The Mechanisms Of PRMT1 Catalyzing The Methylation Of YBX1 Protein To Promote Epithelial-mesenchymal Transition And Metastasis Of Esophageal Squamous Cell Carcinoma

Esophageal cancer is a highly malignant tumor and is one of the leading causes of cancer-related death.Esophageal cancer can be divided into two histological types: esophageal adenocarcinoma and esophageal squamous cell carcinoma(ESCC),the latter of which is the most important type.Although great progress has been made in the diagnosis and treatment of esophageal squamous cell carcinoma,the overall survival rate is still low.Tumor invasion and metastasis are important reasons for the low cure rate and high treatment difficulty of cancer.During the metastasis process,cancer cells invade adjacent tissues and then spread to the blood system before invading the secondary organs,thereby affecting the whole body.Most cancers come from epithelial organs,and epithelial-mesenchymal transition of cancer cells is thought to contribute to their invasion and metastasis.Epithelial cells change into elongated fibroblasts with concomitant loose cell-cell adhesion and higher motility,facilitating their invasion into nearby tissues.Metastatic cancer cells have interstitial properties that are clearly associated with poor clinical outcomes.In the past decade,more and more studies have shown that EMT can promote the invasion,metastasis and anti-apoptosis of various tumor cells,such as breast cancer,colorectal cancer and prostate cancer.Protein arginine methyltransferase is a kind of enzyme widely expressed in mammalian tissue cells.It mainly contains 9 family molecules,which mediates methylation of protein arginine residues and participates in cell signal transduction,gene transcription,DNA damage repair,mRNA scission and other cellular processes,affecting the development of various diseases such as tumor,inflammation and metabolism.PRMT1,the most important member of the PRMTs family,can be involved in mediating more than 70% of protein methylation in cells.Many studies have shown that abnormal expression of PRMT1 is closely related to tumor progression,such as lung cancer,breast cancer,and colorectal cancer.In the present study,we found that the expression of PRMT1 in esophageal squamous cell carcinoma cells was significantly increased.Elevated expression of PRMT1 is significantly associated with poor prognosis in patients with esophageal squamous cell carcinoma.Functional experiments showed that PRMT1 is closely related to EMT,invasion,migration,and tumor formation of esophageal squamous cell carcinoma.With the development of antibody and proteomics technology,research on PRMTs and arginine methylation modification has been widely carried out.In particular,the method of immunoaffinity purification mass spectrometry(IAP-LC-MS/MS)reveals many methylated proteins and methylation sites catalyzed by PRMTs in vivo.However,the role of methylated protein and methylation sites catalyzed by PRMT1 and its mediated protein methylation on ESCC progression has not been reported.We first constructed a PRMT1 overexpressing ECA109 cell line by lentiviral transfection.The PRMT1-bound protein was purified and collected by immunoprecipitation.Subsequently,we used an anti-arginine monomethylation motif antibody to precipitate the peptides rich in methylation sites in the PRMT1 immunoprecipitate eluate.After collection,liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to identify the protein of interest and methylation sites.Finally,the above identified proteins were compared with the CO-IP results and the YBX1 was finally selected,which proved that it directly binds to PRMT1 in vivo and identified the site where PRMT1 catalyzes its methylation.The results of mass spectrometry confirmed that YBX1 directly binds to PRMT1 and identified R199 as a methylation site of PRMT1.So,what is the effect of methylation of the R199 site on the development and progression of esophageal squamous cell carcinoma? In combination with former studies,we constructed methylation site mutants of YBX1-R199 K,YBX1-R200 K,and established ECA109 and TE1 stable cell lines with lentivirus.In vivo methylation experiments confirmed R199 is the PRMT1 methylation site of YBX1.Functional experiments further confirmed the promotion of EMT,invasion and migration of esophageal squamous cell carcinoma by methylation of R199.Finally,we found that the high expression of PRMT1 and YBX1 was significantly associated with poor prognosis in patients with esophageal squamous cell carcinoma.Taken together,in this study,we confirmed the role of PRMT1 in promoting the progression of esophageal squamous cell carcinoma,and identified the methylation protein and methylation site of PRMT1 in esophageal squamous cell carcinoma.We also reveal the mechanism of EMT,invasion and migration of esophageal squamous cell carcinoma and provide a new perspective for the study of PRMT1.What's more,we also provide a potential target for the treatment of esophageal squamous cell carcinoma.