The Effect Of FAT10 On Cell Proliferation In Cancer Cells By Regulating Expression Of Eukaryotic Translation Elongation Factor 1A1 And Its Mechanisms

Objective: 1.To investigate the effects of FAT10 on biological activity in cancer cells through the regulation of eEF1A1 expression.2.To analyze the effects of FAT10 on ubiquitination and degradation of eEF1A1.3.To confirm the Lys in eEF1A1 are required for FAT10 of ubiquitin interaction.4.To clarify the mechanism of FAT10 and ubiquitin regulate eEF1A1.Finally,to identify the molecular mechanism of FAT10 regulate eEF1A1 and the effects of it on biological activity in cancer cells through the regulation of eEF1A1 expression.Methods: 1.MHCC-97 H,Huh7,PANC-1,MB231 and SW620 cell lines were transfected with si FAT10 or sieEF1A1.Reverse transcriptase polymerase chain reaction(RT-PCR)and Western blot assays were used to detect the FAT10 and eEF1A1 m RNA and protein levels,respectively.Luciferase assay detect the effect of FAT10 on eEF1A1 promoter activity in Hep3 B cells.The fluorescence resonance energy transfer(FRET)was performed to confirm the interaction of FAT10 and eEF1A1.MHCC-97 H and Huh7 cell proliferation was measured by CCK8 assay,change of cell cycle was detected by PI staining method through flow cytometry and apoptosis was demonstrated by Annexin V apoptosis kit,the change of cell invasion ability was detected by Transwell assay.2.In vivo ubiquitination assay and cycloheximide chase analysis determine the effect of FAT10 on ubiquitination and degradation of eEF1A1.To confirm the interaction motif of eEF1A1 to FAT10,the truncated mutants of p CMV-N-HA-eEF1A1 were generated by standard molecular cloning methods.The Lys in eEF1A1 motif binding to FAT10 and Ub were mutated to arginine by site directed mutagenesis kit,which to confirm the Lys in eEF1A1 binding to FAT10 and Ub.3.The competitive binding of FAT10 and Ub to eEF1A1 was determined by the competition assay,co-immunoprecipitation and GST pull down assay.GST pull down assay,co-immunoprecipitation and colocalization by confocal microscopy was used to confirm the FAT10/Rpn10 and eEF1A1/Rpn 10 interactions in vivo.In vitro protein expression,competition assay,co-immunoprecipitation and GST pull down assay determine the correlation of Rpn10 binding to FAT10 and eEF1A1.Results: 1.The expression levels of FAT10 and eEF1A1 both noticeably decreased in si FAT10 transfected MHCC-97 H,Huh7,PANC-1,MB231 and SW620 cells.In sieEF1A1 transfected cells,only eEF1A1(not FAT10)expression noticeably decreased.The eEF1A1 expression was restored by eEF1A1 transfection in the sh FAT10 cells.Luciferase assay detected that FAT10 or FAT10 si RNA transfection did not influence eEF1A1 promoter activation.The FRET confirmed the interaction of FAT10 and eEF1A1.The down-expression of FAT10 and eEF1A1 by si RNA interference both could significantly inhibit the proliferation and induce cell cycle arrest in MHCC-97 H and Huh7(P <0.05),whereas the upregulation of eEF1A1 rescued proliferation ability and cell cycle arrest induced by FAT10 knockdown in MHCC-97 H and Huh7 cells(P <0.05).Transwell assay showed that the cell invasion ability was significantly inhibited(P <0.05)in sh FAT10 or sheEF1A1 transfected cells compared with control cells,and this inhibition did not recover by the upregulation of eEF1A1.2.FAT10 affected the ubiquitination and degradation of eEF1A1.Overexpression of FAT10 increased eEF1A1 ubiquitination level,while the interference of FAT10 promotes ubiquitination and degradation of eEF1A1.Co-immunoprecipitation confirmed that only domains II and III of eEF1A1,probably within a region comprising residues 284-332 and 394-462,bound to FAT10 or Ub but not I Domain of eEF1A1.Further,study showed that the eEF1A1 290,313,318,330,395,408,439,443,444,450,453,457,460 and 462 fourteen lysine sites play an important role in binding to FAT10 or Ub by site-directed mutagenesis and co-immunoprecipitation.3.Competitive assay showed that FAT10 compete with Ub for binding to the same sites of eEF1A1.The study confirmed that the Ub-eEF1A1 complex bind to 26 S proteasome subunits Rpn10,but FAT10-eEF1A1 complex does not recognized by Rpn10,so as to protect eEF1A1 from ubiquitin-proteasome degradation.Conclusion: FAT10 ylation antagonizes ubiquitination to stabilize eEF1A1.FAT10 effects biological activity in cancer cells through the regulation of eEF1A1 expression.FAT10/substrate complexes recognized by Rpn10,is a critical step in the response to FAT10 stabilizing or degrading substrate.These provide a new theoretical foundation to clarify FAT10 involved in the mechanism of development and progression of cancer cells,and provide substantial rationale for cancer diagnosis and treatment.