腺癌转移相关蛋白质将为寻找能预测转移的分子标志物或临床治疗靶标奠定基础。
为了筛选肺腺癌转移相关的蛋白质,依据临床诊断选取无转移的原发性肺腺癌组织和有转移的原发性肺腺癌组织作为研究对象,首先采用激光捕获显微切割(laser capture microdissection,LCM)技术对两组原发性肺腺癌组织中的癌细胞进行纯化,再利用双向荧光差异凝胶电泳技术(two-dimensional fluoresence difference gel electrophoresis,2D-DIGE)分离无转移肺腺癌组和有转移肺腺癌组中纯化的癌细胞总蛋白,通过Decyder软件分析两组差异表达的蛋白质点,质谱(mass spectrometry,MS)技术对差异表达的蛋白质点进行鉴定,Western blot验证部分差异蛋白质S100A9,B23,annexin A1,annexin A2和annexin A3的表达。为探讨部分差异蛋白质表达的临床病理意义,采用免疫组化染色检测部分差异蛋白质在存档的石蜡包埋组织中的表达,统计学分析差异蛋白质的表达水平与肺腺癌临床病理因素及患者复发和预后的关系。
本研究建立了LCM纯化的无转移和有转移肺腺癌组织中癌细胞的2D-DIGE图谱,质谱鉴定了20个非冗余差异蛋白质。与无转移肺腺癌组相比,13个蛋白在有转移肺腺癌组表达上调,7个蛋白在有转移肺腺癌组中表达下调。Western blot验证分析显示,差异蛋白S100A9,annexin A1,annexin A2和annexin A3的表达水平在有转移肺腺癌组中较无转移肺腺癌组增高;B23的表达水平在有转移肺腺癌组中较无转移肺腺癌组降低。免疫组化进一步证实:与肺腺癌原发癌组织相比,annexin A1,annexin A2,annexin A3的表达水平在淋巴结转移癌组织中表达上调。统计学分析发现annexin A1,annexin A2和annexin A3的表达水平与肺腺癌的淋巴结转移和临床分期有关。Kaplan-Meier曲线和Cox回归分析进一步发现,annexinA1,annexin A2和annexin A3的表达水平与肺腺癌患者的复发和预后有关,annexin A1,annexin A2或annexin A3高表达的肺腺癌患者易复发,且生存率低,预后差。
本研究首次应用LCM方法联合2D-DIGE及MS技术,分析、鉴定出人肺腺癌转移相关蛋白质,为研究人肺腺癌转移的分子机制、筛选能预测人肺腺癌转移的分子标志物奠定了基础。
Lung cancer is one of malignant tumors with very high morbidity and mortality.According to histological types,lung cancer is divided into four subtypes including lung squamous carcinoma,lung adenocarcinoma,small cell lung cancer and large cell lung cancer.In recent years,the incidence rate of human primary lung adenocarcinoma(AdC) has clearly been on the increase.And the patients of lung AdC have very low survival rate and very poor prognosis.Cancer cell metastasis is the major cause of high mortality and poor prognosis in lung AdC.In clinic,early-finding and controlling cancer cell metastasis is effective strategy to decrease the mortality.However,cancer cell metastasis is a very complicated process which is involved with many factors, many steps and many genes.A variety of positive and negative genes may be involved in this highly sophisticated process,which play important role in promoting or inhibiting cancer cell metastasis.Furthermore,these genes should be translated into proteins to exert the function of controlling cancer cell metastsis.Therefore,screening metastasis-related proteins directly is to provide the foundation for finding predictive metastatic biomarkers and clinical treat targets.
