The Research On The Mechanism Of PGE₂ Upregulates β1-integrin Expression Via The EP1 Receptor/NF-κB Pathway To Promote The Cell Invasion And Metastasis In Non-small-cell Lung Cancer Cells

Background:Malignant tumor is one of the main causes of serious threat to human health. Because of the lack of effective treatments, in malignant tumors, lung cancer has the highest fatality rate. Therefore, to explore the pathogenesis of lung cancer and to seek treatment targets is especially important.Prostaglandin E2(PGE2) is one of the predominant metabolic products of arachidonic acid.After binding with its four kinds of E prostanoid receptors in the cell membrane, named EP1 receptor 、 EP2 receptor 、 EP3 receptor and EP4 receptor.PGE2 exerts diverse biological effects by triggering cell signal transduction and regulating gene expression.Previous studies revealed that PGE2 has a significant role in a number of cancer types during tumor cell growth, migration and invasion.β1-integrin is one of the integrin family members.It is mainly transduce signals from the extracellular matrix that modulate cell growth, differentiation, migration and invasion.It has been reported that fibronectin stimulates human lung carcinoma cell proliferation, and this effect is mediated through β1-integrin and is associated with COX-2 expression and PGE2 biosynthesis. However, the exact mechanism remains to be elucidated. In this study,we aim to investigate the effect of PGE2 on the expression of β1-integrin and the probable signal transduction pathway in NSCLC. Objectives:To investigate the effect of PGE2 on the expression of β1-integrin and its mechanism in NSCLC. Methods:1、The non-small-cell lung cancer cell line A549 was cultured in conventional conditions.2、A549 cells were treated with different doses of exogenous PGE2.Western Blot test was conducted to observe the alteration of β1-integrin.3、A549 cells were treated with 17-PT-PGE2、Butaprost、Sulprostone and Alcohol respectively.Western Blot test was conducted to observe the alteration of b 1-integrin.4、A549 cells were treated with sc51322、AH6809、L798106 and AH23848 respectively,then treated with exogenous PGE2. Western Blot test was conducted to observe the alteration of β1-integrin.5、A549 cells were treated with different doses of 17-PT-PGE2.Western Blot test was conducted to observe the alteration of β1-integrin.6 、 A549 cells were transfected with the EP1R-si RNA, then treated with exogenous PGE2.Western Blot test was conducted to observe the alteration of β1-integrin.7、34 cases of non-small-cell lung cancer tissue and 10 cases of normal lung tissue were collected to detect the relationship between the expression of EP1 receptor and β1-integrin by immunohistochemistry.8、A549 cells were treated with 17-PT-PGE2 and 17-PT-PGE2+ITGB1m Ab. Transwell method was apply to measure the cell migration.9、A549 cells were treated with 17-PT-PGE2 for 0min、15min、30min、60min、120min respectively,Western Blot test was conducted to observe the alteration of Phospho-IkBa and Phospho-P65.10、A549 cells were treated with NF-κB inhibitor PDTC. Western Blot test was conducted to observe the alteration of β1-integrin. Results:1、After treated with 0-5μmol/L PGE2 for 24 h,the expression of β1-integrin in A549 cells was detected by Western Blot.The results showed that the levels of β1-integrin were increased in a PGE2 concentration-dependent manner.1、3、5μmol/L PGE2 treatment could increase its expression to 16.23%、62.83%、88.12%of control.2、After treated with 5μmol/L EP1 receptor agonist(17-PT-PGE2) for 24 h,the expression of β1-integrin in A549 cells was detected by Western Blot.The results showed that the treatment could increase its expression to 110.85% of control.3、A549 cells were pre-treated with 10μ mol/L EP1 receptor inhibitor(sc51322) for 1h followed by stimulation with 5μ mol/L PGE2 for 24 h. The Western Blot results showed that the expression of b 1-integrin in pre-treated group decreased 53.63% of ―PGE2‖group.4、After treated with 0-10μmol/L 17-PT-PGE2 for 24 h,the expression of β1-integrin in A549 cells was detected by Western Blot.The results showed that the levels of b 1-integrin were increased in a 17-PT-PGE2 concentration-dependent manner.1、2.5、5、10μ mol/L PGE2 treatment could increase its expression to56.69%、172.96%、290.48%、106.68% of control.5、After transfected with the EP1 R si RN A,the expression of β1- integrin in A549 cells was detected by Western Blot.The results showed that the depletion of EP1 receptor did not reduce the basal levels of β1- integrin in A549 cells. However, after treated with PGE2, the expression of β1- integrin in EP1 R si RNA transfected group decrease to 85.83% of N.C si RNA+ PGE2 group.6 、 Immunohistochemical staining of serial sections of NSCLC tissues demonstrated that the EP1 receptor and β1-integrin expression exhibited a positive correlation of evident significance in the 44 samples. >3/4 of the NSCLC samples exhibited significantly increased EP1 receptor and β1-integrin expression levels, compared with the benign sample groups.7、A549 cells were pre-treated with 3 μg/ml β1-integrin monoclonal antibody(m Ab) for 30 min followed by stimulation with 17-PT-PGE2. Transwell assay showed that the cell migration was increased by 30% whe n the cells were treated with 5μmol/L 17-PT-PGE2. The pre-treatment of β1-integrin m Ab inhibited the 17-PT-PGE2-mediated cell migration completely.8、After treated with 5μmol/L 17-PT-PGE2 for 0-120 min,the expression of p-IkBa and p-P65 in A549 cells was detected by Western Blot.The results showed that the expression of p-IkBa increased 23.80% of control at 30 min and the expression of p-P65 increased 98.28%、131.25%、115.11% of control at 30、60、120min.9、A549 cells were pre-treated with 5μ mol/L NF-κB inhibitor(PDTC) for 1h followed by stimulation with 5μ mol/L 17-PT-PGE2 for 24 h. The Western Blot results showed that the expression of b 1-integrin in pre-treated group decreased 59.62% of ―17-PT-PGE2‖group. Conclusion:PGE2 can obviously increase the expression of β1-integrin through the EP1 receptor / NF-κB pathway to promote the tumor cell migration in A549 cells.