OBJECTIVETo clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity.

METHODSThe full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint. The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA.

RESULTSCOUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter. Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity.

CONCLUSIONThe human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.

COUP Transcription Factor II ; COUP Transcription Factors ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA-Binding Proteins ; genetics ; pharmacology ; E-Box Elements ; genetics ; HeLa Cells ; Humans ; Promoter Regions, Genetic ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Steroid ; genetics ; Telomerase ; biosynthesis ; genetics ; metabolism ; Transcription Factors ; genetics ; pharmacology ; Transcription, Genetic ; Yeasts ; genetics

Increasing attention is focused on the down-regulation of miRNAs in cancer process. Nuclear receptor subfamily 2 (NR2F2, also known as COUP-TFII) is involved in the development of many types of cancers, but its role in gastric cancer remains elusive. In this experiment, oncomine and Kaplan-meier database revealed that NR2F2 was up-regulated in gastric cancer and that the high NR2F2 expression contributed to poor survival. MicroRNA-27b was targeted and down-regulated by NR2F2 in human gastric cancer tissues and cells. The ectopic expression of miR-27b inhibited gastric cancer cell proliferation and tumor growth in vitro and in vivo. Assays suggested that the overexpression of miR-27b could promote MGC-803 cells' migration and invasion and retard their metastasis to the liver. In addition, down-regulation of miR-27b enhanced GES-1 cells' proliferation and metastasis in vitro. These findings reveal that miR-27b is a tumor suppressor in gastric cancer and a biomarker for improving patients' survival.
Animals ; Biomarkers, Tumor ; genetics ; metabolism ; COUP Transcription Factor II ; genetics ; metabolism ; Cell Line, Tumor ; Female ; Genes, Tumor Suppressor ; Heterografts ; Humans ; Male ; Mice, Nude ; MicroRNAs ; genetics ; metabolism ; Neoplasm Metastasis ; Neoplasm Proteins ; genetics ; metabolism ; Neoplasm Transplantation ; RNA, Neoplasm ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology