Confirmation of 42-bp deletion within the ERCC1 5' UTR.
Our previous studies revealed a splicing variant (lacking a 42 base pair segment) within the 5'-UTR of the ERCC1 gene, a critical component of the nucleotide excision repair (NER) pathway that plays an important role in the development of chemoresistance in platinum-based anticancer therapy. This 42-bp segment seems to possess a regulatory function in ERCC1 expression and representing the level of clinical response to platinum-treatment in ovarian cancer patients. To confirm the existence of the 42-bp deletion and to investigate the 42-bp function, we performed several experiments and assays. Northern blot analysis and RNase protection assay provide evidence that the 42-bp deletion occurs at RNA level of ERCC1 5'-UTR in both ovarian cancer cell lines and ovarian cancer tissues. Luciferase assay suggests that this gene fragment possesses a regulatory function as an enhancer of ERCC1 gene expression in ovarian cancer cells. In Electrophoretic Mobility Shift Assay (EMSA), a shift band present in the ovarian cancer cell line extracts is consistent with the presence of an intracellular protein that recognizes this specific 42-bp sequence. Further, specific EMSA results with 42-bp probe mutated at the site of RFX-1 indicate different putative-DNA binding proteins, rather than RFX-1. We conclude that the 42-bp sequence within the 5'-UTR influences the expression of ERCC1 and hence can influence response to cisplatin in ovarian cancer therapy.
Li
Tiyuan
T
Mary Babb Randolph Cancer Center, Robert C Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506-9300, USA.
Song
Yuping
Y
Liang
Xiaobing
X
Guo
Ying
Y
Reed
Eddie
E
Yu
Jing Jie
JJ
D020121:5' Untranslated Regions / Q000235:genetics*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D004720:Endonucleases / Q000235:genetics*; D017353:Gene Deletion; D005809:Genes, Regulator / Q000502:physiology; D006801:Humans; D000071701:Regulatory Factor X Transcription Factors; D000071716:Regulatory Factor X1; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; C000605537:RFX1 protein, human; D000071701:Regulatory Factor X Transcription Factors; D000071716:Regulatory Factor X1; D014157:Transcription Factors; D004247:DNA; C071447:ERCC1 protein, human; D004720:Endonucleases