The Study On The Deletion Of SMAD4 Protein Expression And SMAD4 Gene Mutation In Pancreatic Ductal Adenocarcinoma

Research background and purpose: Pancreatic ductal adenocarcinoma(PDAC)is one of the malignant tumors in digestive system.90% of the patients with pancreatic cancer belong to this type.Only 15% to 20% of the patients can get the opportunity of surgical treatment.The incidence of pancreatic cancer in China is increasing year by year,and the 5 year survival rate is lower than the world average.SMAD4 was first be found protein expression deficient in 50% of pancreatic cancer,and its main function is to mediate the signal transduction in the transforming growth factor beta(TGF-beta)pathway.There is a certain difference in the mutation rate of SMAD4 gene in PDAC patients from different countries and nationalities,and whether SMAD4 can be used as a biomarker for predicting the prognosis of PDAC patients remains controversial.The hot spots of SMAD4 mutation in PDAC patients are located in exon 8 and exon 11.There are few studies on the relationship between SMAD4 mutation and PDAC,and the reports of the protein expression and gene mutation in the region of Shandong are rare.Therefore,we studied the relationship between the expression of SMAD4 protein and clinicopathological features and prognosis in PDAC patients in Shandong.To detect the mutation of SMAD4 total exon in the PDAC patients in Shandong and investigate the effect of the newly discovered SMAD4 Y353 C mutation on the proliferation and migration of SW1990 and PANC-1 cell line cells to clarify the effect of the mutation on the development of PDAC.Methods:(1)95 cases of PDAC paraffin embeded specimens were collected,matched with adjacent normal tissues.The expression of SMAD4 protein was evaluated by immunohistochemistry and then its realationships with clinicopathological features and prognosis were analyzed.(2)The genomic DNA in 85 cases of PDAC specimens were extracted,then the whole exons of SMAD4 gene were screened by Sanger sequencing method.(3)The effects of the SMAD4 mutation on the proliferation and migration of pancreatic cancer cells were evaluated by the(CCK-8 method)and wound healing experiment.Results:(1)In 95 PDAC patients,SMAD4 negative expression of PDAC tissues accounted for 75%(72/95),SMAD4 negative expression in normal tissues accounted for 25%(24/95),and the difference was statistically significant(c2=48.511,p<0.001).The expression of SMAD4 protein in PDAC was correlated with the diameter,TNM staging,lymphatic invasion and differentiation degree.The median survival time of patients with positive SMAD4 expression was same as that of SMAD4 negative patients.The expression level of SMAD4 is not related to the overall survival time(P=0.053).(2)The mutation rate on the exons of SMAD4 was 11.8%,6 cases occurred at the first exon,1 cases occurred at the secend exon,2 cases occurred at the mutation hotspot region eighth exon,and 1 cases occurred in the ninth exon.(3)The SMAD4 Y353 C functional assays showed that SMAD4 Y353 C had great influence on the cell migration ability but not on the proliferation of PDAC cells.Conclusion: Deletion of SMAD4 protein is closely related to the malignancy of PDAC,but it has nothing to do with the overall survival of PDAC patients.The integrity of SMAD4 plays an important role in inhibiting the migration of tumor cells.The missense mutation SMAD4 Y353 C promotes the cells metastasis ability.