Prognostic Role Of Integrin ?v?6 And MMP-9 And Their Mechanism In Promoting Invasion And Migration In Gastric Cancer

Gastric cancer is one of the most common malignant tumors in humans and the prognosis is poor. In our country, the morbidity and mortality rates are the highest in all malignant tumors. Most patients have had an invasive and metastatic disease in the hospital.In the malignant tumor cells in the process, as the cell adhesion molecules expression changes, tumor cell adhesion ability between tumor cells is abate, and tumor cell adhesion ability with basement membrane is enhanced; On this basis,malignant tumor cells secrete and release proteolytic enzyme (such as matrix metalloproteinase) to degrade the extracellular matrix and destroy the integrity of the basement membrane. Once the basement membrane is destroyed, malignant cells pass through the defect in amoebic movement, melting and roaming the interstitial connective tissue, eventually to blood vessels or the lymphatic tube wall and in the same way again, pass through blood vessels or the lymphatic vessels of basement membrane into the circulatory system, so as to realize metastasis. It can be seen that invasion and metastasis are different manifestations of the same biological behavior(migration and dissolution) of malignant tumor cells, and there is no obvious difference in nature; Among them, the change of cell adhesion ability and the enhancement of matrix solubility are the two most important links, which can promote the continuous migration and dissolution of malignant tumor cells.The family of integrins is a transmembrane protein receptor that is formed by non-covalent bonding between alpha and beta. Among them, the extracellular part mediates the adhesion and signal transduction of the extracellular matrix and the extracellular matrix by identifying the RGD sequence in the ligand. And the intracellular part of beta subunit contains the binding sites of intracellular signaling molecules. when integrins accept extracellular stimuli,convergent clusters occur,following by changes of intracellular region conformationally, activaton of adhesion kinase FAK and raising of cytoplasm protein talin cohesion, etc., which mediates the transmission of intracellular signaling pathways. Pilot study confirmed that integrin alpha V beta 6 argument is the only integrin containing beta 6, and expressed in malignant lesions epithelial tissue, which was found exxpressed in colon cancer,pancreatic cancer, breast cancer, oropharyngeal cancer and other malignant tumors .Besides alpha V beta 6 can be used as a indicator factors of poor prognosis in patients with cancer of the stomach and colon cancer.Matrix metalloproteinases (MMPs) was first discovered in 1962 and was named because of the need for metal ions such as Zn2 + to be used as auxiliary factors.MMPs is a proteolytic enzyme family, constituting signal peptide, propeptide domain,catalytic domains, hinge area and heme binding protein sample or fiber combined with hormone kind of COOH terminal domain structure,and play an important role in the process of tissue remodeling. The MMPs can degrade almost all protein components other than polysaccharides in ECM and disrupt the various histological barriers encountered during cell migration. Therefore, the role of MMPs in the invasion and metastasis of malignant tumor cells is increasingly valued and is believed to play a major role in the process of proteolytic enzymes. MMP-9 as a key member of MMPs, through degradation of gelatin and collagen type ?, plays an important role in malignant tumor invasion and metastasis. Previous studies have shown that MMP-9 is associated with adverse outcomes of pancreatic cancer and non-small-cell lung cancer.Based on this theory, we assume that, since integrin V beta 6 is an important cellular adhesive molecular and expressed in malignant tumor cells, and MMP - 9 as an important member of the family of MMPs, is upregulated in malignant tumor cells and participate in the extracellular matrix dissolution, synergistic action between each other in the invasion and metastasis of gastric cancer may exist. MMP-9 is a downstream target of alpha V beta 6 and is involved in the malignant behavior of colon cancer, but the role of the combined expression in the prognosis of gastric cancer has not been studied. Based on the above background, we wonder whether the combination of alpha V beta 6 and MMP-9 can predict the prognosis of gastric cancer more effectively.Part ? Integrin ???6 and MMP-9 Correlate With Survival in Gastric CancerObjectiveThis study was to investigate the expression of integrin ???6 and MMP-9, their association with prognostic factors and to assess their predictive role in gastric cancer patients.Methods1. Immunohistochemistry was used to determine the expressions of integrin ???6 and matrix metalloproteinase 9 (MMP-9) in 126 specimens from patients with primary gastric carcinoma.2. Associations between immunohistochemical staining and various clinic pathologic variables of tissue specimens were evaluated by the chi-squared test and Fisher's exact test.3. Expression correlation of avP6 and MMP-9 was assessed using bivariate correlation analysis.4. The patients were followed-up every 3 months in the first two years and at least every 6 months afterwards, with a median follow-up of 56 months (ranging from 2 months to 94 months).5. Four different combinations of ???6 and MMP-9 levels (that is, both markers positive, both markers negative, ???6 positive with MMP-9 negative, and ???6 negative with MMP-9 positive) were evaluated for their relative effect on survival.6. The difference