Establishment Of Stable Overexpression Of HER2 Gene Breast Cancer EMT6 Cell Line And The Effect Of HER2 On The Biological Function Of Breast Cancer

BackgroundBreast cancer is one of the three most common cancers worldwide.Human epidermal growth factor receptor 2(HER2),a member of the tyrosine kinase receptor(HER1-4)family,is overexpressed in 25% to 30% of breast cancers,which have a poor prognosis.The development of HER2 drugs has improved the treatment effect of breast cancer to some extent.However,HER2-positive breast cancer is still an incurable disease due to its high metastatic and aggressive nature,and new treatment regimens are urgently needed.For HER2-positive breast cancer,a better understanding of the mechanisms of resistance to existing HER2-targeted drugs,the tumor microenvironment,and the role of the associated signaling pathways are at the heart of clinical trials to explore new drugs and protocols.Therefore,it is particularly important to construct breast cancer cell lines with stable overexpression of HER2 gene for drug screening and mechanism study.ObjectiveThe main purpose of this study is to build stable expression of HER2 gene in breast cancer cell lines and explore the effects of HER2 on the proliferation,migration,invasion and other biological behaviors of breast cancer cells,so as to provide a more rigorous and reasonable cell model for targeted HER2 therapy.MethodsThe plasmid carrying HER2 gene was integrated into breast cancer cells(Experimental Mammary Tumour-6,EMT6)by liposome transfection technique.HER2 stable EMT6 monoclonal cell lines were obtained by G418 screening.The differences of HER2 expression in selected cell lines were detected by q RT-PCR and Western blot.The experimental cell lines were divided into three groups,namely EMT6 group,HER2 low expression group and HER2 high expression group.The expression of HER2 in the stable cell lines was detected by immunofluorescence method.CCK-8 assay was used to determine the proliferation ability of cell lines with low expression of EMT6 and HER2 and high expression of HER2.The migration ability of cell lines with low expression of EMT6 and HER2 and high expression of HER2 were determined by scratch test.Transwell assay was used to determine the invasion ability of cell lines with low expression of EMT6 and HER2 and high expression of HER2.The expression of m TOR and AKT in cells with low expression of EMT6 and HER2 and high expression of HER2 were determined by Western blot.Results1.The expression of HER2 in the five EMT6-HER2 stable cell lines detected by q RTPCR showed high and low differences.Using EMT6-H1 as control group,the expression of EMT6-H3 was the highest(P< 0.001).EMT6-H4 and H5 were higher than EMT6-H1,P<0.01;The expression of EMT6-H2 was lower than that of EMT6-H1,P < 0.001.2.The expression of HER2 protein in cells was consistent with that of HER2 m RNA by Western blot.Taking EMT6 as reference,the expression of HER2 in EMT6-H2 cell line was higher than that in EMT6(P< 0.05),and the expression of HER2 in EMT6-H1 cell line was higher than that in EMT6(P< 0.01).The expression of HER2 in EMT6-H3,EMT6-H4 and EMT6-H5 cell lines was significantly higher than that in EMT6(P< 0.001).Based on the results of q RT-PCR and Western blot,the stable HER2 cell line EMT6-H1 was in the low expression group,and EMT6-H3 was in the high expression group.3.The expression of HER2 in stable cell lines by immunofluorescence staining.Fluorescence of HER2 protein expression can be seen in both low-expression and highexpression cells,and it is mainly distributed on the cell membrane.In stable HER2 cell lines,both the low expression group and the high expression group were higher than the EMT6group(P< 0.001).4.CCK8 assay was used to detect the proliferation ability of cell lines with low expression of EMT6 and HER2 and high expression of HER2.The light absorption values of HER2 low expression and HER2 high expression cells were higher than those of EMT6cells(P< 0.001).The light absorption value of the cell lines with high HER2 expression was higher than that of the cell lines with low HER2 expression(P< 0.001).5.The migration ability of cell lines with low expression of EMT6 and HER2 and high expression of HER2 were detected by scratch test.The migration rates of both HER2 low expression and HER2 high expression cell lines were higher than those of EMT6 cell lines(P< 0.001),and the migration rates of HER2 high expression cell lines were higher than those of HER2 low expression cell lines(P< 0.01).6.Transwell assay was used to detect the invasion ability of cell lines with low expression of EMT6 and HER2 and high expression of HER2.The number of cells passing through the stromal gel was higher in both HER2 low expression and HER2 high expression lines than in EMT6 cell lines(P< 0.001),and the number of cells passing through the stromal gel was higher in HER2 high expression lines than in HER2 low expression lines(P< 0.001).7.The expression of m TOR and AKT in the low expression of EMT6 and HER2 and the high expression of HER2 cells were detected by Western blot.The results showed that the expression of m TOR and AKT in both HER2 low expression and HER2 high expression cell lines were higher than that in EMT6(P < 0.001).The expression of m TOR and AKT in EMT6 cells transfected with HER2 were up-regulated.Conclusions1.There was a difference between high and low expression of HER2 m RNA in stable EMT6 strain,and HER2 receptor existed on the cell membrane.2.The proliferation,migration and invasion ability of EMT6 cells are related to the level of HER2 expression.High expression of HER2 can promote the proliferation,migration and invasion of EMT6 cells.3.High expression of HER2 can promote the up-regulation of m TOR and AKT expression in EMT6 cells,and activate PI3K-m TOR-Akt signaling pathway.