A Study On Regulation Of CD74 Acetylation In Human Breast Cancer Cells

BackgroundsCancer,also known as malignant tumor,is one of the most lethal diseases in human.Metastasis is the basic biological character of malignant tumors,as well as the main death factor of clinical patients with tumor.In all cancer types,breast cancer has become a serious threat to women’s health as it is the type with the highest morbidity in women.The leading cause of death in breast cancer patients is the multiple organ disease triggered by proliferation of tumor cells.Therefore,the study of the mechanism in breast cancer metastasis is of great significance for its treatment.Cluster of differentiation 74(CD74)is the invariant chain of major histocompatibility complex(MHC)Ⅱ,which has four isoforms in human:p33,p41,p35 and p43.The major isoform we detected in breast cancer is p35.CD74 has a variety of post-translational modifications,such as N/O glycosylation,phosphorylation and palmitoylation.But the acetylation of CD74 has not been reported yet.CD74 was originally identified as a molecular chaperone of MHC Ⅱ,which played an important role in antigen presentation.Subsequent studies found that CD74 also had some functions that did not depend on MHC Ⅱ.For example,CD74 was a cell surface receptor of macrophage migration inhibitory factor(MIF)which triggered signal cascades to regulate dendritic cell migration.The previous data in our laboratory showed that CD74 could regulate the metastasis of breast cancer cells.However,the molecular mechanisms of regulating the expression of CD74 is still unclear.Y-Box binding protein 1(YB1)is structurally composed of a non-conserved mutant N-terminal domain,a highly conserved cold-shock domain and a C-terminal domain.The N-terminal domain is rich in alanine and proline,controlling transcriptional activation,while the C-terminal domain is composed of acidic residues and alkaline residues alternately,controlling regulation of proteins and protein-related interactions.YB1 was originally found as a transcription factor which could bind to the promoter region of MHC II,playing important roles on DNA,mRNA and protein levels,such as DNA repairing,precursor mRNA cleavage,mRNA stability maintaining,and protein-protein interactions.Though software analysis,we found that the regulation sites of YB1 may exist in both CD74 promoter region and CD74 mRNA.So we will focus on finding new chemotherapeutic targets through exploring the mechanism of YB1 affecting cell metastasis via regulating CD74.Methods1.To detect the effects of YB1 siRNA interference on the expression of CD74 and cell movement related proteins with western blot2.To detect the effects of YB1 siRNA interference on the transcription level of CD74 with RT-PCR3.To detect the effects of YB 1 siRNA interference on the protein stability of CD74 with western blot and to explore the effect of YB1 siRNA interference on CD74 ubiquitination by immunoprecipitation4.To explore whether CD74 has the acetylation modification with immunoprecipitation experiments,and then using mass spectrometry to analyze the potential acetylation sites of CD745.To explore the regulation of CD74 acetylation by YB1 with immunoprecipitation experiments6.To explore the interaction between YB1 and CD74 as well as other related proteins with immunoprecipitation experiments7.To explore the regulation of CD74 acetylation by SIRT1 with immunoprecipitation experiments8.To explore the changes in the ability of combination between YB1,SIRT1,CD44 and CD74 3KR with immunoprecipitation experiments9.To detect the effect of CD74 acetylation on cell invasion with transwell assay10.To explore the relationship between CD74 acetylation and CD74 ubiquitination with immunoprecipitation experimentsResults1.Cells are transfected with small interfering RNA of YB1 and the protein level of CD74 and the cell movement related proteins are down-regulated2.YB1 regulates the stability of CD74 at the protein level rather than the transcription level3.CD74 has interactions with YB1,SIRT1 and HDAC14.CD74 has acetylation modification and the deacetylase SIRT1 plays major roles in this process5.Both YB1 and SIRT1 can affect the acetylation of CD746.Mass spectrometry analyzation showed that the potential acetylation sites of CD74 were Lys80,Lys99 and Lys106,and the acetylation level of CD74 was reduced after we mutated lysine to arginine7.The mutation of CD74 acetylation sites affects the combination between YB1,SIRT1,CD44 and CD748.The mutation of CD74 acetylation sites affects cell invasionConclusionIn human breast cancer cells,we found that YB1 affected the acetylation and stability of CD74 through impacting the interactions between CD74 and deacetylase SIRT1.After mutating the possible acetylation sites of CD74,the acetylation and ubiquitination level of CD74 were reduced,the ability of interacting with YB1 and SIRT1 were enhanced and the interaction between CD74 and CD44 was decreased.Finally,the mutation of CD74 acetylation sites decreased the invasion ability of cancer cells.