The Effect Of The Novel Splice Variant ER-α30 On Malignant Biological Behavior In ER-negative Breast Cancer And Its Potential Mechanisms

Objective: By exploreing the effect of ER-α30 on proliferation,migration and invasion and the molecular mechanism of promoting invasion and metastasis in ER-negative breast cancer cells for future provide more potential explanations for the role of ER-negative breast cancer in the diagnosis,treatment and prognosis.Methods: 1.43 cases of ER-negative breast cancer and adjacent tissues were collected and the expression levels of ER-α30 in tissue samples were detected with immunohistochemistry.2.ER-α30 was overexpressed or knockdown in ER-negative breast cancer cells by lentivirus infection and liposome transfection,respectively.Western blot assay proves whether the overexpression and silent construction is successful.3.Overexpression or silencing ER-α30,respectively,then the effect of the ER-α30 on proliferation,migration and invasion in ER-negative breast cancer by CCk-8 assay,transwell migration assay and wound healing assay.4.The effect of ER-α30 on the expression levels of MMP9,VEGF-D,N-cadherin,vimentin and E-cadherin in ER-negative breast cancer cells were detected by Western blot assay.5.The effect of overexpression ER-α30 on transcription of ER-negative breast cancer cells was analyzed by the transcriptional sequencing.6.Six differentially expressed lnc RNA were screened and RT-PCR verified the effect of ER-α30 on the expression level of lnc RNA.7.The relationship between lnc RNA ZEB1-223 and ER-α30 by RIP.Results: 1.IHC results showed that ER-α30 mainly expressed in the nucleus of ER-negative breast cancer,the expression of ER-α30was a difference in ER-negative breast cancer and adjacent tissues(P < 0.01).2.After up-regulation the expression level of ER-α30 in MDA-MB-231 and BT-549 cancer cells,the ability of cell proliferation,migration and invasion increased.On the contrary,ER-α30 was knockdown in ER-negative breast cancer cells,the ability of cell proliferation,invasion and metastasis significantly decreased.3.In MDA-MB-231 and BT-549 breast cancer cells,up-regulation of ER-α30 expression,the expression levels of MMP9,VEGF-D,N-cadherin and vimentin significantly increased,and the expression level of E-cadherin significantly decreased.On the contrary,knockdown of ER-α30expression,the expression levels of MMP9,VEGF-D,N-cadherin and vimentin significantly decreased,and the expression level of E-cadherin significantly increased.4.Differentially expressed genes were obtained by transcriptome sequencing,and Go analysis showed that the differentially expressed genes were mainly enriched in extracellular matrix,cell adhesion and growth factor activity and KEGG pathway analysis showed that the differentially expressed genes were mainly concentrated in ECM-receptor interaction,Hypertrophic cardiomyopathy and Intestinal immune network.5.RT-PCR result showed that up-regulation of ER-α30 expression increased the expression levels of CHCHD6-207 and ENTPD3-AS1-20 and decreased the expression levels of SPIDR-225,RERE-213,ZEB1-223 and EXOC4-219,knockdown of ER-α30expression increased the expression levels of SPIDR-225,RERE-213,ZEB1-223 and EXOC4-219 and decreased the expression levels of CHCHD6-207 and ENTPD3-AS1-20.6.RIP assay result showed that ZEB1-223 was the target of ER-α30 in MDA-MB-231 and BT-549 breast cancer cells.Conclusions: ER-α30 help boost the migration proliferation and invasion of ER-negative breast cancer cells,while the mechanism of promoting the invasion and metastasis of ER-negative breast cancer may be ER-α30 participates in the occurrence of EMT process by regulating the level of ZEB1-223.