Studies On Functions Of New Estrogen Receptor Regulation Factor GA5

Estrogen receptor(ER)is one of the members of the nuclear receptor superfamily,which contains two subtypes ERαand ERβ.ER plays an important role in the development of breast cancer.Recently ER is thought to act as a target of the therapy for breast cancer and one of indexes to evaluate prognosis. Until now,the endocrine therapy of mammary carcinoma is achived mainly by decreasing ER transcriptional activity.Therefore,it is of great significance to discover and characterize the proteins that regulate ER activity in developing new drugs to treat the diseases related to estrogen.It is a complex process for ER to regulat its downstream genes' expression, as multiple factors,such as co-regulators distributing in many different tissues, are involved in the process.These co-regulators(including Co-activators and coinhibitors) can affect transcription activation of ER through binding to it. Relatively,the discovery of ERβis much later than ERα,and the number of its co-regulators even much less than that of ERβ.For further exploring the role of ER in transcription regulation of the genes corresponding to estrogen, also for discovering new co-regulators of ERβ,we found a new candidate gene GA5 from a human gland cDNA library using ERβAF-2 sector as a bait.To further understand the function of GA5,a series of experiments were carried out, and some good results were obtained.(1)prokaryotic expression vector pGEX-KG-GA5 was successfully constructed,and GST-GA5 fusion protein was induced to express in E.coli BL21.Purified GST-GA5 fusion protein was inoculate BLAB/c mouse for three times,and polyclonal anti-GA5 antibody was preparied.Western blotting analysis showed that the antibody was specific to GA5 protein.(2)GA5 expression in several different breast cancer cell lines was determined using the above polyclonal anti-GA5 antibody.The result showed that GA5 expression in those breast cancer cell lines was different and that GA5 protein may be modified by other molecules.(3)The expression vectors of GA5 siRNAs were constructed and confirmed by DNA sequencing.Western blotting analysis showed that GA5 siRNA could effectively inhibit the expression of GA5 gene.It is helpful for further study the functions of GA5 in tumor.(4)The Yeast double-hybrid experiment had proven that GA5 and ER interact each other in vitro and in vivo,therefore,it is expected that GA5 is involved in regulation of the estrogen signaling pathway through the interaction. The eukaryotic expression vector pIRESpuro2-Flag-ERαand pIRESpuro2-Flag-ERβwas successfully constructed,westen blotting assay illustrated that Flag-ERαand Flag-ERβfusion protein were expressed in 293T cells.The ranscription activities of pIRESpuro2-Flag-ERαand pIRESpuro2-Flag-ERβwere determined by luciferase ERE-luc.The results showed that,in the presence of strogen,the transcription activities of pIRESpuro2-Flag-ERαand pIRESpuro2-Flag-ERβincreased by 7%and 6% respectively.