【作者】 曾谷清；

【导师】 肖志强；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2012， 博士

【摘要】 肺癌是全球发病率和死亡率最高的恶性肿瘤。尽管肺癌的诊断和治疗水平已大大提高,但病人的生存率仍然很低,五年生存率低于15%。肺癌患者预后差的主要原因,一方面是由于肺癌发病机理仍然不是很清楚,另方面是缺乏有效的肺癌早期诊断标志物。因此,筛选肺癌早期诊断标志物对于提高疗效、改善预后具有重要意义。肺鳞癌起源于支气管上皮,是一个多基因参与、多阶段的复杂病理学过程。在致癌因素作用下,支气管上皮细胞的癌变过程一般要经过基底细胞增生、鳞状化生,进而进展为不典型增生、原位癌,再发展为浸润癌。为了筛选新的肺鳞癌早期诊断标志物,揭示肺鳞癌癌变的机制,我们首先采用激光捕获显微切割(laser capture microdissection, LCM)技术纯化正常支气管上皮组织、鳞状化生、不典型增生、原位癌、浸润癌组织,然后再用同位素标记相对和绝对定量(iTRAQ)技术结合二维液相色谱串联质谱(2D LC-MS/MS)鉴定支气管上皮癌变过程中各阶段的差异表达蛋白质。本研究共鉴定了102个差异表达蛋白质,在这些差异蛋白质中,有的在支气管上皮癌变过程中进行性上调,有的在支气管上皮癌变过程中进行性下调,有的呈阶段特异性改变。分层聚类分析显示：102个差异表达蛋白质聚集成8个簇,3大群。GO功能注释显示：第1群差异蛋白质主要涉及发育、凋亡、翻译和代谢等生物学过程；第2群差异蛋白质主要涉及器官发育、凋亡、对生物刺激的应答和肌动蛋白丝束的形成等生物学过程；第3群差异蛋白质主要涉及代谢、细胞凋亡、运输、细胞大分子定位、细胞增殖和体液免疫应答等生物学过程。KEGG通路分析显示：这三群差异蛋白质涉及与肿瘤相关的一些信号通路,如MAPK信号通路,凋亡、细胞周期、p53信号通路、以及ErbB信号通路等。同一群差异蛋白为共同调节蛋白,它们在支气管上皮癌变的过程中可能具有相似的生物学功能,并可能通过这些信号通路发挥作用,从而促使支气管上皮癌变。在102个差异蛋白质中,HSPB1的表达在支气管上皮癌变过程中呈进行性上调,GSTP1和CKB的表达在支气管上皮癌变过程中呈进行性下调。因此,我们选择HSPB1、GSTP1和CKB进行深入研究,采用Western blotting检测这三个蛋白质在支气管上皮癌变过程中各阶段组织中的表达,结果与蛋白质组学结果一致；采用免疫组织化学染色检测这三个蛋白质在石蜡包埋的正常支气管上皮、鳞状化生、不典型增生、原位癌、浸润癌组织标本中的表达,并且采用ROC曲线分析评估它们早期诊断肺鳞癌的能力,结果显示：三个蛋白组合判别正常支气管上皮和癌前病变的敏感性和特异性分别为96%和92%、判别正常支气管上皮和肺鳞癌的敏感性和特异性分别为99%和98%、判别癌前病变和肺鳞癌的敏感性和特异性分别为92%和91%,结果表明：HSPB1、GSTP1和CKB联合检测能区分正常支气管上皮、癌前病变和肺鳞癌。为探讨GSTP1在肺鳞癌发病中的作用,我们以永生化的人支气管上皮细胞株16HBE为研究对象,采用siRNA干扰技术建立了稳定沉默GSTP1表达的16HBE细胞系(16HBE/PLO.1-GSTP1-shRNA)及空白载体转染的对照细胞系；采用MTT细胞增殖测定、平板集落形成实验、软琼脂集落实验、流式细胞术和Hoechest33258染色等观察GSTP1表达沉默对苯并芘(B[a]P)转化16HBE细胞的影响。结果显示：与对照细胞比较,在B[a]P的诱导下,GSTP1表达沉默的16HBE细胞的增殖速度明显增快、平板集落数和软琼脂集落数均显著增加、细胞凋亡率显著减少。结果表明：GSTP1表达沉默显著增加16HBE细胞对B[a]P诱导细胞转化的敏感性。提示GSTP1表达下调参与了支气管上皮的癌变,GSTP1表达下调有望成为预测吸烟人群对肺鳞癌易感性的指标。本研究采用LCM结合定量蛋白质组学技术开展支气管上皮癌变各阶段组织的比较蛋白质组学研究,鉴定了102个支气管上皮癌变相关的蛋白质,深入挖掘了102个差异表达蛋白质的生物学功能以及涉及的信号通路；发现HSPB1、GSTP1和CKB联合检测对正常支气管上皮、肺鳞癌癌前病变和肺鳞癌组织具有良好的判别能力,HSPB1、GSTP1和CKB可作为肺鳞癌早期诊断的潜在标志物；首次发现GSTP1表达沉默增加支气管上皮细胞对化学致癌物B[a]P诱导细胞转化的敏感性,提示GSTP1表达下调在支气管上皮癌变中具有重要作用。更多还原

