The Study Of C-Src On Epithelial-mesenchymal Transition In Breast Cancer MDA-MB-231 Cells Induced By EGF

Objective: Distant metastasis is one of the leading causes of death in patients with breast cancer,many of studies have shown that the expression level of EGFR in cancerous tissue.And EGFR is closely related in that the metastatic potential and the progress degree in tumor.Previous research shows that Epithelial-mesenchymal transition plays an important role in tumorigenesis,malignant transformation and chemotherapy of tumor resistance.In recent years,literatures report c-Src of Src kinase family members is most significant expressed in metastatic breast cancer tissue samples.This experiment was to study the expression of epidermal growth factor receptor(EGFR)in breast cancer is related to its transfer potential,whether EGF causes breast cancer cell metastasis through Epithelial-mesenchymal transition,whether the c-Src is related to the Epithelial-mesenchymal transition and migration of breast cancer cells induced by EGF.Method:(1)Screening for highly EGFR expressed breast cancer cell lines: the expression EGFL-7 and EGFR of mammary epithelial cells(MCF-10A),Luminal A breast cancer cells(MCF-7),triple negative breast cancer(MDA-MB-231)were test by RT-qPCR and Western blot.High expression EGFR cell lines were used for subsequent experiments.(2)The study of EMT and migration ability on high expression of EGFR cell lines under the influence of EGF.After giving EGF and EGF inhibitor of lidocaine,RT-qPCR and Western blot test EMT marker proteins(ZO-1,MMP-9),wound healing detected the migration ability of change in breast cancer cells.(3)to identify the role of c-Src in EMT and migration process,dividing breast cancer cells into controls,c-Src inhibitor PP2,PBS,EGF,EGF+PP2,EGF+PBS six groups.Western blot tested EMT marker proteins and wound healing detected the migration ability.Results:(1)The expression of EGFR was found in all of MCF-10 A,MCF-7 and MDA-MB-231 cells.Breast cancer MDA-MB-231 cells as TNBC with high metastatic potential,and the level of EGFR mRNA and protein is higher than MCF-10 A and MCF-7.(2)EGFR can lead to EMT in breast cancer cells after activation,After intervention EGF with 48 h,the morphology of MDA-MB-231 will obviously change.Intercellular separation signifying loss of intercellular adhesion,and increased formation of pseudopodia observed emanating from the cell membrane.Added 500 μM lidocaine to block the EGFR activation,the change morphology of cell line will disappeared which will similarity to the control group.RT-qPCR and western blot to detected the level expression of ZO-1 and MMP-9,it were expression high if the EGFR was activated.The wound healing results showed that EGF induced cell migration was significantly higher than the control group and lidocaine intervention group.(3)Before EGF induce the MDA-MB-231 cell line EGFR activated to treat cell line with c-Src inhibitor PP2,the MDA-MB-231 will still maintain the morphology of epithelial cells.Western blot was used to compare to expression level of ZO-1 and MMP-9,MDA-MB-231 cell line treated with PP2 will induce ZO-1 higher than untreated PP2 group,while the mesenchymal cell marker MMP-9 expression level is higher in untreated PP2 group contrasted with treated PP2 group.The experimental results of wound healing show that PP2 intervention group the group without PP2 cells migration ability decline.Conclusion: Normal breast epithelial cells and breast cancer cells express of EGFR,one of triple negative breast cancer MDA-MB-231 express the highest,implying EGFR may become one of the markers to predict potential of breast cancer metastasis.Epidermal growth factor and the corresponding EGFR can induce EMT in MDA-MB-231 breast cancer,which can promote the invasion and metastasis of breast cancer cells.Noreceptor tyrosine kinase c-Src plays an important role in MDA-MB-231 breast cancer in EMT and migration induced by EGF,the c-Src protein may be a potential therapeutic targets for breast cancer.