Study On The Expression, Clinical Significance And Regulatorymechanism In Non Small Cell Lung Cancer

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 .