The Effect Of MMP-11 On The Biological Behavior Of Pancreatic Cancer Cells And Its Diagnostic Value

BackgroundPancreatic ductal adenocarcinoma(PDAC)is recognized as one of the most aggressive Gastrointestinal malignancy with dormant onset,rapid progression,low resection rate,resistance to radiation and chemotherapy,which is difficult for early diagnosis and correlated with poor prognosis.Consequently,the hotspots of PDAC research include improving diagnosis rate and treatment efficacy,as well as exploring mechanisms associated with tumorigenesis and development of pancreatic cancer.Matrix metalloproteinases are zinc-dependent endopeptidases.Matrix metalloproteinase-11(MMP-11,stromelysin-3)is a member of this big family.Different from other members of MMPs family,it has some characteristic functions.Firstly,MMP-11 does not appear to degrade fundamental substrates of extracellular matrix(ECM).In addition,it is intracytoplasmically processed and secreted as an active enzyme,which indicate special functions of MMP-11.A closed relationship between MMP-11 and neoplasms was observed.It has been proved that MMP-11 is mainly expressed in adjacent fibroblasts of tumor.However,MMP-11 was also observed expressed in some certain types of tumor recently,including pancreatic cancer cells.On clinical significance,some researchers reported that serum MMP-11 levels in cancer patients were elevated,and overexpression of MMP-11 in tumor tissue was associated with poor prognosis.Some studies demonstrated the expression of MMP-11 was related to biological behaviors of tumor in vitro and in vivo,though some conclusions are still controversial and unclear.Therefore,we designed and developed this research based on the characteristic functions of MMP-11,the unclear relationship between it and biological behaviors of pancreatic cancer cells with the underlying mechanisms,and the correlation between serum MMP-11 levels of PDAC patients and their clinical characteristics.This study intends to explore the effects that MMP-11 exerts on pancreatic cancer cells and its clinical significance through in vitro experiment,and detection of serum MMP-11 levels of PDAC patients and controls.Objectives1.To identify the localization and levels of MMP-11 in human pancreatic cancer cells;2.To explore the impact of MMP-11 on the biological behaviors of pancreatic cancer cells;3.To detection the serum MMP-11 levels in patients of pancreatic diseases and healthy controls,and to explore its roles in diagnosis and differential diagnosis of pancreatic cancer;Methods1.The baseline expression levels of MMP-11 in human pancreatic cancer cell lines are detected by Western blot and Quantitative Real-time PCR.The localization and expression levels of MMP-11 are observed by immunofluorescent staining.2.Transfect MiaPaCa-2 and AsPC-1 cell lines with siRNA by Lipofectamine 3000 in order to downregulate the expression levels of MMP-11,which is proved by Western blot and Quantitative Real-time PCR.Transwell migration and invasion models are applied to determine the migration and invasion capabilities of cells.CCK8 assay is used to detect cell proliferation.Apoptosis and cell cycle are measured through flow cytometry.The expression levels of associated proteins and signaling molecules are detected by Western blot.3.Serum samples from patients of pancreatic diseases,including PD AC,pancreatic neuroendocrine tumors(PNET),pancreatic cystic neoplasms,and chronic pancreatitis,as well as healthy controls were collected previously.The concentrations of MMP-11 in serum samples are detected by enzyme-linked immuno-specific assay(ELISA).Results1.MMP-11 was expressed in all human pancreatic cancer cells including AsPC-1,MiaPaCa-2,Panc-1,Su-86.86,and T3M4.The expression levels of MMP-11 are higher in Panc-1,AsPC-1,and MiaPaCa-2 among these cells.2.Most of MMP-11 were located in cytoplasm/membrane instead of nucleus.3.MMP-11 could promote proliferation,migration and invasion in pancreatic cancer cells,while no effect from MMP-11 on apoptosis and chemosensitivity of gemcitabine was observed.4.The underlying mechanisms by which MMP-11 regulates cell proliferation is that phosphorylation of Akt is regulated by MMP-11.Phosphorylated-Akt could influence the expression of CyclinD1 and CDK4,which are key molecules during G1/S conversion in cell cycle.5.Serum MMP-11 could be detected in all patients with pancreatic diseases and healthy controls.Expression levels of serum MMP-11 in all patients with pancreatic diseases are significantly higher than those of healthy controls.Expression levels of serum MMP-11 in PD AC patients are significantly higher than those of healthy controls and patients with pancreatic cystic neoplasms.6.It is demonstrated that serum MMP-11 expression levels show good sensitivity and specificity in differential diagnosis between PDAC patients and healthy controls,as well as between PD AC patients and PDAC patients.Conclusions1.MMP-11 is extensively expressed in pancreatic cancer cells,which acts as a tumor enhancer by promoting cell proliferation,migration and invasion.2.Serum MMP-11 expression levels in patients with PDAC and those with other pancreatic disease are elevated,which could assist in diagnosing pancreatic cancer.It could be a potential biomarker with good sensitivity for screening pancreatic cancer in healthy population.