YTHDF3敲低前后三阴性乳腺癌细胞非标记定量蛋白质组学差异分析OA

Objective This study aimed to analyze the protein expression differences of triple negative breast cancer(TNBC)cells before and after YTHDF3 knockdown and explore the possible mechanisms by which YTHDF3 regulates protein function in TNBC cells.Methods The YTHDF3 stably knocked down MDA-MB-231 cell lines were constructed using molecular cloning,target design and viral vector packaging techniques and further validated by Western blot.We cleaved the proteins and obtained the differentially expressed proteins caused by knockdown of YTHDF3 using Ultra Performance Liquid Chromatography(UPLC)and Liquid Chromatography-Tandem Mass Spectrometry(LC-MS/MS)methods.Then,protein-protein interaction(PPI)networks were mapped and analyzed using the String database and Cytoscape 3.10.1 software,and core targets were screened.The core targets were analyzed for Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway and Gene Ontology(GO)functional enrichment through the David database.RNA interference was applied to knock down YTHDF3 or UBB gene in MDA-MB-231 and HS-578T cells,respectively.The expression of core target proteins in the two TNBC cells with YTHDF3 knockdown was verified by Western blot.Finally,the impact of UBB(Ubiquitin B)knockdown on the invasion and migration of TNBC cells was detected by Transwell assay.Results We successfully constructed a TNBC cell model with stably YTHDF3 knockdown and quantified 2 295 proteins as well as screened 261 differentially expressed proteins(126 down-regulated and 135 up-regulated).GO analysis showed that the differentially down-regulated proteins were mainly involved in translation and protein stabilization,while the differentially up-regulated proteins were mainly associated with negative regulation of cell population proliferation.KEGG results indicated that the differentially down-regulated proteins were mainly enriched in spliceosome-related signaling pathways,whereas differentially up-regulated proteins were mainly relevant to apoptosis-related pathways.The PPI analysis revealed that six core targets(RPS27A,UBA52,UBC,UBB,RPL4,and RPS2)were all down-regulated.Western blot analysis uncovered decreased expression of both YTHDF3 and UBB proteins in YTHDF3 knockdown MDA-MB-231 and HS-578T cells.Transwell results indicated that deficient UBB inhibited the invasion and migration levels of TNBC cells.Conclusions YTHDF3 plays a crucial role in the invasion and metastasis of TNBC cells by regulating the downstream core target UBB.