| Background Breast cancer is the highest incidence of malignant tumors in women,the proportion of the incidence of breast cancer accounts for twenty-nine percents of all the female cancer,and the proportion of young patients increased year by year.The mortality of breast cancer is fifteen percents.And it is the second highest fetal cancer.Breast cancer has a significant threat to physical and mental health of women.With the progress of biomedical research,in recent years,in addition to surgical removal of tumor tissue,the comprehensive treatment of breast cancer has formed the clinical consensus of standardized treatment of breast cancer,including surgery,radiotherapy,chemotherapy,endocrine therapy,Chinese medicine treatment and Molecular targeted therapy and more.Among them,chemotherapy of breast cancer has an important significance.Preoperative neoadjuvant chemotherapy can reduce the primary lesion of cancer and increase the chance of surgical treatment of breast cancer patients.Postoperative adjuvant chemotherapy can prevent the recurrence and metastasis of cancer,prolong the survival of patients and improve the quality of life of patients. Doxorubicin is a classical breast cancer chemotherapy drugs,recent clinical studies have confirmed that doxorubicin mono-therapy can be used to achieve a good curative effect,and mono-therapy is superior to the paclitaxel.Its combination with paclitaxel can increase patient benefit.However,in the clinical application of doxorubicin,there is a common problem: resistance of chemotherapy drug.The resistance of chemotherapy drug are closely linked to tumor progression and recurrence of patient.Currently,the mechanism of the resistance of chemotherapy drug is complicated.Including tumor cells apoptosis,cancer stemness,tumor microenvironment,DNA damage repair and other factors.However,the mechanism has not been fully elucidated.Deciphering unknown mechanisms may help identify new treatment strategies for breast cancer patients.Protein arginine methyltransferase 5(PRMT5)belongs to the family of protein arginine methyltransferases(PRMTs).PRMTs can catalyze the transfer of methyl groups to the protein arginine,based on their way to catalyze arginine,PRMTs can be divided into two main categories,type I PRMTs and type II PRMTs.PRMT5,as the earliest identified member of type II PRMTs,has attracted the attention of many researchers.In recent years,a large number of studies have shown that PRMT5 is a potential oncogene which overexpression and abnormal activation can promote breast cancer.And it plays an important role in regulating the tolerance of tumor cells to radiotherapy.Cancer stem cells(CSCs)are a kind of tumor cells with high self-renewal and high differentiation ability.They play an important role in tumor growth and development and tumor recurrence.Recent findings indicate that cancer stem cells play an important role in chemo-resistance in a variety of tumors and that the levels of the stemness of tumor cells in doxorubicin-resistant breast cancer cells MCF-7/ADR are increased.At the same time there is literature that PRMT5 is important in maintaining the stemness in mouse embryonic stem cells and neural stem cells.However,the role of PRMT5 in the regulation of tumor cells has not been elucidated.The analysis of GEO database and previous experimental results show that the effect of doxorubicin on breast cancer cells can significantly increase the expression of PRMT5 in cells.Compared with normal breast cancer cells,the expression of PRMT5 in doxorubicin-resistant breast cancer cells was significantly increased.However,the function of the increased PRMT5 on doxorubicin resistance in breast cancer cells and the underlying molecular mechanism have not been clarified.Methods and results 1.The expression of PRMT5 in breast cancer cell lines and clinical breast cancer samples and its effect on the biological behavior of breast cancer cells(1)The expression of PRMT5 in human breast normal epithelial cells MCF10 A and five human breast cancer cell lines was detected by RT-q PCR and Western Blot.(2)Western Blot and immunohistochemistry were used to detect the expression level of PRMT5 in the tumor tissue samples and adjacent normal tissue samples of clinical breast cancer patients.(3)MDA-MB-231 and MCF-7 which over-expressing PRMT5 were constructed by lentivirus infection.(4)MTT assay was used to detect the change of proliferation of MDA-MB-231 and MCF-7 over-expressing PRMT5.(5)Transwell assay was used to detect the migration of MDA-MB-231 cells after PRMT5 overexpression.The results showed that the expression of PRMT5 in human breast cancer cell lines and human breast cancer tissues was significantly increased,the overexpression of PRMT5 promoted breast cancer cell proliferation and migration.These results suggesting that PRMT5 may function as an oncogene in breast cancer.2.PRMT5 promotes the resistance of doxorubicin of breast cancer cells(1)The expression level of PRMT5 in MCF-7 and doxorubicin-resistant MCF-7/ADR cells was analyzed by GEO database.