The Role And Molecular Mechanism Of SETDB1-dependent CRM1 Nucleoplasmic Shuttle In Inducin Epithelial-mesenchymal Transformation Of Breast Cancer

Objective: To study the protein expression level of SETDB1 in breast cancer and its relationship with the clinicopathological characteristics of breast cancer patients,and to further explore the mechanism of promoting the invasion and migration of breast cancer by SETDB1,so as to provide a new idea for clinical targeted therapy and improving the prognosis of patients.Methods: 1.Wax pieces of breast cancer tissue and normal breast tissue were collected to make tissue microarray,and the expression level of nuclear plasma of SETDB1 was detected.The relationship between the expression level of nuclear plasma of SETDB1 and the prognosis of patients was statistically analyzed by scoring,and the expression levels of SETDB1,ER,PR and HER-2 were detected by immunohistochemistry.Chi-square test was used to analyze the relationship between SETDB1 and clinicopathological characteristics of breast cancer patients.Kaplan-Meier survival curve and log-rank were used to analyze the relationship between SETDB1 and patients’ survival.2.Nuclear plasma distribution of SETDB1 in breast cancer cell lines was detected by immunofluorescence,and the relationship between nuclear plasma expression level of SETDB1 and invasion and migration ability of breast cancer cells was analyzed.SETDB1 vectors with lentivirus interference and lentivirus overexpression were constructed respectively,and the expression level and distribution of SETDB1 in nuclear plasma were detected by immunofluorescence.MCF7/SETDB1 overexpressed cell lines were treated with CRM1 inhibitor LMB,and the expression level and distribution of SETDB1 in the nuclear plasma were detected by immunofluorescence,and the relationship between SETDB1 and invasion and migration ability of breast cancer cells was detected by Transwell assay.Western blot and RT-PCR were used to verify the overexpression of lentivirus and the expression level of SETDB1 after LMB treatment.Wnt ligand Wnt3 A was used in BT549 cell lines to detect the expression level and distribution of SETDB1 in the nucleoplasma by immunofluorescence.Results: 1.Immunohistochemical results showed that compared with normal breast tissue and adenopathy tissue,the expression of SETDB1 was significantly higher in breast cancer,and its plasma expression was significantly positively correlated with lymph node metastasis and negatively correlated with ER expression.2.Immunofluorescence and Western blot analysis showed that SETDB1 was mainly expressed in the plasma of highly invasive breast cancer cell lines BT549 and MDA-MB-231.After Lentivirus interference with SETDB1,the plasma expression of SETDB1 decreased,while exogenous overexpression of SETDB1 showed significant increase in the plasma expression level.After CRM1 inhibitor LMB treatment,the signal mainly accumulates in the nucleus.Western blot and RT-PCR confirmed that the expression of SETDB1 was significantly increased after lentivirus overexpression,while the expression of SETDB1 in plasma was significantly decreased after LMB treatment.After treatment with Wnt ligand Wnt3 A,its expression in plasma was obviously enhanced.Conclusions: 1.The expression level of SETDB1 in cell nucleus and cytoplasm of breast cancer patients is significantly positively correlated with lymph node metastasis,negatively correlated with ER expression,and closely correlated with molecular typing of breast cancer;2.Activation of Wnt/β-catenin signaling pathway by Wnt3 A can promote the nucleation and plasma entry of SETDB1-dependent CRM1 protein,which may induce breast cancer EMT,and ultimately promote the proliferation,migration and invasion of breast cancer.