Relationship Between CD44v16 And Chemotherapy Resistance In Breast Cancer And Its Mediating Mechanism

OBJECTIVE:To explore the effect of a new CD44 variant 16(CD44v16)on doxorubicin resistance in breast cancer cells and explore the mechanism involved in influencing doxorubicin resistance in breast cancer cells.METHODS:CD44v16 overexpressed MCF7 cell line MCF7-CD44v16 and CD44v16 silenced MCF7/Adr cell line MCF7/ Adr-sh CD44v16 were constructed by lentivirus infection.CD44v16 antiserum was designed and prepared.The expression levels of CD44v16 protein and m RNA in MCF7,MCF7-CD44v16,MCF7/Adr,MCF7/Adr-sh CD44v16 cells were detected by western blot and real-time fluorescence quantitative PCR.CCK-8 assay was used to detect the semi-inhibitory concentration(IC50)of doxorubicin in MCF7,MCF7-CD44v16,MCF7/Adr,MCF7/ Adr-sh CD44v16 cells and the growth of the four groups of cells treated with doxorubicin at the same concentration(10 μg/m L).Rhodamine retention assay was used to test the ability of rhodamine retention in four groups of cells to express their resistance to chemotherapy.Real-time fluorescence quantitative PCR was used to detect the expression levels of MRP1 and other genes related to drug resistance of breast cancer in four groups of cells.Beclin-1 and LC3 II in four groups of cells were detected by western blot.Four groups of cells were treated with the autophagy inhibitor chloroquine(30nmol/L).Then we set up a control group.CCK-8 and real-time fluorescence quantitative PCR were used again to detect the IC50 value and the expression changes of drug-resistant genes of each cells.RESULTS:The expression levels of CD44v16 protein and m RNA in MCF7,MCF7-CD44v16,MCF7/Adr,MCF7/ Adr-sh CD44v16 cells were significantly different(P<0.05).The expression level of CD44v16 protein and m RNA in MCF7-CD44v16 cells was significantly higher than that in MCF7 cells,while the expression level of MCF7/ Adr-sh CD44v16 cells was lower than that in MCF7/Adr cells.The IC50 values of MCF7,MCF7-CD44v16,MCF7/Adr and MCF7/Adr-sh CD44v16 cells were 5.51±0.52 g/m L,110±9.23 g/m L,256±15.69 g/m L,164±7.69 g/m L.With the presence of doxorubicin,MCF7-CD44v16 cells grew better than MCF7 cells,while MCF7/ Adr-sh CD44v16 cells grew worse than MCF7/Adr cells.The mean rhodamine fluorescence intensity of MCF7 cells was higher than that of MCF7-CD44v16 cells,while that of MCF7/Adr cells was lower than that of MCF7/ Adr-sh CD44v16 cells.The relative expression levels of several drug resistance related genes were different in the four groups.Among them,MRP1,LRP and ERCC1 showed significant changes(P<0.05),while TOPOII,BCRP,Cyclin D1 and AIB-1 showed no significant changes(P>0.05).Specifically,GST-π gene was not significantly differentially expressed in MCF7 and MCF7-CD44v16 cells(P>0.05),but were differentially expressed in MCF7/Adr and MCF7/Adr-sh CD44v16 cells(P<0.05).Beclin-1 and LC3 II were also significantly different in the four different group of cells(P<0.05),the expression level of autophagy protein in MCF7-CD44v16 cell line was higher than that in MCF7 cell line,while the expression level of autophagy protein in MCF7/ Adr-sh CD44v16 cell line was lower than that in the control.After treatment with the autophagy inhibitor chloroquine(30n M),IC50 values of cells in each group were decreased compared with those in the control group without chloroquine(P<0.05).The expression levels of drug-resistance related genes MRP1,LRP and ERCC1 were also significantly reduced(P<0.05).Conclusion:1.The abnormal expression of CD44v16 can affect the chemotherapy resistance of breast cancer cells MCF7 to doxorubicin.The higher the expression level of CD44v16,the stronger the resistance of breast cancer cells to doxorubicin and vice versa.2.Abnormal expression of CD44v16 led to changes in three breast cancer-related drug resistance genes MRP1,LRP and ERCC1.3.CD44v16 can induce autophagy in breast cancer cells.After treatment with chloroquine,an autophagy inhibitor,the drug resistance of cells in each group were weakened compared with the control groups.CD44v16 may enhance the chemotherapy resistance of breast cancer MCF7 cells to doxorubicin throμgh the autophagy pathway.