Identification Of Transcription Regulatory Elements Within STGC3Gene Core Promoter Region

OBJECTIVE: To predict the potential transcription factor binding sites of STGC3gene within the core promoter region, we applied bioinformatics method; usingelectrophoretic mobility shift analysis and chromatin immunoprecipitation, toidentificate the transcriptional regulatory elements in the gene core promoter; utilizingRT-PCR and Western blotting techniques, to detect the expression level of transcriptionfactor Sp1in NPC cells, in order to reveal the molecular mechanisms of transcriptionalregulation of STGC3gene.METHODS: Based on our preliminary findings, using bioinformatics software topredict the possible transcription factor binding sites in the core promoter region(-934bp/-653bp) of STGC3gene; according to forecasts, we select transcription factorSp1site sequence to prepare three kinds of probe respectively, including biotin-labeledwild-type probe and mutant probe and unlabeled wild-type probe; extract nuclearprotein of CNE2cells, and identified the ranscription factor binding sites in vitro andin vivo through EMSA and ChIP; RT-PCR and Western blotting techniques to detectthe Sp1expression at mRNA and protein level in different cells.RESULTS: The prediction results of Alibaba2.1software show that there weretwenty one transcription factor binding sites, such as AP-1, Sp1, GAL4, HNF-3B andC/EBPalp and so on, in the region of-934bp to-653bp, of which including nine Sp1transcription factor binding sites; however, the results only contain five transcriptionalregulatory elements after strict setting parameters of this software, two of them wereSp1transcription factor binding sites; electrophoretic mobility shift assay (EMSA) andchromatin immunoprecipitation (ChIP) experiments confirmed that CNE2cell nucleiexists the protein, which could Combine the transcription factor Sp1, and thiscombination with specificity; RT-PCR test results showed that the mRNA level of Sp1expression in CNE2cells (STGC3gene high expression) was obviously increased, compared to NP69cells (STGC3low gene expression), the difference was significant,with statistical significance(p<0.01); Western blotting analysis showed that the proteinexpression levels of Sp1in CNE2cells was significantly higher than in NP69cells,with a statistically significant difference (p <0.05).CONCLUSION:1. The presence of transcription factor Sp1specific binding sites in STGC3gene corepromoter region.2. Transcription factor Sp1negative regulation of STGC3gene expression innasopharyngeal carcinoma CNE2cells.