TMEFF2 Inhibits Pancreatic Cancer Cells Proliferation,Migration,and Invasion By Suppressing Phosphorylation Of The MAPK Signaling Pathway

Background:Pancreatic cancer is a malignant tumor with high degree of malignancy and mortality in human digestive system.Due to the highly malignant proliferation of tumor cells,the malignant invasion and migration,and the special anatomical location,the clinical course develops rapidly and the surgical resection rate is low,resulting in a poor clinical prognosis.Therefore,it is urgent to deeply study the pathogenesis of high proliferation,malignant invasion and metastasis of pancreatic cancer,and explore the key pathogenic factors and signal transduction pathways,which is of great significance for early diagnosis,molecular targeted therapy and improvement of prognosis of pancreatic cancer patients.The TMEFF2 gene is a type I transmembrane protein that contains one epidermal growth factor(EGF)-like domain and two follistatin-like domains.Studies have shown that TMEFF2 is significantly underexpressed in esophageal cancer,breast cancer,lung cancer,gastric cancer,colorectal cancer,gallbladder cancer and other malignant tumor tissues,and has a significant role of tumor suppressor gene,affecting the proliferation,apoptosis,migration and invasion of tumor cells.But so far,there is no related research on the expression of TMEFF2 in pancreatic cancer at home and abroad,and the relevant mechanism is still unclear.In this paper,the role of TMEFF2 in the development and progression of pancreatic cancer and its possible mechanism were investigated from the clinical pancreatic cancer tissue specimens,in vitro cytology experiments and subcutaneous tumor transplantation model in nude mice,in order to provide a new theoretical basis and molecular target for the pathogenesis and clinical treatment of pancreatic cancer.objective:1.To investigate the expression levels of TMEFF2 gene in five human pancreatic cancer cell lines and clinical pancreatic cancer tissue specimens;To investigate the correlation between the expression level of TMEFF2 and the clinicopathological parameters and stages of patients.2.The effects of TMEFF2 gene on the proliferation,apoptosis,migration and invasion of pancreatic cancer cells and EMT(epithelial-mesenchymal Transition)were investigated through in vitro and in vivo.3.To investigate whether the TMEFF2 gene influences and regulates the molecular mechanism of pancreatic cancer cell proliferation,invasion and migration through the MAPK signaling pathway.Methods:1.Using q RT-PCR and Western Blot in turn to detect the expression levels of TMEFF2 gene in five human pancreatic cancer cell lines and human normal pancreatic ductal epithelial cells from m RNA and protein levels,respectively.It was also detected by q RT-PCR,Western Blot and immunohistochemistry in the samples,the correlation between the expression level of TMEFF2 and the clinicopathological parameters of the patients(36 cases)and their stages was studied.2.Transfection of pancreatic cancer cell lines with overexpressing TMEFF2 plasmid up-regulates the expression of TMEFF2,and the pancreatic cancer cell lines were divided into overexpressing TMEFF2 group and negative control group(NC)transfected with empty vector.CCK-8 cell proliferation assay,colony formation analysis,q RT-PCR,Western Blot and Transwell cell migration and invasion assay were used to detect the effects of overexpression of TMEFF2 on the biological behavior of pancreatic cancer cells.The transfected pancreatic cancer cell lines were used to construct a subcutaneous transplanted tumor model in nude mice,and the effect of overexpression of TMEFF2 on the growth and proliferation of pancreatic cancer tumor was verified in vivo.3.The effects of overexpression of TMEFF2 on the phosphorylation and expression levels of JNK and P38 proteins in the MAPK signaling pathway were preliminarily detected by Western blot.A stably transfected TMEFF2 si RNA pancreatic cancer cell line was constructed,the cells were treated with SB203580,and the effects of p38 MAPK inhibitor on the biological behavior of pancreatic cancer cells were detected by CCK-8 cell proliferation assay,colony formation assay,and Transwell cell migration and invasion assay,respectively.The effects of overexpression of TMEFF2 on the expression levels and phosphorylation of major proteins in Ras/Raf/MEK/ERK signaling pathway were detected by Western blot.Results:1.In all the five human pancreatic cancer cell lines,the TMEFF2 gene showed different levels of low expression(p<0.001).The expression level of TMEFF2 gene in pancreatic cancer tissues of clinical patients was significantly lower than that in paracancer normal pancreas tissues(p<0.001).The level of TMEFF2 gene expression was highly correlated with the size of tumor foci,clinicopathologic stage,and tumor differentiation degree of the patients,but was not significantly correlated with the age,gender,or presence of distant metastasis of the patients.The expression level of TMEFF2 gene in tumor tissues gradually decreased with the increase of tumor malignancy and clinical staging.2.After transfection with lentivirus plasmids overexpressing TMEFF2,the cloning and proliferation of the two pancreatic cancer cell lines(ASPC1 and Panc1)were significantly inhibited.The ability of cell migration and invasion was obviously weakened.In addition,compared with the NC control group,TMEFF2 overexpression significantly up-regulated the expression of E-cadherin and inhibited the expression of mesenchymal phenotypic marker related proteins(Snail,Vimentin,MMP-2 and MMP-9).Further animal in vivo experiments confirmed that compared with the negative control group(NC),TMEFF2 overexpression group significantly reduced tumor body weight(p<0.001),significantly decreased cell proliferation in tumor tissues(p<0.001),and significantly increased cell apoptosis(p<0.001).3.Compared with the NC group,the ratio of p-JNK / JNK and p-P38 / P38 in the MAPK signal pathway of the overexpressed TMEFF2 group was significantly reduced,and Ras,p-Raf / Raf,P-MEK / MEK,p ERK / ERK expression levels in the Ras / Raf / MEK / ERK signal pathway were significantly reduced.When knocked down the expression of TMEFF2,the proliferation,cloning ability,and cell migration and invasion ability of pancreatic cancer cells were significantly up-regulated,and the expression levels of E-cadherin protein in the cells decreased significantly,while the expression levels of MMP-2,MMP-9,Snail and Vimentin protein molecules were significantly increased.However,after treatment with p38 MAPK inhibitors,cells that knocked down TMEFF2 expression significantly decreased their proliferation,cloning ability,and cell migration and invasion ability,while the average expression level of E-cadherin protein was significantly increased,and the expression levels of MMP-2,MMP-9,Snail and Vimentin protein molecules were significantly decreased.Conclusions:1.The expression of TMEFF2 gene is low in clinical pancreatic cancer specimens and five human pancreatic cancer cell lines;The later the clinicopathologic stage of pancreatic cancer patients is,the lower the expression of TMEFF2 gene is,and it is related to the size of pancreatic cancer tumor and the degree of tumor differentiation.2.In vitro experiments,overexpression of TMEFF2 inhibited the cloning,proliferation,migration,invasion and epithelium-mesenchymal transformation of two pancreatic cancer cell lines(Panc1 and ASPC1).Overexpression of TMEFF2 can inhibit the proliferation level of pancreatic cancer tissues in animals and promote the apoptosis of cancer cells.3.TMEFF2 gene inhibits the proliferation level,migration and invasion of pancreatic cancer cells by blocking the phosphorylation process of MAPK signaling pathway.