Research Of Function Of A Novel Co-regulator Of Estrogen Receptor(ER)

Estrogen receptor is a member of a large superfamily of nuclear receptors, including ERαand ERβ, which is a transcriptional factor regulating transcription of genes related to estrogen by combinition with specific responsive elements of target genes. The action of ER requires coregulators(such as AP1 and SP1). Currently, many coregulators of ERαhave been identified, including coactivators and corepressors. Since ERβis discovered more recently, very few coregulators for ERβhave been reported. ER plays a crucial role in the occurrence and development of breast cancer, which could be key target molecule for treatment and prognostic indicator of breast cancer. Taken together, the research of coregulators will facilitate the elucidation of the mechanism of ER, and will contribute to the invention of related target drugs of breast cancer.GA9 was screened with ERβfrom cDNA library of human breast cancer by using a yeast two-hybrid system, which have not been reported(we named it GA9). Through GST pull-down and immunoprecipitation assays, we obtained the same result to that of yeast two-hybrid system, which validated the specific interation of GA9 and ER. To further explore the biological function of GA9, many approaches of molecular biology were employed. Through the luciferase assays, the breast cancer cells were transfected with the reporter plasmid containing the responsive element of ER. The inhibition of GA9 on the ER-mediated transcriptional activity both in vitro and in mammaliam cells was demonstrated, whereas the GA9 point mutant repressed the effect of suppression on ER-mediated transcriptional activity. To further investigate the function of GA9, we purified protein of GA9 in inclusion body pattern. And then, we immuned Balb/c mice to produce polyclonal antibody using the purified inclusion body. Though Western blot assay, the produced antibody could specifically and effectively identify GA9. Then, the expression level of GA9 in a variety of breast cancer cell lines can be detected using the produced antibody of GA9. We also construct small interfering RNA of GA9. Through luciferase assays, we found that siRNA of GA9 evidently enhanced the ER-mediated transcriptional activity. Similarly, the result also was confirmed by Western blot with anti-GA9 antibody that we have produced. Through crystal violet assay, GA9 could inhibit the breast cancer cell growth, while knockdown of endogenous GA9 could enhance the breast cancer cell growth. According to the analysis of the homology, GA9 possibly is the methylase of DNA or mRNA of ER. Then, luciferase assays were performed with treatment with the AZA of different concentration gradients, GA9 increased the ER-medicated transcriptional activity and it is dose-dependent.Taken together, GA9 probably is a novel coregulator in estrogen signaling pathway. Further exploration of GA9 will contribute to reveal the regulation mechanism of GA9 and the therapy of breast cancer. It will be a brand new target for the treatment of GA9.