肺癌是全球癌症相关死亡的首要原因。近年来，肺癌的发病率和死亡率在中国还在不断升高。由于肺癌不易进行筛查和早期诊断，肺癌患者在确诊时通常已经是晚期，在过去的三十年中，肺癌的生存率并没有得到明显提高，如何早期诊断肺癌仍然是目前研究的重点。肺癌有2个主要类型：小细胞肺癌（SCLC）和非小细胞肺癌（NSCLC），其中非小细胞肺癌占所有肺癌的85％左右。近三十年来，在NSCLC中腺癌仍然是妇女最主要的亚型，并且也已经超越鳞癌成为全球男性肺癌的主要亚型。 蛋白酶激活受体（Protease-activated receptors, PARs）是一种多功能的G蛋白偶联型受体。该家族有四个成员（PAR1、PAR2、PAR3、PAR4），其中PAR1和PAR2已被证明与多种肿瘤的生长、侵袭和转移相关，但对PAR3和PAR4的研究还很少。PAR4在人体组织中广泛表达，并已发现在关节炎症、肝细胞癌细胞的迁移、结肠癌进展等过程中发挥一定作用。在前期本实验室的研究中发现PAR4在胃癌组织中表达下调，并与胃癌的分化程度和进展密切相关，首次报道了PAR4这一特殊的G蛋白偶联受体在胃癌进展中可能发挥负性调控的作用，提示PAR4或许是一个潜在的肿瘤抑制基因。 抑制素（Prohibitins，PHBs）属于SPFH家族，是在真核生物中广泛表达且高度保守的具有多种生物学功能的蛋白质。哺乳类的抑制素蛋白目前包括两种，即抑制素蛋白1（PHB1）与抑制素蛋白2 （PHB2），它们与细胞的代谢、增殖、发育、衰老、转化等相关。尽管PHB1有抑制细胞增殖的作用，但在多种肿瘤中均发现其过度表达。PHB1与肿瘤的复杂关系可能与其亚细胞定位有关。 本实验室前期在以血小板为模型的研究中，发现PHBs与PAR1在血小板上分布一致，并可以与PAR1相互作用，首次揭示PHBs与PAR1的联系以及PHBs蛋白对PAR1信号通路具有调节作用。在乳腺癌细胞系上进一步的实验结果表明PHB1蛋白可能参与了PAR1的蛋白降解过程。提示PHBs与PARs之间可能存在密切联系。 四种PARs均在人的正常肺组织中存在并与肺部的炎症、哮喘、慢性阻塞性肺病和肺癌等疾病相关。其中PAR4在肺组织中大量表达但它在肺上皮细胞中的生理和病理学意义还不明确，其与肺癌的的相关性也还没有深入研究。PHB1在肺癌中的研究不多且结果具有争议。因此，我们在前期研究的基础上，进一步选取肺癌来进行相关研究。收集一定数量的肺癌组织样本，结合患者的临床病例资料，运用分子生物学的方法分别检测PAR4和PHB1在肺癌和癌旁正常肺组织以及肺癌细胞系中的表达情况。根据研究内容不同，本论文分为两个部分。 对肺腺癌组织中PAR4的检测结果显示PAR4的mRNA和蛋白在正常肺组织中阳性表达，但当与配对的癌旁正常肺组织相比时，肺腺癌组织中mRNA的表达水平普遍下降（67.7％），并且与低的分化程度（P = 0.017）和远处转移（P = 0.048）相关。Western blotting及免疫组化分析也显示大部分肺腺癌组织中PAR4的蛋白水平下降（61.3％），并与分化程度（P=0.035）和临床分期（P = 0.027）相关。此外，与BEAS-2B细胞相比，肺腺癌NCI-H157细胞中PAR4的表达也减少。提示PAR4可能在肺腺癌的发生发展中起到负性调控的作用。 对非小细胞肺癌（NSCLC）中PHB1的检测结果显示，PHB1 的mRNA和蛋白在肺癌组织中的表达普遍高于配对的癌旁组织，并且PHB1的增高程度与肺癌组织的分化程度和淋巴结转移等密切相关。与BEAS-2B细胞相比，PHB1的表达水平在三种肺癌细胞系（SK-MES-1，NCI-H157，NCI-H292）中也是增加的。此外，在不同的肺癌细胞中，PHB1的亚细胞定位有所不同。并且三种肺癌细胞细胞膜上的PHB1含量均显著减少甚至缺如。结果提示：PHB1的表达上调与NSCLC的进展相关。肺癌细胞系中膜相关PHB1的缺失可能发挥着重要角色。PHB1的不同亚细胞定位提示不同亚型的NSCLC具有不同的发生机制和特征， PHB1在NSCLC的不同亚型中可能扮演不同的角色。 PAR4与PHB1在肺癌的发生、发展中均扮演了一定角色。PAR4可能具有肿瘤抑制的作用，或许是一种潜在的肿瘤抑制基因。肺癌中PHB1总表达量的增加与肺癌的分化程度和进展相关，但细胞膜上的PHB1在肺癌中发挥的怎样的作用以及它的不同亚细胞定位是否与不同亚型肺癌的发生相关这还有待进一步探索。基于实验室的前期研究成果，还需要进一步思考和研究PHB1和PAR4之间是否也存在相互作用。这将有利于进一步深入理解PAR4受体信号的传导及其与PHB1蛋白的关系，理解它们在肿瘤生长与转移中的分子病理意义。; Lung cancer remains the leading cause of cancer-related deaths worldwide and the morbidity and mortality of lung cancer is increasing recent years in China. Because early screening strategies are not available for this malignancy, lung cancer patients typically present at an advanced stage at the time of diagnosis, and there has been little improvement in lung cancer survival over the past 3 decades. There are 2 main types of lung cancer, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 85% of all lung cancer. Of the total, adenocarcinoma has remained the most prevalent lung cancer subtype among women over the past 3 decades, and has surpassed squamous cell carcinoma as the leading subtype of lung cancer in men in the world. Protease-activated receptors (PARs) are multifunctional G protein–coupled receptors and four PAR members (PAR1, PAR2, PAR3 and PAR4) have been cloned and characterized in many animal species. PAR1 and PAR2 have been demonstrated to be implicated