Promoter Cloning And Transcriptional Regulation Of Tongue Cancer Resistance Protein 1 (TCRP1)

Background & Objective:Tongue cancer is the most common in oral and maxillofacial cancers, clinically treated with conventional surgery in combination with radiotherapy or chemotherapy. Because of chemotherapy often leads to drug resistance in cancer cells,and the mechanism of drug resistance has not been clarified,the treatment effectiveness of tongue cancer has not been so satisfactory presently. TCRP1,a drug-resistance-related gene, was identified by our laboratory from multidrug-resistance cell (Tca8113/pym) employing a strategy of EST-mediated sscreening.In vivo and in vitro studies preliminary confirmed, TCRP1 gene of tongue cancer cells can induce resistance to cisplatin specificly.However,the promoter sequence and transcriptional regulation mechanism of TCRP1 gene is not clear.This study will base on cloning of TCRP1 promoter and prediction of transcription factor,and carry on related reseach of the transcriptional regulation of TCRP1 to provide new theoretical evidences for its mechanism of drug-resistance in tongue cancer.Methods:(1) Bioinformatics analyzed the TCRP1 promoter region in the 5'end of humans,predicted the potential promoter region and transcription start site of TCRP1 gene.To creat a series of deletion luciferase report constructs which were transfected into tongue cancer cells,and to determine the TCRP1 promoter region by detecting luciferase activity;(2)Bioinformatics analyzed transcription factors and their binding sites of TCRP 1 promoter,and to validate transcription factor c-Myc with chromatin immunoprecipitation(CHIP);(3)The luciferase report construct pGL4+322/+709 were co-transfected into Tca8113 cells with the expression plasmid of c-Myc,or the construct pGL4+322/+709 were transfected into Tca8113/PYM cells that c-Myc were interfered or inhibited,to detect activity changes of TCRP1 promoter using the luciferase activity analysis;(4)To detect the difference of expression of c-Myc in Tca8113 and Tca8113/PYM cells using Western Blot. The expression plasmid of c-Myc were transfected into Tca8113 cells,or c-Myc were interfered or inhibited in Tca8113/PYM cells, then the expression of TCRP1 were detected usingRT-PCR and Western Blot, the sensitivity to cDDP of tongue cancer cells were detected using MTS.Results:(1)A region spanning from positions-939 to+700 bp was predicted as potential promoter region of TCRP1 gene,and it had several transcription start sites.The insults of luciferase report constructs tests determined that the region spanning from positions+322 to+709 bp is the promoter of TCRP1 gene,and the TCRP1 promoter activity in Tca8113/PYM cells was higher than it in Tca8113 cells; (2) Bioinformatics analysis showed that no canonical TATA or CAAT boxes were found,while several GC boxes and putative transcription binding sites for AP2,ADR1,Spl,NF-1,RAF and c-Myc were found in TCRP1 promoter region.The results of CHIP revealed the specific binding of transcription factor c-Myc with its corresponding binding sites in TCRP1 promoter;(3)The rusults of luciferase report constructs tests certified that c-Myc could up-regulate the activity of TCRP1 promoter;(4)The expression of c-Myc in Tca8113/PYM cells was higher than it in Tca8113 cells.The expression of mRNA and protein of c-Myc exogenously expressing cells (Tca8113/c-Myc) were increased, the sensitivity to cDDP of Tca8113/c-Myc cells were increased.The expression of mRNA and protein of c-Myc silence expression cells (Tca8113/pym-i) were decreased, the sensitivity to cDDP of Tca8113 /pym-i cells were decreased.Conclusion:(1) A region spanning from positions -322 to +709 bp is located as promoter region of TCRP1 gene;(2) No TATA or CAAT boxes are found,while several GC boxes and transcription binding sites for AP2,ADR1,Sp1,NF-1,RAF and c-Myc are found in TCRP1 promoter region;(3) The transcription factor c-Myc can bind with TCRP1 promoter specificly,and enhance activity of TCRP 1 promoter and reduce sensitivity to cDDP of tongue cells.