The Effect Of TSTA3 On Invasion And Migration Of Esophageal Squamous Cell Carcinoma By Regulating α-1,2 Fucosylation Of ERBB2

Objectives:Based on the whole genome sequencing of the esophageal squamous cell carcinoma,we found that the copy number amplification,High expression of TSTA3 gene encoding the key enzyme of GDP-L-fucose synthesis in ESCC were correlated with lymph node metastasis and poor prognosis of the patients.We found that TSTA3 can observably promote the invasion and migration of ESCC cells in vitro.Based on the previous work,this study aims to further clarify the role of TSTA3 in ESCC and the mechanism of abnormal fucosylation mediated by TSTA3 on the invasion and migration of ESCC.Methods:1.Stable overexpression of TSTA3 gene(TSTA3-WT)in KYSE150 cells and negative control cells(KYSE150-NC)were injected into BALB/ C-NU mice through tail vein,and the metastatic nodules of various organs in nude mice was observed by small animal PET/CT imaging technology.After 6 weeks,the mice were killed by an overdose of 2% pentobarbital sodium,calculate the number and size of metastatic nodules on the surface of the lung and liver.At the same instant,HE staining was used to further verify the metastasis of the tumor.The study aims to determine the effect of TSTA3 overexpression on ESCC metastatic ability at animal level.2.Protein and glycoprotein expression levels in negative control and TSTA3-overexpressed KYSE150 cells were compared using LC-MS/MS techniques featuring proteomics and N-glycoproteomics.Ensure high levels of confidence,the standard of localization probability > 0.75 was used to filter the data and the quantified values of the filtered glycosylation modification sites were normalized by protein quantification After being filtered by proteomic data,the differentially expressed glycoproteins were further screened.3.We collected control and overexpression groups of KYSE150 cells.Total proteins were extracted and enriched with UEA-I lectin for α-1,2 fucosylated proteins.After elution and lyophilization,the enriched total fucosylated proteins were dissolved and added into the upper chamber of transwell chamber together with ESCC cells to observe the ability of invasion of ESCC.Then,fucosylated protein was treated with α-L-fucosylsidase to observe the change in invasion ability,Meanwhile,UEA-I lectin affinity enrichment combined with LC/MS/MS analysis of enzymatic digestion in the whole gel was to screen the differential proteins between TSTA3-WT and NC groups.Then,the identified differential N-glycoproteomics data were combined with gel mass spectrometry to screen the α-1,2 fucosylated proteins targeted by TSTA3.4.In KYSE150 and KYSE450 cells,TSTA3 and NC groups were used to extract the total protein for UEA-I lectin affinity enrichment,then Western blot analysis was performed on ERBB2,Meantime,different concentrations of L-fucose were added into the affinity chromatography system to observe whether different concentrations of L-fucose affected the enrichment of ERBB2 protein by UEA-I5.UEA-I western blot after ERBB2 immunoprecipitation was used to observe the binding ability of UEA-I lectin and ERBB2 protein in NC and TSTA3 groups with or without PNGase treatment.6.TSTA3 overexpressed ESCC cells was infected by small interfering RNA(si RNA)targeting ERBB2 to investigate the effect of ERBB2 knockdown on the invasion and migration of ESCC cells.7.After TSTA3 overexpressed ESCC cells were treated with glycosylation inhibitor 2-F-fuc,the invasion and migration of cells were detected by transwell chamber assay,and Western blot observation of fucosylated ERBB2 and ERBB2 total protein expression8.The target gene ERBB2-specific si RNA was transfected into ESCC cell lines of TSTA3-WT,and the interference efficiency was verified by western blot.The effection of ERBB2 in ESCC cell lines was observed by MTT,clone formation,Transwell,flow apoptosis and other experiments.Results:1.The results of two independent experiments show that small animal CT imaging,lung metastases in the TSTA3-WT group were significantly more than those in the NC group.Anatomy also showed that compared with the NC group,mice in the TSTA3-WT group had poor physical state and more metastatic pulmonary nodules,the difference was statistically significant(P<0.05).HE staining was further confirmed the results.2.After proteomic data filtering,a total of 1100 N-glycosylation sites in 575 glycoproteins were identified in N-glycoproteomics.Among these glycoproteins,we found that 37 glycoproteins were up-regulated and 87 were down-regulated with fold change over 2.0.The most significant enrichment of motif is the classic N-glycosylation sequence: N-X-S.Differentially expressed glycoproteins are mainly concentrated in phagocytes,ECM receptor interactions,galactose metabolism and other pathways.3.UEA-I lectin enriched fucosylated proteins in the TSTA3-WT group promoted cell invasion more than those in the NC group,and the difference was significantly reduced after treatment of fucosylated proteins with α-L-fucosylase.4.UEA-I lectin affinity enrichment followed by western blot showed significantly increased fucosylated ERBB2 levels in TSTA3-WT compared with NC transfected KYSE150 and KYSE450 cells,while input showed no differences in the expression level of total ERBB2 protein.Furthermore,with the increase ofα-L-fucose concentration and competitive binding to lectin,the protein content of ERBB2 enriched by UEA-1 also decreased correspondingly.Consistently,immunoprecipitation(IP)of ERBB2 followed by UEA-I blot showed increased UEA-I binding to ERBB2 proteins in TSTA3-WT group compared with NC group.The observed differences also disappeared upon PNGase treatment,despite equal amounts of IP input of each condition5.Knockdown of ERBB2 in TSTA3 overexpressed ESCC cells can reverse the effect of TSTA3 overexpression on promoting the invasion and migration of ESCC cells(P < 0.05).6.Treatment with fucosylation inhibitor 2-F-fuc significantly inhibited the invasion and migration of ESCC cells(P < 0.05).After 2-F-fuc treatment and enrichment of UEA-I lectin,western blot showed that the level of fucosylated ERBB2 was significantly decreased,and the expression level of total ERBB2 protein was not different.2-F-FUC decreased the expression of TSTA3 protein and the level of ERBB2 fucosylation to a certain extent.7.MTT and clone formation assay showed that ERBB2 knockdown significantly reduced the proliferation and clone formation ability of ESCC cells with overexpression of TSTA3(P < 0.05).Flow flow assay showed that compared with the control group,the early and late apoptosis rates of ERBB2 interference group were lower,the difference was statistically significant(P < 0.05).Conclusion:1.Overexpression of TSTA3 gene may promote invasion and metastasis of ESCC cells by promoting α-1,2 fucosylation of ERBB2.2.The glycation inhibitor 2-F-fuc can reduce the protein expression of TSTA3 and the α-1,2 fucosylated modification of ERBB2,thereby inhibiting the invasion and migration of ESCC cells.