Eya1 Promotes Proliferation Through Up-regulation Of Cyclin D1 In Breast Cancer Cells

Objective To evaluate the role of Eyal in the breast cancer cell proliferation. Methods We used the lentivirus system to establish the stably expressed Eya1, Eya1 phosphatase mutant Eya1D327A and vector control in MDA-MB-453, SK-BR3 and BT474 breast cancer cell lines. Also, we knocked down the endogenous Eyal by lentivirus shRNA (small hairpin RNA) system in the MDA-MB-231 breast cancer cell line. We evaluated the role of Eya1 in the breast cancer cell proliferation through MTT assay, cell growth curve, 3H-TdR incorporation and contact dependent/independent colony formation assay. Results We got the stable cell lines over-expressed Eya1 in MDA-MB-453, SK-BR3 and BT474 breast cancer cell lines, as well as the Eya1 knockdown breast cancer cell line MDA-MB-231. The MTT assay, cell growth curve and 3H-TdR incorporation showed that those cells which were forced over-expressed Eya1 showed an advantage on cell proliferation compared to vector control, while knockdown of the endogenous Eya1 in MDA-MB-231 breast cancer cell line attenuated cell proliferation. Also, we got the same pattern in the contact dependent/independent colony formation assay. Forced over-expression of Eya1 increased the number of colony, that is, over-expression of Eya1 showed a 1.5-3 fold increase compared to control, while knockdown of Eya1 showed a 50%decreased number of colony formation. Conclusion Eya1 promotes breast cancer cell proliferation. Objective To elucidate the related mechanisms by which Eya1 promotes breast cancer cell proliferation. Methods We performed gene expression microarrays analysis and got the gene expression profile after Eya1 overexpressed in MDA-MB-453 cells. We confirmed the microarray data by quantitative PCR and western blot assay in MDA-MB-453 and SK-BR3 cells. We chose the potential target genes and confirmed it by promoter luciferase assay and chromatin immunoprecipitation (ChIP) assay. Also, we did some further functional studies such as MTT, cell growth curve and contact dependent or independent colony formation assay. Results We found that Cyclin D1 was upregulated in MDA-MB-453 cells overexpressed Eya1 by microarrays analysis and then confirmed it by quantitative PCR and western blot assay. Knockdown the endogenous Eya1 in MDA-MB-231 cells caused reduced expression of Cyclin D1. Next, we showed that Eya1 increased the activity of Cyclin D1 promoter. There was a about 2-4 fold increase of the Cyclin D1 promoter activity in the Eya1 group compared to vector control, while Eya1 phosphatase mutant Eya1D327A group didn't show any advantage on the Cyclin D1 promoter activity compared to vector control. Chromatin immunoprecipitation (ChIP) assay showed that Eya1 binds to Cyclin D1 promoter. Also, we found that knockdown of endogenous Cyclin D1 attenated the effect of Eya1 on the proliferation in MDA-MB-453 and SK-BR3. We also found Eya1 interacted with Six1 and that was involed in Eya1 signaling pathway. Conclusion Eya1 promotes breast cancer cell proliferation in a Cyclin D1 dependent manner.