【作者】 黎联；

【导师】 梅同华； 周向东；

【作者基本信息】 重庆医科大学 ， 内科学， 2009， 博士

【摘要】 肺癌是当前人类发病率最高的恶性肿瘤,肺癌的侵袭与转移是其恶性标志和特征,也是造成病人死亡的主要原因。而基底膜和细胞外基质(主要成分是IV型胶原、层粘连蛋白、纤维连接蛋白等)的降解和破坏是肿瘤转移多阶段过程中的重要步骤。多种蛋白酶家族如基质金属蛋白酶、丝氨酸蛋白酶、半胱氨酸蛋白酶等可能促进各种肿瘤的侵袭转移,基质金属蛋白酶(matrix metalloproteinases,MMPs)是其中最主要的酶家族。MMPs是一组金属离子锌依赖的、能降解细胞外基质的高度保守蛋白水解酶家族,迄今为止,人类中已识别和定性的有23种。根据作用底物的特异性,MMPs家族可分为胶原酶(MMP-1、MMP-8、MMP-13、MMP-18),明胶酶(MMP-2、MMP-9),间质溶解素(MMP-3、MMP-10、MMP-11)、基质溶解因子(MMP-7、-26),膜接合型(MT)-MMPs (MMP-14、-15、-16、-24、MMP-17、-25)和其他型(MMP-12、-19、-20、-21、-23、-27、-28)。基质金属蛋白酶-26(MMP-26) ,又称基质溶解因子-2(matrilysin-2或endometase) ,是MMPs家族基质溶解因子亚组的新成员,新近从人子宫内膜癌细胞系cDNA文库克隆,呈现一系列不同于MMPs家族其它成员的结构和功能特性。MMP-26可有效裂解纤维蛋白原和ECM蛋白,包括纤维连接蛋白、玻璃体结合蛋白、变性胶原、α1-抗胰蛋白酶,表明其在肿瘤侵袭和血管发生方面有重要而独特的功能。目前已有研究表明, MMP-26在正常组织的表达具有组织特异性,MMP-26在不同癌的表达趋势不同,而已有的研究表明肿瘤相关因子刺激下可促进MMP-26、TIMP-4转录激活,因而我们推测p38MAPK、ERK与MMP-26之间可能存在相互作用。关于MMP-26在癌侵袭转移的可能机制,现有几个研究发现MMP-26可能主要是通过激活MMP-9,改变TIMP-4表达等促进癌细胞的侵袭能力。目前关于MMP-26有限的研究主要集中在前列腺、子宫、乳腺、食管癌,然而不同癌结果相异,作用机制也不很清楚,尚无MMP-26与肺癌相关性的系统研究。本研究拟探讨:1、MMP-26在非小细胞肺癌组织的表达与临床病理相关性;2、MAPK信号通路阻滞剂对肺癌细胞MMP-26蛋白表达的影响;3、肺癌细胞转染含MMP-26cDNA质粒及针对MMP-26mRNA的干扰质粒shRNA后MMP-26、MMP-9、TIMP-4基因、蛋白、细胞侵袭能力变化及MMP-26、MMP-9在细胞内定位情况。通过上述多个层面研究,探讨MMP-26在肺癌发生发展中的作用机制以及作为靶点进行干预的可能性。实验分三部分:第一部分:MMP-26蛋白在非小细胞肺癌的表达及意义目的:检测MMP-26蛋白在侵润性非小细胞肺癌(Non-small cell lung cancer,NSCLC)、侵润前肺癌和正常肺组织中的表达,探讨MMP-26在NSCLC发生发展中的作用及与预后的关系。方法:采用免疫组织化学(SP)法,分别检测72例NSCLC、14例非典型增生和10例正常肺组织中MMP-26蛋白的表达。结果:正常肺组织MMP-26蛋白高表达率0(0/10),非典型增生高表达率14.3%(2/14), NSCLC高表达率59.7﹪(43/72)。MMP-26蛋白在NSCLC中的表达高于非典型增生及正常肺组织(P<0.01),非典型增生MMP-26蛋白表达高于正常肺组织表达,但二者差异无显著性(P >0.05)。MMP-26蛋白表达与分期(P<0.05)和淋巴结转移有关(P<0.05),而与患者年龄、性别以及肿瘤大小、分化无关(均为P >0.05)。Cox比例风险模型进行多因素生存分析显示:MMP-26与临床分期是有意义的预后指标(P<0.05)。MMP-26蛋白高表达的NSCLC患者无复发生存期和总生存期低于阴性表达的患者(分别为log-rank=19.34、23.2,P<0.001、0.001 )。结论:MMP-26蛋白高表达与NSCLC发生、淋巴结转移、临床分期及预后有关,有可能作为判断NSCLC进展和预测预后的一种指标。第二部分:ERK信号通路与MMP-26在肺腺癌表达的关系目的:研究细胞外信号调节激酶(extracelluar signal-regulated kinase ,ERK)通路与基质金属蛋白酶-26(matrix metalloproteinase-26,MMP-26)在肺腺癌表达的相关性及意义。方法:应用免疫组织化学(SP)法检测pERK、pp38MAPK及MMP-26在44例肺腺癌组织中的表达。Western blot检测U0126阻断ERK信号通路后肺腺癌A549细胞MMP-26蛋白表达水平的变化。分析pERK和MMP-26表达与肺腺癌预后的关系。结果:pERK、pp38MAPK及MMP-26蛋白在肺腺癌组织中高表达率分别为59.1%、20.4%、54.5%,pERK与MMP-26表达存在正相关(r=0.63,P<0.05),并与肺腺癌的临床分期及淋巴转移状况相关(P<0.05),而与患者年龄、肿瘤大小无明显相关性(P>0.05)。pp38MAPK与MMP-26、患者年龄、淋巴转移、肿瘤大小、临床分期均无明显相关性(P>0.05)。用ERK阻断剂U0126阻断ERK信号通路可降低MMP-26蛋白表达水平,并且随U0126浓度升高MMP-26蛋白表达水平逐渐降低。肺腺癌组织pERK、MMP-26蛋白的表达与肿瘤的预后显著相关(分别为log-rank=5.52、7.01,P=0.019、0.008)。结论:ERK信号通路可能通过上调MMP-26的表达促进肺腺癌的恶性进展, ERK信号通路可能是肺腺癌侵袭和转移的重要途径,pERK和MMP-26的表达可辅助用于肺腺癌的预后评估,为ERK信号通路作为肺腺癌治疗的靶点提供理论依据。第三部分:MMP-26 cDNA表达质粒和针对MMP-26 mRNA的shRNA干扰质粒的构建及其对肺癌细胞生物学特性的影响目的:构建含MMP-26 cDNA的表达质粒,构建含针对MMP-26 mRNA的shRNA干扰质粒,观察转染上述质粒对细胞生物学特性的影响。方法:构建含MMP-26 cDNA的表达质粒,转染到MMP-26低表达的95-C细胞,并筛选稳定表达的含MMP-26 cDNA的细胞,Western blot检测MMP-26、MMP-9、TIMP-4蛋白表达,RT-PCR检测MMP-26mRNA、MMP-9mRNA、TIMP-4mRNA表达,明胶酶谱分析检测过表达MMP-26对95-C分泌MMP-9的影响,Transwell试验检测细胞体外侵袭力,双荧光免疫细胞化学染色检测MMP-26、MMP-9蛋白在细胞内定位。构建含针对MMP-26 mRNA的shRNA干扰质粒,转染高表达MMP-26的A549细胞,并筛选稳定表达的含MMP-26 shRNA的细胞,Western blot检测MMP-26、MMP-9、TIMP-4蛋白表达,RT-PCR检测MMP-26mRNA、MMP-9mRNA、TIMP-4 mRNA表达,明胶酶谱分析检测过表达MMP-26对A549细胞分泌MMP-9的影响, Transwell试验检测细胞体外侵袭力。结果:转染含MMP-26 cDNA表达质粒的95-C细胞MMP-26、MMP-9蛋白表达上调( p<0.05 ), TIMP-4下调( p<0.05 ); MMP-26mRNA、MMP-9mRNA表达上调(p<0.05),TIMP-4mRNA表达下调p<0.05)。明胶酶谱分析显示过表达MMP-26的95-C分泌MMP-9能力明显增强(p<0.05),体外侵袭力增强(p<0.05),双荧光免疫细胞化学染色发现MMP-26、MMP-9在细胞内共定位。转染针对MMP-26 mRNA的shRNA干扰质粒的A549细胞MMP-26、MMP-9蛋白表达下调( p<0.05 ), TIMP-4蛋白上调( p<0.05 ),MMP-26mRNA、MMP-9mRNA表达下调(p<0.05),TIMP-4 mRNA上调(p<0.05),分泌MMP-9能力明显下降(p<0.05),体外侵袭力减弱(p<0.05)。结论:MMP-26可以促进肺癌细胞的侵袭,可能部分通过调节MMP-9、TIMP-4表达促进肺癌细胞的侵袭,MMP-26有望成为肺癌干预的靶点。更多还原

