Study On The Mechanism Of Wnt/β-catenin Signaling Pathway Mediating Epithelial-mesenchymal Transition In Multiple Myeloma

Objective:Multiple myeloma(MM)is a fatal hematologic malignancy with clonal and proliferative plasma cells in the bone marrow and causes devastating bone disease.This study aimed to explore the expression of epithelial-mesenchymal transformation(EMT)markers(E-cadherin,N-cadherin,Vimentin,and β-catenin)in MM and to analyze its relationship with clinical parameters and prognosis.Investigating the regulation of the Wnt signaling pathway on the EMT characteristics of MM by using IWR-1 which has been deemed as Wnt signaling pathway inhibitors.Methods:Bone marrow blood samples from 25 newly diagnosed MM patients were collected from the Department of Hematology,Affiliated Hospital of Qingdao University.All patients had complete clinical data.The expressions of E-cadherin,N-cadherin,Vimentin,and β-catenin in MM specimens and cell lines were detected by real-time quantitative PCR(q RT-PCR).SPSS 22.0 software was used to analyze the relationship between gene expression and clinical parameters.Based on the GEO database,RNA information of MM patients was downloaded to analyze the relationship between genes expressions and prognosis.Gene ontology(GO),Kyoto encyclopedia of genes and genomes enrichment(KEGG)analysis,and protein-protein interaction(PPI)network construction were utilized to further explore the biological function and mechanism of genes.Cell counting kit-8(CCK-8)Assay was used to examine the effect of IWR-1 on the proliferation of RPMI-8226 cells.q RT-PCR and western blot(WB)were used to verify the effects of IWR-1 on EMT-related genes and proteins,respectively.Transwell assay was used to detect the invasion and migration of cells.Result:The expression of E-cadherin in CD138-positive plasma cells from the bone marrow of patients was lower than that in CD138-negative cells(P=0.028),and the expression level of E-cadherin was negatively correlated with bone destruction(r =-0.440,P=0.028)and DS stage(r=-0.409,P=0.042).N-cadherin,Vimentin,and β-catenin were highly expressed in CD138-positive plasma cells,and the differences were statistically significant.Analysis of the prognostic information of 542 patients provided by the GEO database showed that low expression of E-cadherin suggested short overall survival and poor prognosis.Moreover,the reduction of E-cadherin is already present in the precancerous stage(MGUS,SMM)and continues to decrease as the disease progresses.Knockdown of β-catenin in MM cell lines resulted in the expression changes of N-cadherin and Vimentin genes,and the differentially expressed genes were enriched in cell adhesion molecules and cell adhesion regulation.The q RT-PCR outcomes of clinical patients and MM cell lines showed EMT-feature existed in myeloma.Wnt/β-catenin signaling pathway inhibitor IWR-1 could inhibit proliferation and invasion of RPMI-8226 cells,as well as reversed the expression of E-cadherin,N-cadherin,Vimentin,andβ-catenin.Conclusion:EMT characteristics were found in plasma cells of MM patients and MM cell lines.Low expression of E-cadherin suggested that patients might have bone destruction and high clinical DS stage,as well as the possibility of shortened overall survival and disease progression.IWR-1 showed clear inhibition of MM cell proliferation and EMT process by Wnt/β-catenin pathway.Therefore,E-cadherin and Wnt/β-catenin signaling pathways might be considered therapeutic targets for MM.