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Abstract: Non-metastatic cells 5 (NME5), a recently found gene belonging to the NDPK-like molecules gene family, is highly expressed in testis and some types of human cancer. Current studies have revealed diverse potential functions of NME5 and we have reported that NME5 is associated with innate resistance to gemcitabine in human pancreatic cancer cells in previous study. However, the mechanism underlying the transcriptional regulation of NME5 has not been elucidated yet. In this study, we analyzed the 5′-flanking region of the human NME5 gene and revealed its transcription start site (TSS) at −35bp relative to its translation start codon ATG. Using 5′ unidirectional deletion analysis, we demonstrated that the proximal promoter of NME5 is located within −1051bp to +35bp. Two functional GC-boxes (−300bp and −323bp) were identified within the promoter region. Mutation of either GC-box led to significant reduction in NME5 promoter activity, whereas overexpression of Sp1 activated NME5 promoter activity in MIA PaCa-2 and 293T cells. In silico analysis predicted that transcription factor Sp1 binds to both GC-boxes, which were confirmed by EMSA and ChIP. In addition, we found that compared with MIA PaCa-2, Sp1 was highly expressed in PAXC002, a well characterized human pancreatic cancer cell line with innate gemcitabine resistance where NME5 was reported to be highly expressed, indicating that Sp1 induces NEM5 expression in PAXC002 cells. In conclusion, our study characterized for the first time the human NME5 promoter which is controlled by Sp1 transcription factor in pancreatic cancer. [Copyright &y& Elsevier]