【Abstract】 Lung cancer is a common malignant tumor,presenting an increasing trend in morbidity and mortality year by year.Metastasis and recurrenc related to degradation of the extracellular matrix (ECM) mediated by matrix metalloproteinases (MMPs) are the leading causes of death in tumor patients. Degradation of the base membrane and extracellular matrix (ECM) mediated by various types of proteinases such as matrix metalloproteinases, serine proteinases , cysteine proteinases, is the key steps, but the major enzymes are considered to be matrix metalloproteinases (MMPs). Matrix metalloproteinases (MMPs) are a family of zinc metallo-endopeptidases secreted by cells, and are responsible for much of the turnover of matrix components. Currently 23 MMP genes have been identified in humans. Based on domain organization and substrate preference, MMPs are grouped into collagenases (MMP-1, MMP-8, MMP-13 and MMP-18 in Xenopus), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10 and MMP-11), matrilysins (MMP-7 and -26), membrane-type (MT)-MMPs (MMP-14, -15, -16, and -24, MMP-17 and -25) and others(MMP-12,-19,-20,-21,-23,-27,-28).MMP-26 is a new member of the MMP family, initially cloned from human endometrial cancer cell line complementary DNA library in 2000. The genomic gene of MMP-26 is localized at the 11p15.3 locus, and codes a 30 kD protein of 261 amino acid residues. MMP-26 has been shown to cleave multiple components of the ECM,including fibronectin,type IV collagen, vitronectin, gelatins and fibrinogen, as well as non-ECM proteins such as insulin-like growth factor-binding protein-1,α-1 protease inhibitor, suggest that MMP-26 plays a key role in tumor progression and angiogenesis.Studies have shown that MMP-26 expression is tissue specificity in normal tissues , and MMP-26 expression models were different in cancers . Tumor correlation factor may promote transcriptional activation of MMP-26 and TIMP-4 ,thus there may be interaction with p38MAPK, ERK and MMP-26 . The underlying mechanisms of MMP-26 over-expression promoting cancer cell invasion and metastasis, however, remains to be elucidated. Several studies indicated that MMP-26 was involved in invasion-promoting regulation of cancer cells and it may function,at least in part, through activating MMP-9 and effecting of TIMP-4 expression . Till now , the limited studies on MMP-26 are in prostate cancer, uterus cancer, breast cancer and esophageal carcinoma , but MMP-26 expression models were different in cancers, and the mechanism of action remains to be elucidated. Moreover, there is no systemic researches of MMP-26 with lung cancer . This study was to test: 1. the expression of MMP-26 protein and the clinical significance of NSCLC; 2. the effects of MAPK signal pathway inhibitor on expression of MMP-26 protein in NSCLC ; 3. MMP-26 cDNA was introduced into carcinoma cells that