in tumor growth, invasion and metastasis in many kinds of malignancies. However, the role of PAR3 and PAR4 in human cancers required further investigation. PAR4 is widely distributed and thought to be involved in joint inflammation, hepatocellular carcinoma cell migration and colon cancer progression. In our previous study, we have found that PAR4 expression is frequently down-regulated in gastric cancer and closely associated with tumor differentiation and clinical progression. It is the first report that the unique GPCR，PAR4，has a negative role in gastric cancer progression and might be a candidate of tumor suppressor. Prohibitins(PHBs) belongs to SPFH family and contains two subtypes： PHB1 and PHB2. They are highly conserved and ubiquitous eukaryotic protein and involved in cell metabolism, proliferation, development, aging and transformation. Despite PHB1 can inhibit cell proliferation, it has been found overexpression in a variety of tumors. The relationship of PHB1 and tumor is complex and related to its subcellular localization . Early study about the platelet in our laboratory have found that the distribution of PHBs on platelets is consistent with PAR1 and PHB1 interact with PAR1. Its the first time to reveal the interaction of PHB1 and PAR1 and PHB1 protein has a regulatory role to PAR1 signaling. Further study on breast cancer cell lines showed that PHB1 may be involved in PAR1 protein degradation. These results prompt that there may be close relationship between PHBs and PARs. The human respiratory tract appears to express all four PAR subtypes and PARs may play roles in the development of inflammation, asthma, chronic obstructive pulmonary disease (COPD) and cancer. Although PAR4 is preferentially expressed in human lung tissues, the physiological and potential pathological functions of PAR4 in lung epithelial cells have yet to been defined and the possible role of PAR4 in lung cancer also has not been defined. There are conflicting data regarding the involvement of PHB1 in tumorigenesis and few studies regarding the role of PHB1 in lung cancer. So, based on early results, the studies reported herein used a combination of clinical observations and molecular methods to investigate the possible role of PAR4 and PHB1 in lung cancer. The results of adenocarcinoma showed PAR4 mRNA expression was generally decreased in lung adenocarcinoma tissues as compared with matched noncancerous tissues (67.7%) and was associated with poor differentiation (P=0.017) and metastasis (P =0.048). Western blotting and immunohistochemical analysis also showed that PAR4 protein levels were mostly decreased in lung adenocarcinoma tissues (61.3%), and were also associated with poor differentiation (P=0.035) and clinical stage (P =0.027). Moreover, PAR4 expression was decreased in NCI-H157 cells as compared with BEAS-2B cells. The results suggest that PAR4 may acts as a tumor suppressor in lung adenocarcinoma.
修改评论