【作者】 张扬；

【导师】 李才； 房学迅；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 近年来肺癌的发病率和死亡率在世界范围内呈不断上升的趋势,其中80%表现为NSCLC。因此,从分子水平了解NSCLC侵袭与转移的过程,寻找可靠的标志物来预测患者复发或不良预后是目前国内外研究的热点。MMP-26广泛表达于正常和肿瘤组织,但在NSCLC中的表达及其生物学作用的研究尚未见报道。本研究采用原位杂交、免疫组化、RT-PCR、Western blot和明胶酶谱分析等方法,检测了MMP-26及其特异性抑制因子TIMP-4在NSCLC组织和细胞中的表达情况;应用RNAi技术抑制MMP-26基因在人肺癌细胞中的表达,体外观察基因沉默对细胞生物学行为的影响,并对其作用机制进行初步探讨。研究表明,MMP-26和TIMP-4在NSCLC组织中协同表达;MMP-26可能通过降解ECM或激活MMP-9参与NSCLC的早期侵袭过程,检测MMP-26的表达有助于预测NSCLC患者的预后;RNAi介导的MMP-26基因表达沉默可有效降低肺癌细胞浸润能力,其作用可能是通过与MMP-9的协调来实现的,故而MMP-26有望成为抗NSCLC侵袭的分子靶点。随着MMP-26在肿瘤发生发展中作用研究的深入,MMP-26作为靶基因可能为肿瘤的治疗开辟新的有效途径,其具体应用价值还有待进一步探寻。更多还原

【Abstract】 Lung cancer remains the most common cause of cancerrelated death in the world. Non-small cell lung cancer (NSCLC) accounts for appoximately 80% of all lung cancers. The 5-year survival of patients with NSCLC remains among the lowest of all major human cancers despite recent advances in surgical, radiation, and medical treaments. The majority of the dignosed patients with NSCLC died of local recurrence and distant metastasis. Therefore, there is intense interest in gaining a better understanding of the molecular and cellular processes involved in this aggressive disease and searching for reliable biomarkers to predict relapse and poor outcome.MMP-26 is a recently discovered and only partially characterized human proteinase. It has several structural features of MMPs, including the signal sequence, the prodomain domain and the catalytic domain, but it lacks the hemopexin-like domain. A unique“cysteine switch”sequence in the prodomain, PHCGVPD as opposed to the conserved PRCGXXD sequence found in many other MMPs, keeps the enzyme latent. MMP-26 mRNA is specifically expressed in epithelial cancers, such as breast, endometrial and prostate carcinomas, in their corresponding cell lines and in normal adult tissues, such as the uterus, placenta and kidney. This protein hydrolyzes various components of the ECM, including fibronectin, type IV collagen, vitronectin and fibrinogen, as well as non-ECM proteins, indicating that MMP-26 is a potent enzyme with a wide substrate specificity. MMP-26 is also able to activate progelatinase B. These data suggest that MMP-26 may plays a key role in tumor progression. Previous studies have showed that TIMP-4 is most potent against MMP-26 compared with other MMPs tested. To date, detailed function and expression patterns of MMP-26 in NSCLC tissues and cells have not been reported. The aim of the study is to determine if MMP-26 may be responsible for the invasive and metastasis behaviour of NSCLC and to future investigate the mechanism of MMP-26 in tumor process.The main methods of this study were as follows:1. In situ hybridization was performed to identify MMP-26 and TIMP-4 mRNA location in NSCLC. MMP-26 and TIMP-4 expressions were determined in NSCLC tissues, paracancerous tissues and benign pulmonary diseases tissues by immunohistochemical technique. Furthermore, we studied the relationship between expression of MMP-26 and TIMP-4 proteins and clinic pathological feature in NSCLC. The expression of MMP-26 and MMP-9 were analyzed immunohisto- chemically in NSCLC tissues.2. The expression levels of MMP-26 and TIMP-4 mRNAs and proteins in A549 cells and SPC-A-1 cells were detected by semi-quantitative reverse transcription- polymerase chain reaction (RT-PCR), Western blot and zymography.3. MMP-26 mRNA targeted hairpin siRNA was devised and three pairs of oligonucleotides encoding the above siRNA were synthesized. After annealing of the complementary strands, the DNA fragments were inserted into plamid pSUPERIOR.puro to generate siRNA eukaryotic expression vectors, followed by amplificaton and DNA sequencing.4. The recombinant plasmids were transiently transfected into A549 cells. The expression levels of MMP-26 mRNA and protein in A549 cells were detected by semi-quantitative RT-PCR, Western blot and immunfluorescence. A549 cells stably transfected with the recombinant plasmids were selected by puromycin. 