lack expression of endogenous MMP-26, and MMP-26 shRNA was introduced into carcinoma cells that high expression of endogenous MMP-26, the expression of mRNA and protein of MMP-26, MMP-9 and TIMP-4 were detected , In vitro invasion assay and intracellular location of MMP-26, MMP-9 in cancer cell were studied , too. With sevral aspects of studies of MMP-26 , we explored the mechanics in carcinogenesis of lung cancer and posibility of being inervented as target .There are three parts in the experiment:Part one: Expression and Clinical Significance of MMP-26 Proteinin in Non-small Cell Lung CancerObjective: To investigate the expression of MMP-26 protein in invasion non-small cell lung cancer (NSCLC), pre-invasive lung cancer and normal lung tissues, and to explore its relation to the progression and prognosis of NSCLC.Methods: SP immunohistochemistry was used to test the expression of MMP-26 protein in 72 specimens of NSCLC, 14 specimens of atypical hyperplasia and 10 specimens of normal lung tissues. Result: The high expression rate of MMP-26 was 0(0/10)in normal lung tissues, 14.3%(2/14)in atypical hyperplasia and 59.7﹪(43/72) in NSCLC .The expression rate of MMP-26 protein was significantly higher in NSCLC than in atypical hyperplasia and normal lung tissues(P <0.01),and higher in atypical hyperplasia than in normal lung tissues,but the difference was not significant(P >0.05).. The high expression rate of MMP-26 protein was significantly related to stages( P <0.05 ) and lymph node metastassis(P <0.05 ), but not to age , gender , tumor size and differentiation(P>0.05). Multivariate analysis showed that MMP-26 and stage were independent prognostic indicators(P<0.05). NSCLC patients with high expression of MMP-26 had shorter disease-free survival and overall survival than did those with low expression (log-rank=19.34,23.2, P<0.001,0.001, respectively ).Conclusion:MMP-26 protein high expression is related to the carcinogenesis, lymph node metastasis, clinical stage and prognosis of NSCLC. MMP-26 expression may be served as a tumor marker monitoring progression and predicting prognosis of NSCLC.Part two: Correlation of ERK signal transduction pathway with MMP-26 expression in lung adnocacinomaObjective: To investigate the correlation of extracelluar signal-regulated kinase (ERK) with matrix metalloproteinase-26(MMP-26)and its significance in lung adnocacinoma. Method: SP immunohistochemistry was used to test the expression of pERK , pp38MAPK and MMP-26 in 44 specimens of lung adnocacinoma . Western blot was used to detect the protein levels of MMP-26 in lung adnocacinoma A549 cells after blocking ERK signal transduction pathway by U0126. The correlation of pERK and MMP-26 expression with prognosis of lung adnocacinoma was also analyzed.Results: The high expression rates of pERK, pp38MAPK and MMP-26 proteins in lung adnocacinoma tissues were 59.1%,20.4%and 54.5%,respectively.The expression of pERK was positively correlated to the expression of MMP-26(r=0.63,P<0.05),and the expression of pp38MAPK was not related to MMP-26 expression ( r=0.01 ,P>0.05). The expression of pERK was correlated to lymph node metastasis and TNM stage(P<0.05),but not to age and tumor size(P>0.05). pp38MAPK was not