LIM Protein Ajuba Activates ERα Mediated Transcriptional Regulation Of Breast Cancer Cells

Objective:To explore the role of LIM protein Ajuba in the transcription regulation of estrogen receptor α(ERα)signaling pathway.Methods: The interaction between Ajuba and ERα was demonstrated by endogenous as well as exogenous immunoprecipitation techiniques and GST-pull down assay.RNA high throughput sequencing analysis was performed in T47 D cell lines with stable knockdown/overexpression of Ajuba so as to find out ERα downstream target genes regulated by both estrogen and Ajuba.The finding was validated by RT-qPCR.Luciferase reporter assay was used to examine the activation of Ajuba on the promotor of TFF1,one downstream target gene of ERα,and find out the exact Ajuba binding site.Cell counting Kit 8 assay(CCK8)was performed to identify the combined effect of Ajuba and estrogen on cell growth activity in T47 D cells with stable overexpression/knockdown Ajuba and MCF7 cells with stable overexpression of Ajuba,respectively.The sensitivity to tamoxifen was also determined in the above T47 D and MCF7 cell lines.Results: Direct combination of Ajuba with ERα,independently of estrogen,can be found,but this combination can be further enhanced by the influence of estrogen.A series of ERα target genes regulated by Ajuba were screened out by RNA-seq,and the findings were reliably validated by the RT-qPCR technique.The complex Ajuba/ERα activated the transcriptional activity of ERαdownstream target gene TFF1 by binding the estrogen responsible element(ERE)on its promotor.Ajuba and ERα have a synergistic promoting effect on the growth of ER positive breast cancer cells.Ajuba declined the sensitivity to tamoxifen in ER positive breast cancer cell lines.Conclusion: Ajuba,an auxiliary activating protein,can positively regulate the signaling pathway mediated by ERα.