(2)The expression level of PRMT5 in MCF-7 and MCF-7/ADR was detected by Western Blot.(3)Downregulating PRMT5 in MCF-7/ADR cells by using a lentiviral infection.(4)MTT assay was used to detect the doxorubicin resistance in MCF-7/ADR-sh PRMT5 cells.(5)Western Blot was used to detect the expression of apoptosis related molecules in MCF-7/ADR-sh PRMT5 cells.(6)The apoptosis of MCF-7/ADR-sh PRMT5 cells was detected by TUNEL assay.(7)MTT assay was used to detect the doxorubicin resistance in MDA-MB-231-PRMT5 cells and MCF-7-PRMT5 cells.(8)The apoptosis related molecules expression in MDA-MB-231-PRMT5 cells and MCF-7-PRMT5 cells were detected by Western Blot.(9)The apoptosis in MDA-MB-231-PRMT5 cells and MCF-7-PRMT5 cells were detected by TUNEL assay.The results showed that PRMT5 levels were significantly increased in MCF-7/ADR cells compared to MCF-7 cells.The resistance to doxorubicin of MCF-7/ADR-sh PRMT5 cells was significantly reduced.In MCF-7/ADR-sh PRMT5 cells,the expression of Bcl-2 decreased while the expression of Bax increased,the number of apoptotic cells increased significantly,indicating that the apoptosis level was increased.However,the resistance to doxorubicin was increased in breast cancer cells which over-expressing PRMT5.The overexpression of PRMT5 increased the expression of Bcl-2 and decreased the expression of Bax,and number of apoptotic cells decreased after PRMT5 overexpression,indicating that the level of apoptosis in cells decreased.Thus,PRMT5 promotes the resistance of doxorubicin of breast cancer cells.3.PRMT5 promotes the resistance to doxorubicin by governing stemness in breast cancer PRMT5 can promote the resistance of breast cancer cells to chemotherapeutic drugs,and previous studies have shown that PRMT5 can maintain the stemness of embryonic and neural stem cells.In addition,the increase of stemness in breast cancer cells can lead to its resistance to chemotherapeutic drugs.Therefore,the changes of cell stemness were detected by using mammosphere formation assay and flow cytometry respectively in those cells.The results showed that overexpression of PRMT5 can enhance the stemness of breast cancer cells.PRMT5 promotes the resistance of doxorubicin to breast cancer cells by regulating the stemness of breast cancer cells. 4.A study about the mechanism of PRMT5 decreased stemness and sensitivity of breast cancer cell to doxorubicin(1)Western Blot was used to detect the expression of C-MYC,OCT4/A and KLF4 in MDA-MB-231-PRMT5,MCF-7-PRMT5,MCF-7/ADR,MCF-7/ADR-sh PRMT5 cells.(2)The expression of C-MYC,OCT4/A and KLF4 in breast cancer tissues and adjacent normal tissues were detected by Western Blot and analyzed the correlation with PRMT5.(3)The lentiviral infection method was used to construct the cell lines of MDA-MB-231 and MCF-7 overexpressing methylation-inactivating mutant PRMT5(R368A).(4)The m RNA levels of C-MYC,OCT4/A and KLF4 in these cells were detected by RT-q PCR.(5)The changes of C-MYC,OCT4/A and KLF4 expression in MDA-MB-231 and MCF-7 overexpressing PRMT5 or PRMT5(R368A)were detected by Western Blot.(6)The levels of cell stemness of MDA-MB-231 and MCF-7 cells overexpressing PRMT5(R368A)were detected by mammosphere formation assay and flow cytometry respectively.(7)The MTT assay was used to detect the differences of the resistance to doxorubicin among MDA-MB-231 and MCF-7 parents,respectively,over-expressing PRMT5 and PRMT5(R368A).(8)Western Blot was used to detect MDA-MB-231 and MCF-7 parents,overexpression of PRMT5 or PRMT5(R368A)apoptosis-related molecules expression.(9)The apoptosis of MDA-MB-231 and MCF-7 cells overexpressing PRMT5(R368A)was detected by TUNEL assay.The results showed that overexpression of PRMT5 could up-regulate the expression of C-MYC,OCT4/A and KLF4,but only increased the expression of OCT4/A and C-MYC at m RNA level.The expression of PRMT5 protein in breast cancer tissues and adjacent normal tissues was correlated with the protein expression of C-MYC,OCT4/A and KLF4.PRMT5 regulates cell stemness to promote breast cancer cell resistance to doxorubicin,which depends on its methylation ability.There is no significant difference in resistance to doxorubicin between parental cells and cells overexpressing PRMT5(R368A) cell line.Conclusion In this study,we found that PRMT5 is abnormally highly expressed in the tumor tissues and breast cancer cells,which is a potential oncogene.Meanwhile,the expression of PRMT5 in doxorubicin-resistant breast cancer cell line MCF-7/ADR was significantly increased.PRMT5 can promote the resistance of breast cancer to chemotherapeutic drug doxorubicin by regulating the stemness of breast cancer cells.Inhibiting PRMT5 can reduce the resistance of doxorubicin to breast cancer cells.In summary,our study showed for the first time that the methyltransferase PRMT5 determines sensitivity to doxorubicin in breast cancer cells through induction of cell stemness.Our study may provide a new therapeutic target to overcome resistance to doxorubicin and suggests cell stemness induction as a critical event in chemotherapeutic resistance driven by the PRMT5 pathway. |