in survival curves was evaluated with a log-rank test. Survival analysis was conducted using the Kaplan-Meier survival and Cox proportional hazards model analysis.Results1. The expressions of integrin ???6 and MMP-9 were investigated in 126 cases,among which 34.92% were positive for ???6 expression, and 42.06% for MMP-9 expression.2. The expression of ???6 was associated with Lauren type,differentiation,N stage,and TNM stage(the P ralues were 0.006, 0.038, 0.016, and 0.002, respectively).While MMP-9 expression was associated with differentiation, T stage, N stage,and TNM stage (the P values were 0.039, 0.014, 0.033, and 0.008, respectively).3. The positive correlation between ???6 and MMP-9 in gastric cancer was confirmed by a correlation analysis.4. The Kaplan-Meier survival analysis showed that patients with expression of ???6 or MMP-9 alone died earlier than those with negative expression and that patients who were both ???6 and MMP-9 positive had a shorter overall survival than those with the opposite pattern (both ???6 and MMP-9 negative) (P=0.000).5. A Cox model indicated that positive expression of avP6 and MMP-9, diffuse Lauren type, as well as a senior grade of N stage, M stage, and TNM stage were predictors of a poor prognosis in univariate analysis. Only ???6 and MMP-9 retained their significance when adjustments were made for other known prognostic factors in multivariate analysis (RR = 2.632, P = 0.003 and RR =1.813, P = 0.007).ConclusionsThe expression of ???6 and MMP-9 are closely correlated, and the combinational pattern of ???6 and MMP-9 can serve as a more effective prognostic index for gastric cancer patients.Part ? Influence of Integrin ???6 on MMP-9 and their Role in Invasion and Migration of Gastric CancerObjectiveThis study was to investigate the influence of integrin ???6 on MMP-9 and their role in invasion and migration of gastric cancer cells.Methods1. Human normal gastric mucosa GES-1 cells were divided into three groups:control group, mock transfected and ?6 transfected. Western blot was used to examine the expression of ?6 integrin in the three groups to test the result of our transfection for the next step of experiments.2. Human gastric cancer AGS cells were divided into three groups: control group,?6 siRNA negative transfected and ?6 siRNA transfected. Western blot was used to examine the expression of (36 integrin in the three groups to test the result of our transfection for the next step of experiments.3. Quantitative Real-time PCR was used to test the expression of MMP-9 mRNA in each group.4. ELISA was used to test the expression of MMP-9 protein in each group.5. Transwell matrigel invasion assay and Wound healing assay were used to test the invasion and migration of gastric cancer AGS cells in each groups.Results1. Western blot showed that ?6 integrin was effectively up-regulated in?6-transfected normal gastric mucosa GES-1 cells, while mock transfected cells did not show obvious change.2. Western blot showed that ?6 integrin was effectively down-regulated in ?6 siRNA transfected gastric cancer AGS cells, while ?6 siRNA negative transfected cells did not show obvious change.3. Quantitative Real-time PCR showed that MMP-9 mRNA expression was highly increased in ?6-transfected GES-1 cells compared with the mock transfectants and highly decreased in ?6 siRNA transfected AGS cells compared with the ?6 siRNA negative transfectants.4. ELISA showed that MMP-9 protein expression was highly increased in?6-transfected GES-1 cells compared with the mock transfectants and highly decreased in ?6 siRNA transfected AGS cells compared with the ?6 siRNA negative transfectants.5. Transwell matrigel invasion assay and Wound healing assay showed that the invasion and migration of gastric cancer AGS cells were highly weakened in ?6 siRNA transfectants compared with the ?6 siRNA negative transfectants.ConclusionsThis part of the study showed that integrin ???6 regulated the expression of MMP-9 at the level of both mRNA and protein, through which integrin ???6 had a large contribution to the invasion and migration of gastric cancer AGS cells. The result provides theory for prognostic role of integrin ???6 and MMP-9 in gastric cancer.Part ? Integrin ???6 Induces MMP-9 via ERK/MAPK Pathway in Gastric CancerObjectiveThis study was to investigate the role of ERK/MAPK pathway in Integrin???6-mediated MMP-9 up-regulation..Methods1. ERK inhibitor PD98059 was used to ?6-transfected normal gastric mucosa GES-1 cells and ?6 siRNA transfected AGS cells. Western blot was used to examine the expression of ERK to test the result of our inhibition for the next step of experiments.2. Quantitative Real-time PCR was used to test the expression of MMP-9 mRNA in each group.3. ELISA was used to test the expression of MMP-9 protein in each group.Results1. Western blot showed that ERK was effectively deceased in ?6-transfected normal gastric mucosa GES-1 cells and ?6 siRNA transfected AGS cells when compared with corresponding untreated cells.2. Quantitative Real-time PCR showed that MMP-9 mRNA expression was highly decreased in ?6-transfected GES-1 cells and p6 siRNA transfected AGS cells when compared with corresponding untreated cells.3. ELISA showed that MMP-9 protein expression was highly decreased in?6-transfected GES-1 cells and ?6 siRNA transfected AGS cells when compared with corresponding untreated cells.ConclusionsThis part of the study showed that Integrin ???6 regulated the expression of MMP-9 at the level of both mRNA and protein via ERK/MAPK pathway in gastric cancer. The result indicates the direction for Integrin avP6 related targeting therapy for gastric cancer.