UBE2T Regulates Multidrug Resistance In Breast Cancer Cells Through P-gp

Breast cancer is a common malignancy in the female population with a high incidence.With the continuous advances in medical technology,there are more choices to treat breast cancer including chemotherapy,target therapy,radiotherapy and surgery,chemotherapy is currently still dominated.Although research on cancer chemotherapy continues to progress,only a portion of breast cancer cases are sensitive to chemotherapy.Even more,the majority of cancer patients rapidly develop resistance to chemotherapeutic drugs,greatly reducing the effect of chemotherapy.Therefore,reducing or even reversing the multi-drug resistance(MDR)of breast cancer has significant importance.Studying the mechanism of MDR in breast cancer can provide a theoretical basis for weakening or even reversing the MDR of breast cancer,and then increase the effect of chemotherapy.UBE2T(ubiquitin-conjugating enzyme 2T)is a member of ubiquitin-conjugating enzyme(ubiquitin-conjugating enzyme,E2)family.A large number of studies have confirmed that the high expression of UBE2T is closely related to the occurrence and development of various cancers such as bladder cancer,prostate cancer,and gastric cancer.Our previous studies have confirmed that UBE2T can enhance the characteristics of breast cancer stem cells,and a large number of studies have shown a close relationship between cancer stem cells and MDR.However,there is no research to explore the role and mechanism about UBE2T in regulating MDR in breast cancer cells.This study is aimed to explore the role of UBE2T on breast cancer cell MDR and the possible underlying mechanism in order to provide new ideas to weaken or even reverse MDR in breast cancer.ObjectiveThis study was aimed to explore the role and underlying mechanism of UBE2T in regulating MDR of breast cancer cells.Methods1.Endogenous UBE2T expression was analyzed by western blotting in different human breast cancer cell lines(BT549?MDA-MB-231?MCF-7?SK-BR-3 and MDA-MB-468)in order to select proper cell lines to establish the below cell lines.2.UBE2T overexpressing and silencing plasmids and their corresponding control plasmids were transfected into breast cancer cell lines to establish stable over-expressed UBE2T and silenced UBE2T cell lines.Western blotting was used to confirm the establishment of the above cell lines.3.Soft agar and sphere formation assays were performed to detect the change of characteristics of breast cancer stem cells induced by UBE2T.4.MTT assay,colony formation assay and wound healing assay were performed to detect the potential role of UBE2T in regulating MDR of breast cancer cells.5.To further explore the potential mechanism of UBE2T regulating MDR in breast cancer cells,western blotting and immunofluorescence staining assays were performed to detect the expression of P-gp.Results1.The results of western blotting showed that the endogenous expression of UBE2T in MA-MB-231 is much higher than other breast cancer cell lines,the expression of UBE2T in BT-549 is lower than MDA-MB-231,MCF-7 and MDA-MB-468.2.Western blotting was performed to detect the expression of UBE2T in established breast cancer cell lines:1)Over-expressed UBE2T cell line and its control one(designated as BT549-pLVX-AcGFP-UBE2T and BT549-pLVX-AcGFP)were successfully established.2)Silenced UBE2T expression cell lines and their control one(designated as MDA-MB-231-pLKO.1-shUBE2T.2,MDA-MB-231-pLKO.1-shUBE2T.3 and MDA-MB-231-pLKO.1)were successfully established.3.The results of soft agar assay and sphere formation assay suggested that over expression of UBE2T could enhance the characteristics of breast cancer stem cells,and knock down of UBE2T expression could decrease the characteristics of breast cancer stem cells.4.The results of MTT assay suggested that over expression of UBE2T could increase IC50 value of anti-tumor drugs,and knock down of UBE2T could decrease IC50 value of anti-tumor drugs.5.The results of colony formation assay suggested that over expression of UBE2T could significantly reduce the rate of extinction of the breast cancer cell clones and knock down of UBE2T could significantly increase the rate of extinction of the breast cancer cell clones under the same acting conditions of the same anti-tumor drug.6.The results of wound healing assay suggest that over expression of UBE2T could significantly reduce the rate of recovery and knock down of UBE2T could significantly increase the rate of recovery under the same acting conditions of the same anti-tumor drug.7.The results of western blotting and immunofluorescence staining assays suggested that over expression of UBE2T could increase the expression of P-gp.ConclusionOur study found that UBE2T could regulate the MDR of breast cancer cells through P-gp.As far as we know,this is the first time to reveal the correlation between UBE2T and MDR in breast cancer cells.This finding may provide a new idea for reversing MDR in breast cancer clinically and a potential target for breast cancer therapy.