Protein Expression Assay Of Down-Regulated STGC3Gene In NP69Cell Line

Objective The molecular network and mechanism of STGC3gene wereinvestigated in carcinogenesis of NPC. The STGC3gene was knocked-down byRNAi, and analysed the differentially expressed proteins with mass spectrometry afteridentifying the stable low expression of STGC3gene in NP69cell line, providedstrong experimental basis for clarifying the molecular mechanism of NPC.Methods Designed the specificity shRNA targeted to STGC3gene and constructedthe eukaryotic expression vector of pRNAi-U6.1/shRNA/STGC3and pRNAi-U6.1/scramble/STGC3. The recombinant plasmid was transfected into NP69cell line byLPTM2000and selected by G418to establish the stable transfected NP69cell lineswith low expression of STGC3gene. The mRNA of STGC3was analysed by RT-PCRafter transfection. The total proteins were extracted and proteolysis from thepRNAi-U6.1/shRNA/STGC3/NP69cell, pRNAi-U6.1/srcamble/STGC3/NP69celland NP69cell respectively, obtained information of the protein expression abundanceby iTRAQ coupled with Mass Spectrometry, and then screeninged the differentiallyexpressed proteins by using database searching, constructed the protein interactionnetwork by bioinformatics analysis.Results Successfully constructed the eukaryotic expression vector of pRNAi-U6.1/shRNA/STGC3and pRNAi-U6.1/scramble/STGC3by DNA sequencing and blasting.The transfected cells expressed with GFP under the fluorescence microscopy showedsuccessful transfection of the eukaryotic expression vector. RT-PCR analysis showedthat the expression of STGC3was down-regulated by pRNAi-U6.1/shRNA/STGC3recombinant plasmid. iTRAQ isotope labeling and mass spectrometry showed that atotal of83differentially expressed proteins were identified in NP69cell successfullytransfected with pRNAi-U6.1/shRNA/STGC3, in which there were45up-regulation and38down-regulation differential proteins involved in synthetization of protein, cellproliferation and apoptosis.Conclusions1. Successfully established the pRNAi-U6.1/shRNA/STGC3/NP69cell line withlow expression of STGC3gene.2.83differentially expressed proteins were identified by proteomics technology, inwhich there were45up-regulation and38down-regulation differential proteinsinvolved in synthetization of protein, cell proliferation and apoptosis.