PRRX1 Overexpression Promotes Multidrug Resistance In MCF-7 Breast Cancer Cells

Objective:To investigate the contribution of PRRX1 to MDR and the underlying molecular mechanism in MCF-7 breast cancer cells.Methods:1.Overexpress PRRX1 in MCF-7 cells through transfection.MCF-7 cells with overexpressed PRRX1 were marked as PRRX1+group.MCF-7 cells transfected with blank vector were marked as Negative Control group(i.e.,the N.C group)while MCF-7 cells treated with culture medium were marked as Blank Control group(i.e.,the B.C group).2.The morphological changes and transfection efficiency were observed by inverted fluorescence microscopy.The relative mRNA and protein expression levels of PRRX1 were evaluated by simple western blotting and Rt-qPCR.3.The relative mRNA and protein expression levels of P-gp(a typical MDR related protein),Vimentin(a typical EMT-related protein),N-cadherin(another typical EMT-related protein)were evaluated by simple western blotting and Rt-qPCR,respectively.4.In addition,CCK-8 assay was performed to detect the response of the three groups to docetaxel and cis-platinum treatment.5.Finally,Rt-qPCR assays and simple western blotting were used to detect the mRNA and protein levels of PTEN and the phosphorylation levels of PI3K and AKT.Results:Cells transfected with PRRX1 exhibited green fluorescence,indicating that the transfection efficiency was up to 80%.The mRNA(F=12.91,P<0.001)and protein(F=16.23,P<0.01)levels of PRRX1 were significantly higher in the PRRX1+group than in the control groups.Cells in the N.C and B.C groups exhibited a "paving stone"shape and were tightly clustered,whereas cells in the PRRX1+group showed a "spindle"morphology with loose connections among cells.The relative mRNA(F=10.03,P<0.01)and protein(F=7.081,P<0.05)expressions of Vimentin were significantly increased in the PRRX1+group compared with the other two groups.The relative mRNA(F=8.538,P<0.05)and protein(F=10.81,P<0.05)expressions of N-cadherin were also significantly increased in the PRRX1+group compared with the other two groups.These results indicated that PRRX1 promotes EMT in breast cancer cells.Compared with the N.C group and B.C group,the half-maximal inhibitory concentration of the PRRX1+group was significantly higher than the other two groups after treated with docetaxel(F=26.84,P<0.01)and cisplatin(F=7.081,P<0.01)for 24 hours.The mRNA(F=23.39,P<0.01)and protein(F=20.01,P<0.01)levels of P-gp were significantly higher in the PRRX1+group than in the control groups.These results revealed that PRRX1 can promote MDR in breast cancer cells.Furthermore,levels of phosphorylated PI3K(F=21.51,P<0.01)and AKT(F=25.41,P<0.01)increased substantially.The mRNA(F=21.81,P<0.01)and protein(F=16.66,P<0.01)levels of PTEN decreased significantly in PRRX1-overexpressing MCF-7 cells.Conclusion:PRRX1 overexpression induced EMT process,up-regulated the expression of P-gp and promoted MDR in MCF-7 breast cancer cells.PTEN-PI3K/AKT signaling may be the underlying mechanism of the process.