The Effect And Mechanism Of Icariin On Triple Negative Breast Cancer Cells By Regulating Pim-1

OBJECTIVE: This study was to investigate the effect and mechanism of icariin(ICA)inhibiting triple negative breast cancer cell MDA-MB-231 by regulating Pim-1.METHODS: 1.The effect of icariin on the proliferation ability of MDA-MB-231 cells was detected by CCK-8 proliferation experiment,and the icariin concentration for subsequent experiments was determined.2.To detect the expression level of Pim-1 m RNA and protein in MDA-MB-231 cells under the influence of icariin by real-time fluorescent quantitative PCR(q RT-PCR)and Western blot.3.The expression of Pim-1 in MDA-MB-231 cells was interfered by lentiviral vector transfection technique,and transfection efficiency was verified by Western blot to obtain stable transfected cell lines.4.MDA-MB-231 cells were divided into three groups: blank control group(Ctrl group),icariin group(ICA group)and antagonist group(Pim1+ICA group).The migration ability,invasion ability and apoptotic status of cells in each group after ectopic overexpression of Pim-1 on ICA inhibition of MDA-MB-231 were detected by Transwell migration assay,invasion assay,scratch test,apoptosis experiment and Western blot.5.MDA-MB-231 cells were divided into four groups: blank control group(Ctrl group),icariin group(ICA group),overexpression group(Pim1 group)and antagonist group(Pim1+ICA group).Antagonistic effect of ectopic overexpression Pim-1 on ICA inhibition of MDA-MB-231 cells by tumor-bearing experiments in nude mice.RESULTS: 1.CCK-8 experimental results show that icariin significantly inhibits the growth of MDA-MB-231 cells.As the concentration increases,the drug’s ability to inhibit cell proliferation is stronger.The optimal stimulating concentration of the drug is 75 μM.2.The results of q RT-PCR and Western blot showed that as the concentration of icariin increased,the m RNA and protein expression of Pim-1 in MDA-MB-231 cells decreased.3.The proportion of fluorescent cells ≥90% indicates that the transfection efficiency is better.Western blot results showed that the relative expression of Pim-1 protein in the overexpression group was higher than that in the negative control group(P <0.05)and the blank control group(P <0.05).4.Transwell migration and invasion experiments showed that the permeate cells in Ctrl group(183 ± 9.54,93 ± 10.44)and Pim1 + ICA group(142 ± 20.42,65 ± 10.41)were higher than those in ICA group(48 ± 4.16,23 ± 9.61)The difference was statistically significant(P <0.05).Cell scratch test results showed that the healing rate of the Ctrl group(0.6201 ±0.50)and Pim1 + ICA(0.5286 ± 0.06)was significantly higher than that of the ICA group(0.3140 ± 0.04),and the difference was statistically significant(P<0.05).The results of apoptosis experiments showed that the apoptosis rate in Ctrl group(4.340 ± 3.24)and Pim1 + ICA group(12.563 ± 6.79)was significantly higher than that in ICA group(30.610 ± 6.26),the difference was statistically significant(P <0.05).Western blot showed that the relative expression levels of Bcl-2(0.624 ± 0.04),PCNA(0.518 ± 0.16),and Vimentin(0.549 ± 0.17)in the ICA group were lower than those in the Ctrl group and the Pim1 + ICA group(0.844 ± 0.03,0.871 ± 0.122,1.011 ± 0.14).The difference was statistically significant(P <0.05),which was opposite to the relative expression of E-cadherin protein(ICA group 1.975 ± 0.28,Pim1 + ICA group 1.185 ± 0.167),and the difference was statistically significant(P<0.05).5.The growth rate of transplanted tumor in Ctrl group and Pim-1 group was significantly higher than that in ICA group and Pim1 + ICA group(P<0.05).There was no statistically significant difference in growth rate between Pim1 + ICA group and ICA group.The average weight of transplanted tumor in Ctrl group and Pim-1 group was significantly greater than that in ICA group and Pim1 + ICA group(P <0.05),the average tumor weight in Pim1 + ICA group was significantly greater than that in ICA group(P<0.05),Ctrl group and Pim-1 group The difference was not statistically significant(P = 0.627).CONCLUSION: The experiments in vitro and vivo prove that icariin can promote the apoptosis of MDA-MB-231 cells and inhibit the process of epithelial epithelial-mesenchymal transition of MDA-MB-231 cells,and its mechanism may be to inhibit the expression of Pim-1...