RSK4 Inhibits Doxorubicin Resistance In Breast Cancer By Regulating P13K/Akt Signaling Pathway

Objective: This study aimed to explore the role and molecular mechanism of RSK4 in reversing doxorubicin resistance in breast cancer by regulating PI3K/Akt signaling pathway.Method:1.Use KM plotter and ROC Plotter online tools to analyze the correlation between the expression level of RSK4 and the overall survival rate of breast cancer and the effect on the efficacy of doxorubicin.2.Select breast cancer MCF-7 cells as the research object,and use the increasing DOX concentration method to construct breast cancer doxorubicin-resistant cell line MCF-7/DOX.Real-time fluorescent quantitative PCR(q RT-PCR)and Western blotting were used.(Western blot)was used to detect the expression of RSK4 in these two cells.3.Construct a breast cancer resistant cell line stably overexpressing RSK4 by virus packaging transfection,and divide the cells into three groups:MCF-7/DOX/RSK4 cells transfected with the overexpression of RSK4 lentiviral vector as the experimental group.MCF-7/DOX/NC transfected with blank virus vector was used as a negative control group,and MCF-7 / DOX untransfected cells were used as a blank control group.4.CCK-8 method was used to detect cell proliferation and IC50 changes in each group;cell scratches and Transwell invasion tests were used to detect cell migration and invasion changes in each group;RSK4 overexpression was detected by flow cytometry for resistance to breast cancer Effects of cell line apoptosis.5.The changes of expression of drug resistance-related proteins(ABCG2,ABCB1,MRP2),apoptosis-related proteins(Bax,Bcl-2,Caspase3)and core protein of PI3K/Akt signaling pathway in each group of cells were detected by Western blot to preliminary To investigate the possible mechanism of RSK4 involved in the resistance of human breast cancer cells to doxorubicin.6.MCF-7/DOX and MCF-7/DOX/RSK4 cells were used to construct a subcutaneous drug resistant xenograft model in nude mice.The experiment was divided into 4 groups: MCF-7/DOX group,MCF-7/DOX + DOX treatment group,MCF-7/DOX/RSK4 group,MCF-7/ DOX/RSK4 + DOX treatment group,4 in each group.Observe and compare the tumor growth rate,size and volume of nude mice in each group.Result:1.The overall survival rate of breast cancer patients with high RSK4 expression is significantly higher than that of low expression patients(P <0.05);when breast cancer patients are treated with anthracyclines,the therapeutic effect of RSK4 high expression patients is better than that of low expression groups(P <0.05).2.The expression of RSK4 in MCF-7/DOX was significantly lower than that in parental breast cancer cells MCF-7(P <0.05).3.Successfully constructed RSK4 overexpressing human breast cancer resistant cell line MCF-7/DOX,we used q RT-PCR and Western blot to detect the transfection effect,the results showed that the m RNA and protein expression levels of RSK4 gene in the cells of the experimental group were significant Higher than blank control group and negative control group(P <0.05).4.The experimental results of CCK-8 testing the IC50 value of cells in each group showed that the IC50 value of MCF-7/DOX group was significantly higher than that of MCF-7 cells(P <0.05);after overexpression of RSK4,MCF-7/DOX / Compared with cells in negative control group and blank group,IC50 value of RSK4 group decreased significantly(P <0.05).5.The results of CCK-8 showed that the proliferation activity of MCF-7/DOX cells was significantly higher than that of MCF-7 cells(P <0.05);the proliferation of MCF-7/DOX/RSK4 cells in the experimental group was significantly lower than that in the blank group and negative control Group(P<0.05);the results of cell scratch test and Transwell invasion test showed that the migration and invasion ability of MCF-7/DOX/RSK4 cells in the experimental group were significantly lower than that in the blank group and the negative control group(P <0.05);the results of flow cytometry showed that the apoptosis rate of the experimental group was significantly higher than that of the blank group and the negative control group(P <0.05).6.Western blot results showed that compared with the blank group and the negative control group,the expression levels of anti-apoptosis-related proteins(Bcl-2,Caspase3)in the experimental group were significantly decreased(P<0.05),and the Bax protein levels were significantly increased(P <0.05);the expression levels of resistance-related proteins BRCP,p-gp,MRP2 and PI3K/Akt signaling pathway-related proteins p-PI3 K and p-Akt in the experimental group were significantly decrease(P <0.05).7.In the nude mouse subcutaneous drug-resistant xenograft model,the volume and growth rate of the xenograft tumor in the RSK4 overexpression group were significantly less than the control group(P <0.05);after treatment with doxorubicin,the nude mice in the RSK4 overexpression group The volume and growth rate were significantly reduced compared with the untreated group(P <0.05).Conclusion:1.RSK4 is related to the prognosis and treatment of breast cancer.The higher its expression,the better the prognosis and the better the response to chemotherapy.2.The expression of RSK4 decreases during the induction of breast cancer doxorubicin resistance.Overexpression of RSK4 inhibits the proliferation,migration and invasion of breast cancer resistant cells,increases apoptosis,and inhibits the growth of breast cancer resistant xenografts.3.RSK4 is involved in the regulation of doxorubicin resistance in breast cancer cells,and its mechanism of action is probably related to the PI3K/Akt signaling pathway.