correlated to age , lymph node metastasis , tumor size and TNM stage ( P>0.05 ). U0126 concentration-dependently inhibited ERK pathway and reduced MMP-26 protein expression .The expression of pERK and MMP-26 was negatively correlated to prognosis of lung adnocacinoma patients(log-rank=5.52, 7.01;P=0.019, 0.008) .Conclusion: ERK signal transduction pathway might promote lung adnocacinoma progression by up-regulating MMP-26 expression,and might be an important route in invasion and metastasis of lung adnocacinoma . pERK and MMP-26 might help to evaluate prognosis of lung adnocacinoma.Part three: Establishment of MMP-26 cDNA plasmid and shRNA plasmid and exploring their effection of lung cancer cells on bionomicsObjective: To establish the recombinant plasmid of A full-length cDNA encoding human MMP-26 and construct the recombinant plasmid of small interfering RNA(shRNA) against matrix metalloproteinase-26, and explore their effection on bionomics of lung cancer cells.Methods: A full-length cDNA encoding human MMP-26 was cloned into the eukaryotic expression vector PUC57, This vector was designated PUC57-mmp26 transfected into 95-C. RT-PCR and Western blot were used to detect expression of MMP-26,MMP-9 and TIMP-4 . In vitro invasion assay and localization of MMP-26 and MMP-9 proteins were determined .Construct the plasmid containing short hairpin RNA(shRNA) of MMP-26, and transfected into A549. RT-PCR and Western blot were used to detect expression of MMP-26,MMP-9 and TIMP-4 . In vitro invasion assay and localization of MMP-26 and MMP-9 proteins were also determined.Results: Transfection of MMP-26 expressing plasmid in 95-C led to over-expression of MMP-26 protein(P<0.05),and cell invasiveness in these cells were significantly promoted(P<0.05).The invasiveness of MMP-26-transfected 95-C cells was increased (P<0.05).Meanwhile , the expression of MMP-9 protein was increased (P<0.05)and the expression of TIMP-4 protein decreased (P<0.05)in these MMP-26-transfected 95-C cells . The expression of MMP-26 mRNA was increased (P<0.05), MMP-9 mRNA was increased(P<0.05)and TIMP-4 mRNA decreased (P<0.05)in these MMP-26-transfected 95-C cells .The results of gelatin zymogram reveal overexpression MMP-26 increase the secretion of MMP-9(P<0.05). Double immunofluorescence staining revealed that MMP-26 and MMP-9 co-localized in MMP-26-transfected 95-C cells .Transfection of MMP-26 shRNA plasmid in A549 led to low-expression of MMP-26 protein(P<0.05),and cell invasiveness in these cells were significantly decreased (P<0.05)..Meanwhile , the expression of MMP-9 protein was decreased (P<0.05)and the expression of TIMP-4 protein increased (P<0.05)in these pshRNA-mmp26-transfected A549 cells . The expression of MMP-26 mRNA was decreased (P<0.05), MMP-9 mRNA was decreased(P<0.05)and TIMP-4 mRNA was increased (P<0.05)in these pshRNA-mmp26-transfected A549 cells .The results of gelatin zymogram reveal low-expression MMP-26 decrease the secretion of MMP-9(P<0.05).Conclusion: MMP-26 is involved in invasion-promoting regulating of lung cancer cells ,and it functions ,at least in part, through interaction with MMP-9 and TIMP-4 . MMP-26 is expected to become a target for gene therapy of non small cell lung cancer .更多还原