To screen metastasis-associated biomarkers of lung AdC,laser capture microdissection(LCM) was used to purify the cancer cells from human primary lung AdC with(LNM AdC) and without metastasis(non-LNM AdC) according to clinical diagnosis of lymph node metastasis and distant metastasis.Then two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was performed to isolate the total proteins of the pooled microdissected cancer cells from non-LNM AdC and LNM AdC.The differential proteins between non-LNM AdC and LNM AdC were analyzed by Decyder software and further identified by mass spectrometry(MS).The partial differential proteins including S100A9,B23,annexin A1,annexin A2 and annexin A3 were validated by Western blot.To further study the associations between expression levels of the partial differential proteins with clinical pathological fators and evaluate their clinicopathological significance, immunohistochemical technique was performed to analyze the expression levels of these proteins in archived paraffin imbedded tissues.And statistical analysis evaluated the relationships of their expression levels and clinical pathological factors and patients' relapse and outcome.
In the present study,2D-DIGE patterns of microdissected non-LNM AdC and LNM AdC were established,and 20 differential proteins in the above two tissues were identified,13 out of which were up-regulated and 7 were down-regulated in LNM AdC compared to non-LNM AdC.Western blot results indicated that S100A9,annexin A1,annexin A2 and annexin A3 were significantly up-regulated in LNM AdC compared to non-LNM AdC;B23 was significantly down-regulated in LNM AdC compared with non-LNM AdC.Immunohistochemical analysis further indicated S100A9,annexin A1, annexin A2 and annexin A3 were up-regulated in LNM AdC compared with non-LNM AdC;B23 was down-regulated in LNM AdC compared with non-LNM AdC.Furthermore,annexin A1,annenxin A2 and annexin A3 were up-regulated in positive lymph nodes compared with primary lung adenocarcinoma.Statistical analysis indicated the increase of the three annexins expression levels in these tumors was significantly associated with lymph node metastasis and advanced clinical stage.Kaplan-Meier curve and Cox regression analysis indicated annexin A1,annexin A2 and annexin A3 expression levels were correlated with relapse and survival.This is to say,the three annexins over-expression were associated with increased relapse rate and decreased survival rate.
It was the first time that metastasis-associated proteins were identified in human primary lung AdC by LCM coupled with 2D-DIGE and MS techniques. Our findings will facilitate understanding of human lung AdC metastasis and provide some direct proof for mining markers for predicting metastasis and patients' outcome so as to improve the diagnosis and treatment of lung AdC.
为了筛选肺腺癌转移相关的蛋白质,依据临床诊断选取无转移的原发性肺腺癌组织和有转移的原发性肺腺癌组织作为研究对象,首先采用激光捕获显微切割(laser capture microdissection,LCM)技术对两组原发性肺腺癌组织中的癌细胞进行纯化,再利用双向荧光差异凝胶电泳技术(two-dimensional fluoresence difference gel electrophoresis,2D-DIGE)分离无转移肺腺癌组和有转移肺腺癌组中纯化的癌细胞总蛋白,通过Decyder软件分析两组差异表达的蛋白质点,质谱(mass spectrometry,MS)技术对差异表达的蛋白质点进行鉴定,Western blot验证部分差异蛋白质S100A9,B23,annexin A1,annexin A2和annexin A3的表达。为探讨部分差异蛋白质表达的临床病理意义,采用免疫组化染色检测部分差异蛋白质在存档的石蜡包埋组织中的表达,统计学分析差异蛋白质的表达水平与肺腺癌临床病理因素及患者复发和预后的关系。
本研究建立了LCM纯化的无转移和有转移肺腺癌组织中癌细胞的2D-DIGE图谱,质谱鉴定了20个非冗余差异蛋白质。与无转移肺腺癌组相比,13个蛋白在有转移肺腺癌组表达上调,7个蛋白在有转移肺腺癌组中表达下调。Western blot验证分析显示,差异蛋白S100A9,annexin A1,annexin A2和annexin A3的表达水平在有转移肺腺癌组中较无转移肺腺癌组增高;B23的表达水平在有转移肺腺癌组中较无转移肺腺癌组降低。免疫组化进一步证实:与肺腺癌原发癌组织相比,annexin A1,annexin A2,annexin A3的表达水平在淋巴结转移癌组织中表达上调。统计学分析发现annexin A1,annexin A2和annexin A3的表达水平与肺腺癌的淋巴结转移和临床分期有关。Kaplan-Meier曲线和Cox回归分析进一步发现,annexinA1,annexin A2和annexin A3的表达水平与肺腺癌患者的复发和预后有关,annexin A1,annexin A2或annexin A3高表达的肺腺癌患者易复发,且生存率低,预后差。
本研究首次应用LCM方法联合2D-DIGE及MS技术,分析、鉴定出人肺腺癌转移相关蛋白质,为研究人肺腺癌转移的分子机制、筛选能预测人肺腺癌转移的分子标志物奠定了基础。
