KRT15 Promotes Breast Cancer Cell Invasion And Radiotherapy Resistance And Inhibits Ferroptosis Through The Wnt/β-catenin Signaling Pathway

Objective: Breast cancer is the malignant tumor with the highest incidence in the world.Radiotherapy is an important means for the treatment of breast cancer,which can reduce recurrence,prolong survival time and improve the quality of life.However,many patients do not recover well because of tumor resistance to radiotherapy,so radiotherapy resistance is a clinical problem that needs to be solved in radiotherapy.In recent years,molecular targeting has achieved significant clinical efficacy in the treatment of malignant tumors.Screening genes involved in the regulation of breast cancer cell proliferation,invasion and therapeutic resistance is the current exploration and research focus.The purpose of this study was to explore the effect of keratin 15(KRT15),which has the above-mentioned functional regulatory genes,on malignant proliferation and radiosensitivity of breast cancer,and to verify the related signal pathway.Methods: breast cancer MDA-MB-231 and MCF-7 cells were cultured in vitro,and KRT15 knockout or overexpression cells were established.Lentiviral vectors containing KRT15 targeting sh RNA or c DNA were used to transfer breast cancer MDA-MB-231 and MCF-7 cells.The cells transduced with empty vector were used as negative control.The expression of KRT15 m RNA and protein was detected by q PCR and Western blot.The effects of KRT15 gene knockout and overexpression on the proliferation of MDA-MB-231 and MCF-7 cells were observed by CCK-8 test and clone formation assay.The effects of KRT15 on apoptosis of MDA-MB-231 and MCF-7 cells were observed by Annexin V-APC/PI double staining method.Transwell,scratch test and Western blot were used to observe the effects of KRT15 on the migration and invasion of MDA-MB-231 and MCF-7 cells.Western blot was used to observe the effect of KRT15 on ferroptosis of MCF-7 cells.A tumor-bearing mouse model was established to verify the tumor-promoting function of KRT15.Western blot was used to detect the expression of molecules related to Wnt/β-catenin signaling pathway.Results: the proliferation activity of KRT15 was verified by CCK-8 and clone formation assay.The proliferation ability of KRT15 knock-down cells of MDA-MB-231 and MCF-7 cells decreased(the inhibition rate was 29.62% and 29.38%,respectively,P<0.001),while the proliferation ability of overexpression strains increased(the proliferation rate was 10.06% and 18.26%,respectively,P<0.001).Colony formation assay showed the same results(P<0.01).Flow cytometry analysis showed that overexpression of KRT15 significantly inhibited apoptosis(P<0.001),while knockout KRT15 reversed the inhibitory effect on apoptosis(P<0.05).In order to verify the effect of KRT15 on radiosensitivity,CCK-8 and colony formation assay were used to observe the survival of MCF-7 cells exposed to 4Gy ionizing radiation.The results showed that the survival rate of KRT15 overexpression cells increased significantly(P<0.01).Flow cytometry analysis showed that the apoptosis rate was significantly lower than that in Control+IR group(P<0.05).Western blot showed that KRT15 Promoted EMT;Transwell before and after radiotherapy and scratch test showed that the invasion and migration ability of breast cancer cells with overexpression of KRT15 was significantly increased before and after radiotherapy(P<0.001).Western blot showed that compared with the Control group,the protein levels of GPX4,FTH1 and SLC7A11 in the KRT15 overexpression group before and after radiotherapy were significantly higher,while the DMT1 protein levels were significantly decreased;in the mouse model,the tumor volume and weight of the KRT15 group before and after radiotherapy were lower than those of the Control group;immunohistochemistry showed that the Ki-67 content was higher than that of the Control group.Western blot showed that overexpression of KRT15 could enhance the protein expression of β-catenin,TCF1,MMP7,CCND1 and c-Myc in Wnt/β-catenin signal pathway in vivo and in vitro.This study also showed that the expression of p53 protein in breast cancer cells overexpressing KRT15 decreased before and after receiving ionizing radiation.Therefore,it is predicted that KRT15 overexpression may inhibit the expression or transcriptional activity of p53 through negative feedback regulation.Conclusion: KRT15 promotes the proliferation,migration and invasion of breast cancer,inhibits apoptosis and ferroptosis,and then increases the activity of Wnt/β-catenin signal pathway and inhibits the effect of radiotherapy.