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

The axon initial segment (AIS) is a specialized structure that controls neuronal excitability via action potential (AP) generation. Currently, AIS plasticity with regard to changes in length and location in response to neural activity has been extensively investigated, but how AIS diameter is regulated remains elusive. Here we report that COUP-TFI (chicken ovalbumin upstream promotor-transcription factor 1) is an essential regulator of AIS diameter in both developing and adult mouse neocortex. Either embryonic or adult ablation of COUP-TFI results in reduced AIS diameter and impaired AP generation. Although COUP-TFI ablations in sparse single neurons and in populations of neurons have similar impacts on AIS diameter and AP generation, they strengthen and weaken, respectively, the receiving spontaneous network in mutant neurons. In contrast, overexpression of COUP-TFI in sparse single neurons increases the AIS diameter and facilitates AP generation, but decreases the receiving spontaneous network. Our findings demonstrate that COUP-TFI is indispensable for both the expansion and maintenance of AIS diameter and that AIS diameter fine-tunes action potential generation and synaptic inputs in mammalian cortical neurons.
Action Potentials ; Animals ; Axon Initial Segment ; COUP Transcription Factor I ; DNA-Binding Proteins/physiology* ; Mammals ; Mice ; Transcription Factors

OBJECTIVETo explore the mechanism of inhibitory effect of SLW on estrogen production by endometrial cells of endometriosis.

METHODAfter the model of eutopic primary cultured endometrial cells of endometiosis and hysteromyoma in vitro was successfully established, the changes of steroidgenic factor-1 (SF-1), chicken ovalbumin upstream-transcription factor (COUP-TF), 17-beta-hydroxysteroid dehydrogenase 1 (17-beta-HSD1) and 17-beta-hydroxysteroid dehydrogenase 2 (17-beta-HSD2) mRNA were detected by RT-PCR before and after treatment of medicated serum of SLW. The changes of SF-1 and COUP-TF protein were also observed by western blot synchronously according to the same treatment method mentioned-above. Meanwhile ,the data of hysteromyoma group was obtained from the above experiments.

RESULTThe expression of SF-1 mRNA and protein, 17-beta-HSD1 mRNA was weak, but COUP-TF mRNA and protein, 17-beta-HSD2 mRNA was remarkable in Hysteromyoma endometrium, as compared with those of endometiosis ,which was taken as control group (P<0.01). After the 48 hours' treatment of medicated serum of 5.0, 2.5 g kg(-1) d(-1) of SLW , the expression of COUP-TF mRNA and protein, 17beta-HSD2 mRNA was found significantly increased, but SF-1 mRNA and protein, 17-beta-HSD 1 mRNA was decreased in contrast to the control group (P <0.01 or P <0.05). Although the expresson of COUP-TF mRNA and protein was increased, SF-1 protein and 17-beta-HSD1 mRNA was decreased in 1.25 g kg(-1) d(-1) medicated serum group ,compared with those of the control group (P <0.01), the low dose group had no apparent inhibitory effect on the expression of SF-1, 17-beta-HSD2 mRNA.

CONCLUSIONThe medicated serum of SLW could inhibit the secretion of estradiol in eutopic endometrial cells of endometiosis, and its mechanism might be associated with combined action of inhibiting expression of SF-1, 17-beta-HSD1 and up-regulating expression of COUP-TF, 17-beta-HSD2.

17-Hydroxysteroid Dehydrogenases ; genetics ; Adult ; Animals ; COUP Transcription Factors ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Endometriosis ; blood ; metabolism ; pathology ; Endometrium ; drug effects ; metabolism ; pathology ; Estradiol Dehydrogenases ; Estrogens ; biosynthesis ; Female ; Gene Expression Regulation ; drug effects ; Humans ; In Vitro Techniques ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Rats ; Serum ; chemistry ; Steroidogenic Factor 1 ; 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

OBJECTIVE: To explore the genetic basis for a child with optic atrophy and global developmental delay. METHODS: A child who had presented at the Guangzhou Women and Children's Medical Center in January 2022 was selected as the study subject. Clinical data were collected. Whole exome sequencing (WES) was carried out for the child. Candidate variant was validated by Sanger sequencing and bioinformatic analysis. RESULTS: The child, a nine-month-old female, had manifested dysopia and global developmental delay. Genetic testing revealed that she has harbored a de novo c.425G>C (p.Arg142Pro) variant of the NR2F1 gene, which has been associated with Bosch-Boonstra-Schaaf syndrome. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic (PS2+PM1+PM2_Supporting+PM5+PP3+PP4). CONCLUSION The c.425G>C (p.Arg142Pro) variant of the NR2F1 gene probably underlay the pathogenesis in this child. Above finding has enriched the genotypic and phenotypic spectrum of the NR2F1 gene.
Female ; Humans ; Infant ; Computational Biology ; COUP Transcription Factor I/genetics* ; Genetic Testing ; Genomics ; Genotype ; Optic Atrophy/genetics*