Lung cancer is one of malignant tumors with very high morbidity and mortality.According to histological types,lung cancer is divided into four subtypes including lung squamous carcinoma,lung adenocarcinoma,small cell lung cancer and large cell lung cancer.In recent years,the incidence rate of human primary lung adenocarcinoma(AdC) has clearly been on the increase.And the patients of lung AdC have very low survival rate and very poor prognosis.Cancer cell metastasis is the major cause of high mortality and poor prognosis in lung AdC.In clinic,early-finding and controlling cancer cell metastasis is effective strategy to decrease the mortality.However,cancer cell metastasis is a very complicated process which is involved with many factors, many steps and many genes.A variety of positive and negative genes may be involved in this highly sophisticated process,which play important role in promoting or inhibiting cancer cell metastasis.Furthermore,these genes should be translated into proteins to exert the function of controlling cancer cell metastsis.Therefore,screening metastasis-related proteins directly is to provide the foundation for finding predictive metastatic biomarkers and clinical treat targets.
To screen metastasis-associated biomarkers of lung AdC,laser capture microdissection(LCM) was used to purify the cancer cells from human primary lung AdC with(LNM AdC) and without metastasis(non-LNM AdC) according to clinical diagnosis of lymph node metastasis and distant metastasis.Then two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was performed to isolate the total proteins of the pooled microdissected cancer cells from non-LNM AdC and LNM AdC.The differential proteins between non-LNM AdC and LNM AdC were analyzed by Decyder software and further identified by mass spectrometry(MS).The partial differential proteins including S100A9,B23,annexin A1,annexin A2 and annexin A3 were validated by Western blot.To further study the associations between expression levels of the partial differential proteins with clinical pathological fators and evaluate their clinicopathological significance, immunohistochemical technique was performed to analyze the expression levels of these proteins in archived paraffin imbedded tissues.And statistical analysis evaluated the relationships of their expression levels and clinical pathological factors and patients' relapse and outcome.
In the present study,2D-DIGE patterns of microdissected non-LNM AdC and LNM AdC were established,and 20 differential proteins in the above two tissues were identified,13 out of which were up-regulated and 7 were down-regulated in LNM AdC compared to non-LNM AdC.Western blot results indicated that S100A9,annexin A1,annexin A2 and annexin A3 were significantly up-regulated in LNM AdC compared to non-LNM AdC;B23 was significantly down-regulated in LNM AdC compared with non-LNM AdC.Immunohistochemical analysis further indicated S100A9,annexin A1, annexin A2 and annexin A3 were up-regulated in LNM AdC compared with non-LNM AdC;B23 was down-regulated in LNM AdC compared with non-LNM AdC.Furthermore,annexin A1,annenxin A2 and annexin A3 were up-regulated in positive lymph nodes compared with primary lung adenocarcinoma.Statistical analysis indicated the increase of the three annexins expression levels in these tumors was significantly associated with lymph node metastasis and advanced clinical stage.Kaplan-Meier curve and Cox regression analysis indicated annexin A1,annexin A2 and annexin A3 expression levels were correlated with relapse and survival.This is to say,the three annexins over-expression were associated with increased relapse rate and decreased survival rate.
It was the first time that metastasis-associated proteins were identified in human primary lung AdC by LCM coupled with 2D-DIGE and MS techniques. Our findings will facilitate understanding of human lung AdC metastasis and provide some direct proof for mining markers for predicting metastasis and patients' outcome so as to improve the diagnosis and treatment of lung AdC.
引文
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