5. The changes in cell morphology in routine and three-dimensional culture were observed under a light microscopy. MTT and colony forming assay were used to detect status of cell proliferation. Cell cycle of A549 cells after transfection was evaluated by flow cytometry. The adhesive, migratory and invasive abilities of A549 cells were measured via plate adhesion assay, cell scratch wound model and transwell chamber in vitro.6. The expression levels of MMP-9 mRNA and protein were examined by semi- quantitative RT-PCR, Western blot and double immunofluorescent staining.The main results of this study were as follows:1. In situ hybridization signal localized MMP-26 predominantly in tumor cells and epithelial cells, but TIMP-4 mRNA in the same samples was present mainly in stromal cells.2. There was strong MMP-26 staining of epithelial cells and cytoplasm of tumor cells in carcinoma tissues, but no general stromal staining was seen. In paracancerous lung tissues, weak immunoreactivity for MMP-26 was only occasionally observed in macrophages. The signal intensities of MMP-26 protein in NSCLC were significantly higher than those in benign pulmonary diseases tissues and paracancerous tissues (P<0.01). MMP-26 expression was significantly correlated with histopathology, cell differentiation and advanced TNM stage (P<0.05).3. Immunostaining for TIMP-4 was present mainly in epithelial cells and cytoplasm of tumor cells in carcinoma tissues, and stroma had weakly staining. Immunohistochemistry for TIMP-4 protein was negative in paracancerous lung tissues. The signal intensities of TIMP-4 protein in NSCLC were significantly higher than those in benign pulmonary diseases tissues and paracancerous tissues (P<0.01). The expression of TIMP-4 showed no significance with all clinicobiological varies (P>0.05).4. MMP-26 and MMP-9 proteins were both expressed in the same regions of NSCLC tissue samples.5. MMP-26 mRNA and protein were expressed in these two NSCLC cell strains, but those of TIMP-4 were not detected.6. The DNA fragments encoding MMP-26-targeted siRNA were cloned into the pSUPERIOR.puro and confirmed by restrictive enzyme digestion and DNA sequencing.7. Semi-quantitative RT-PCR showed that the mRNA transcription of MMP-26 gene in A549 cells transfeced with the recombinant plasmid pshRNA-MMP26-C was reduced by nearly 65%. The expression of MMP-26 protein in pshRNA-MMP26-C group was significantly lower than that of other groups (P<0.01).8. We obtained monocloned cell strains, which stably expressed siRNA-MMP26 after selection by puromycin. The results demonstrated that①The adhesive rate was down-regulated in pshRNA-MMP26-C group, when compared to the controls (P<0.05). Silencing of MMP-26 gene significantly retarded the invasiveness of A549 cells in transwell insert invasion assay (P<0.05).②All of the cells could proliferate in the three-dimensional culture system. A549 cells transfected with pshRNA-MMP26-C plasmid mainly developed reticular structure in morphology, and formed few clones with clear and smooth edges as well as tight intercellular junctions. Controls cells formed clones with irregular morphology, unclear edge, and loose intercellular junctions. In addition, the clones also developed a lot of pseudopodia, but developed no reticular structure.③Compared with the controls, other varies showed no difference statistically (P>0.05).9. The mRNA and protein expression of MMP-9 in pshRNA-MMP26-C group were significantly lower than those of the controls (P<0.05). Double immuno- fluorescence labeling and focal laser scanning microscopy revealed that MMP-26 was colocalized with MMP-9 in the controls.The main conclusions of this study are as follows: 1. MMP-26 may participate in NSCLC early invasive, either directly by degrading ECM or indirectly by activating pro-MMP-9.2. Expression silencing of MMP-26 by RNAi could reduce invasion of lung carcinoma cells effectively, and it may function, at least in part, through coordination with MMP-9.更多还原