| BackgroundBreast cancer is the most common malignant tumor in women,and the incidence rate is increasing year by year.Recurrence and metastasis is the main reason for the poor curative effect of Breast Cancer.More than 90% of epithelial malignant tumors developed epithelial-mesenchymal transitions(EMT),such as lung cancer,breast cancer,colon cancer and liver cancer.The study of EMT provides a new way to reveal the mechanism of tumor cell invasion and metastasis and to search for the target of anti-tumor therapy.Early Response Factor 4(EGR-4)is a member of EGR protein family and encodes zinc finger protein transcription factor.Egr-4 is a necessary factor to promote the cell cycle from rest to cell cycle.It is involved in the regulation of cell differentiation and is related to the occurrence and development of some tumorous diseases.Some studies show that EGR protein family is closely related to EMT and Tumor Development! However,the role of EGR4 in breast cancer,there is still a lack of systematic and comprehensive research!ObjectiveInvestigate the expression of EGR-4 in breast cancer and its relationship with clinical parameters and prognosis.To prove EGR-4 could influence the proliferation,migration and invasion of breast cancer cells and induce EMT.To elucidate the specific molecular mechanism of EMT induced by EGR-4 in breast cancer cells.MethodsThis study was divided into three partsThe first part: Selected 100 female breast cancer patients from January 2014 to December 2014 who were diagnosed pathologically in the Department of Breast Cancer,Xuzhou Cancer Hospital,Jiangsu Province,the cancerous tissue samples were collected and matched with normal adjacent tissue.At the same time,human breast cancer cells BT474,SKBr-3,ZR-75-30,zr-7,MDA-MB-453,MDA-MB-231 and normal breast epithelial cells were cultured in vitro.The expression level of EGR-4 was detected by Reverse transcription-Polymerase Chain Reaction(RT-PCR).Theexpression of EGR-4 was detected by Western-blotting and Immunohistochemisty(IHC).The clinical data included tumor size,lymph node metastasis,Her-2 status,histological grade,and recurrence and metastasis.Correlation between expression of EGR-4 and pathological features and prognosis in patients with multiple logistic regression analysis.In the second part,breast cancer cell MCF-7 and SKBr-3 were cultured in vitro.overexpression and underexpression of EGR-4 in breast cancer cell were constructed by Plasmid and small interfering RNA.The expression of EGR-4was detected by RT-PCR and WB,and the activity and proliferative ability of breast cancer cells were detected by CCK-8 and plate clone.Detection of migration ability of breast cancer cells after the change of EGR-4 expression by scratch test.The change of invasive ability of breast cancer cells after changing the expression of EGR-4 by Transwell Chamber.The expression of E-cadherin,Vimentin and snail in breast cancer cells after the change of EGR-4 expression was detected by immunofluorescence and WB.In the third part,CO-IP and liquid chromatography-mass spectrometry(LC-MS)were used to search for specific binding proteins of EGR-4.The changes of the target protein and the key protein during EMT were detected by WB and RT-PCR.ResultsThe expression of EGR-4 was up-regulated in breast cancer tissues and Cells(p<0.05),and the expression of EGR-4 was positively correlated with the histological grade of breast cancer.The expression of EGR-4 was correlated with lymph node metastasis,Her-2status and recurrent metastasis,and the expression level of EGR-4 was higher in patients with local recurrence and distant metastasis than in early stage.The survival rate of patients with low expression of EGR-4 was higher than that of patients with high expression.Overexpression of EGR-4 promoted the proliferation,migration and invasion of breast cancer cells.The results of CCK-8showed that the cell viability was significantly increased in breast cancer cells overexpression EGR-4;The results of plate cloning showed that the cell colonies were significantly increased in breast cancer cells overexpression EGR-4;The results of wound healing showed that the scratch healing was significantly faster in breast cancer cells overexpression EGR-4;The results of transwell chamber showed that the invasion ability was significantly increased in breast cancer cells overexpression EGR-4.It suggested that EGR-4 promoted the proliferation,migration and invasion of breast cancer cells.Low-expression of EGR-4 supressed the proliferation,migration and invasion of breast cancer cells.The results of CCK-8 showed that the cell viability was significantly decreased in breast cancer cells low-expression EGR-4;The results of plate cloning showed that the cell colonies were significantly decreased in breast cancer cells low-expression EGR-4;The results of wound healing showed that the scratch healing was significantly slower in breast cancer cells low-expression EGR-4;The results of transwell chamber showed that the invasion ability was significantly decreased in breast cancer cells low-expression EGR-4.It suggested that low-expression of EGR-4 supressed the proliferation,migration and invasion of breast cancer cells.The results of cellular immune fluorescence experiment showed that the expression of E-cadherin was significantly decreased but the expression of Vimentin was significantly increased in breast cancer cells overexpressed EGR-4.On the contrary,the expression of E-cadherin was significantly increased but the expression of Vimentin protein was significantly decreased in breast cancer cells low-expressed EGR-4.The results of WB showed that the expression of E-cadherin was significantly decreased but the expression of N-cadherin and Snail proteins were significantly increased in breast cancer cells over-expressed EGR-4.On the contrary,the expression of E-cadherin was significantly increased but the expression of N-cadherin and Snail proteins were significantly decreased in breast cancer cells low-expressed EGR-4.EMT was inhibitored by EGCG,when changing the expression of EGR-4,the proliferation,migration and invasion showed no significant change.Co-IP and mass spectrometry analysis revealed that EGR-4 interacted with TGF-β.Reverse Co-IP experiments confirmed that TGF-β1 can specifically bind EGR-4.WB and PCR results showed that overexpression of EGR-4,TGF-β1 protein and m RNA level expression did not change significantly;ELISA results showed that overexpression of EGR-4,TGF-β1 secretion increased.WB and PCR results showed that there was no significant change in the expression of TGF-β1 protein and m RNA levels when EGR-4 expression was interfered;ELISA showed that TGF-β1 secretion was reduced when EGR-4 expression was interfered.WB results showed that overexpression of EGR-4 increased Smad protein expression,decreased E-cad protein expression,and increased N-cad and Snail protein expression in breast cancer cells,suggesting that EGR-4 induces EMT through the TGF-β/Smad pathway.ConclusionThe expression of EGR-4 is associated with the progression of breast cancer and can predict the prognosis.The higher expression level of EGR-4 indicate the worse prognosis.Overexpression of EGR-4 promotes proliferation,migration,invasion and EMT of breast cancer cells.Lowexpression of EGR-4 Inhibits proliferation,migration,invasion and EMT of breast cancer cells EGR-4 promotes proliferation,migration and invasion of breast cancer cells by inducing EMT.EGR-4 induces EMT through interaction with TGF-β1 and promotes proliferation,migration and invasion of breast cancer cells. |
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