【Abstract】 Lung cancer is the most frequently occurring malignancy with increasing incidence and is the leading cause of mortality in cancer-related deaths in China and worldwide. Although great improvement has been made in diagnosis and treatment of lung cancer, the overall patients’ survival is still very low and less than15%. The poor prognosis of this cancer is mainly explained by the fact that the diagnosis is generally made only at advanced stages due to the lack of reliable, early diagnostic biomarkers and the limited understanding of its carcinogenic mechanisms. Therefore, identification of biomarkers for early detection of lung cancer is mandatory, in turn leading to more effective treatment and reduction of mortality.Lung squamous cell carcinoma (LSCC) originated from the bronchial epithelial cells is the most common histological type of lung cancer. It is known that carcinogenesis of LSCC is a multi-stage process and the result of multistep accumulation of genetic and epigenetic alterations. With exposure to environmental carcinogens, bronchial epithelial carcinogenesis often progresses in the following manner: hyperplasia, squamous metaplasia (SM), atypical hyperplasia (AH), cancer in situ(CIS) and invasive cancer. To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with2D LC-MS/MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result,102differentially expressed proteins were identified. Among these differentially expressed proteins, some proteins are progressively upregulation, some proteins are progressively downregulation in human bronchial epithelial carcinogenic process, and the other proteins are upregulation or downregulation in a certain stage of the process of carcinogenesis. To get more insight on the biological significance of the differentially expressed proteins in bronchial epithelial carcinogenic process, hierarchical clustering was performed on102differentially expressed proteins. All differentially expressed proteins were hierarchically grouped into8clusters and3groups. GO analysis showed that Group1(clusters1and2) was dominated by proteins associated with development, apoptosis, translation and metabolic process, group2(clusters6) consisted of proteins related to biological processes in LSCC, such as multi-organism process, regulation and negative regulation of apoptosis, response to biotic stimulus and actin filament bundle formation, group3(clusters3,4,5,7and8) were enriched with catabolic process, cellular process, apoptosis, protein transport, localization, cell proliferation and humoral immune response. KEGG pathway analysis revealed that the proteins in3groups were involved in cancer-associated signaling pathways such as MAPK signaling pathway, apoptosis, cell cycle and p53signaling pathway, and ErbB signaling pathway. The proteins within the same cluster are co-regulated proteins, and may have similar biological functions during bronchial epithelial carcinogenesis. The differentially expressed proteins may play certain role in bronchial epithelial carcinogenesis by these signaling pathways.Among102differentially expressed proteins, HSPB1was progressively upregulation, GSTP1and CKB were progressively downregulation in human bronchial epithelial carcinogenic process. Therefore, we choose HSPB1, GSTP1, and CKB an in-depth study. The expression of these three proteins in various stage tissues of bronchial epithelial carcinogenesis were detected by Western blotting, the results were concordant with that of the proteomics. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including normal bronchial epithelium, squamous metaplasia, atypical hyperplasia, cancer in situ and invasive cancer, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combined detection of HSPB1, GSTP1, and CKB could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of96%and a specificity of92%in discriminating NBE from preneoplatic lesions, a sensitivity of99%and a specificity of98%in discriminating NBE from invasive LSCC, and a sensitivity of92%and a specificity of91%in discriminating preneoplatic lesions from invasive LSCC, respectively.To investigate the role of GSTP1in carcinogenesis of lung squamous cell carcinoma, we took immortalized human bronchial epithelial cell line16HBE as the study object, established a stable silencing GSTP1expression16HBE cell line (16HBE/PLO.1-GSTP1-shRNA) and the empty vector transfected control cell line using siRNA techlique, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation by MTT assay, anchorage dependent and independent colony formation assays, flow cytometry analysis and hoechst33258staining of apoptotic cells. The results showed that cell growth rate was significantly higher, the number of plate colonies and soft agar colonies was significantly increased, the rate of apoptosis was significantly reduced in6HBE cells with knockdown of GSTP1than in the control cells induced by B[a]P. Taken together, these results demonstrated that knockdown of GSTP1increased the susceptibility of human bronchial epithelial cell transformation induced by B[a]P, supporting that GSTP1downregulation involved in human bronchial epithelial carcinogenesis. GSTP1downregulation may be the susceptibility biomarker of prediction lung squamous cell carcinoma for smokers.In this study, Comparative Proteomics Analysis was performed in various stage tissues of bronchial epithelial carcinogenesis using LCM combined with quantitative proteomics technology. We identified102differentially expressed proteins and studied their biological functions and signaling pathway which were related to human bronchial epithelial carcinogenesis. The results demonstrated that the combined detection of HSPB1, GSTP1, and CKB could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC; GSTP1, HSPB1and CKB are novel potential biomarkers for early detection of LSCC. We first found that knockdown of GSTP1in6HBE cells can increase the sensitivity of bronchial epithelial cells to malignant transformation induced by chemical carcinogens B[a]P, GSTP1downregulation plays an important role in lung squamous carcinoma carcinogenesis.更多还原