Research On The Expression Of MFG-E8 In Gastric Cancer And Its Clinical Prognosis

Background:Gastric cancer(GC)is one of the most common malignant tumors in the world.It is the fifth most common cancer in the world and the third leading cause of cancer death.Although great progress has been made in the treatment of GC with the progress of science and technology,the prognosis of patients with GC is often poor because of the lack of understanding of the mechanism of GC and its pre-tumor state.With the development of molecular understanding in recent years,we think that cancer may have unique pathogenic mechanism and rich oncogenic pathway.For these reasons,finding and identifying molecular markers associated with gastric carcinogenesis and development to clarify the clinical features of GC and thereby predict outcomes such as recurrence and prognosis in patients,with an important goal of GC research.Milk fat globule-EGF factor 8(MFG-E8)is a peripheral membrane protein.First found in the mammary glands of lactating mice,it is also found in many other types of cells.The N-terminal of MFG-E8 binds to the integrin protein at the cell membrane surface through the arginine-glycine-L-aspartic acid(RGD)integrin sequence in the epidermal growth factor(EGF)-like domain,the c-terminal contains the F58 C domain of the tyrosine kinase receptor,it binds specifically to phosphatidylserine(PS)on the cell membrane surface.Studies have shown that MFG-E8 is an important factor in the development of various inflammatory diseases,for example,it can delay senescence by targeting chondrocytes through the NF-κB pathway,and it can also promote M2 polarization of macrophages,to prevent osteoarthritis.Recent studies have found that MFG-E8 also plays an important role in cancer,such as colorectal cancer,breast cancer and ovarian cancer.However,its expression and function in GC remain unclear and need to be further studied.Objective:(1)The expression of MFG-E8 in gastric cancer and adjacent tissues;(2)To explore the important function of MFG-E8 in gastric cancer;(3)To analyze the role of MFG-E8 in the diagnosis of gastric cancer and its effect on the survival rate and prognosis of patients with gastric cancer Clinical significance.Method:Using tumor immune assessment resource(TIMER),Gene expression profiling interaction analysis(GEPIA)and Kaplan-meier(KM)to analyze expression and prognosis of MFG-E8 in GC.We used immunohistochemistry,immunofluorescence,real-time fluorescence quantitative PCR(q RT-PCR),Western blot(western blood)and other experiments to further verify.To explore the relationship between MFG-E8 expression and the stage,grade and survival rate of GC,the information of GC patients was analyzed by TCGA database.In addition,we performed MFG-E8 gene mutation analysis by using the c Biprotal online database.To verify the important role of MFG-E8 in gastric cancer,we knocked down MFG-E8 in GC cells and carried out cell function experiments including EDU proliferation experiment,wound healing experiment and Transwell invasion experiment.We further predicted the upstream mi RNA of MFG-E8 via the Targetscan database and validated whether there is a direct effect between the two.We then transfected mi RNA mimics into gastric cancer cells to investigate their important roles in GC.TISIDB and TIMER were used to analyze the correlation between MFG-E8 and tumor immune infiltration.Finally,we performed gene classification(GO)and KEGG enrichment analysis to explore important signaling pathways downstream of MFG-E8 and its associated genes.Results:In this study,we analyzed the differential expression and prognosis of MFG-E8 in GC by online database,and found that MFG-E8 was highly expressed in GC compared with normal tissues,the results were further confirmed by immunohistochemistry,immunofluorescence,western-blood and so on.We collected information on patients with GC from the TCGA database and found that high MFG-E8 expression was associated with clinical stage,grade,and poor survival of GC.Furthermore,we performed mutation analysis of the MFG-E8 gene using the c Biprotal database and found that the mutation frequency of MFG-E8 was 2.57% in patients with GC and that patients with MFG-E8 mutations had a worse prognosis than those without mutations.We found that knockdown of MFG-E8 could significantly inhibit the proliferation,migration and invasion of GC cells.mi R-214-5p upstream of MFG-E8 was predicted by the Targetscan database,and dual luciferase experiments were performed and found that the two could bind directly.At the same time,cell function experiments were performed after transfection of mi R-214-5P mimics and it was found that mi R-214-5p could inhibit the properties of GC,so we hypothesized that mi R-214-5p may play an important role in GC by targeting MFG-E8.The expression of MFG-E8 was significantly correlated with the expression of CD8 + T cells,CD4 + T cells,macrophages,neutrophil and so on,the M2 polarization of macrophages was the most significant.Finally,we performed GO and KEGG enrichment analyses and found that genes similar to MFG-E8 were mostly enriched in extracellular matrix and closely associated with pathways such as focal adhesions.Our results suggest that MFG-E8 is a novel prognostic marker for GC and that it may play a role as an oncogenic factor in GC,which is also critical for exploring therapies targeting MFG-E8.Conclusion:(1)The expression of MFG-E8 was increased in gastric cancer,and correlated with pathological parameters such as depth of invasion and clinical grading.(2)Knockdown of MFG-E8 inhibited the proliferation,migration and invasion of gastric cancer cells.(3)High expression of MFG-E8 is associated with poor prognosis,high frequency of gene mutation and increased level of immune infiltration in gastric cancer patients,which may be a new prognostic marker.