| Breast cancer is considered to be one of the most common forms of malignant cancer in female worldwide.Among all treatments for breast cancer,radiotherapy and chemotherapy are the two main clinical treatments.Although different treatments are used in different types of breast cancer,resistance to drug is common in all breast cancer types and is a major challenge in clinical practice.Sex steroids play a crucial role in cancer development and mammary gland development.Estrogen is a major regulator in the development and progress of breast cancer.Studies have shown that estrogen reduction or anti-estrogen strategies have a promising therapeutic effect on breast cancer treatment,mainly in estrogen receptor-dependent breast cancer.Among the drugs that reduce the incidence of breast cancer,a selective estrogen receptor modulator,tamoxifen,acts as an ER antagonist and is a promising therapeutic agent for ER-positive breast cancer.Although a series of studies have shown that tamoxifen can significantly reduce the risk of recurrence and metastasis in women with breast cancer,resistance to tamoxifen remains a worldwide concern in the treatment of breast cancer.Resistance to endocrine therapy is caused by genetics(changes in DNA sequence)and epigenetic factors.At present,several mechanisms of resistance to tamoxifen have been discovered,which is not enough to understand the development of tamoxifen resistance in breast cancer.Therefore,we need to urgently explore the detailed mechanism of resistance to tamoxifen in breast cancer,and find a new breakthrough for breast cancer treatment.Objective1.To reveal key regulators of tamoxifen resistance in MCF7/TAMR cells,we detected the m RNA and protein levels of NEMP1 in tamoxifen-resistant MCF7/TAMR cells and tamoxifen-sensitive MCF7 cells.In addition,we also detected the protein and m RNA levels of NEMP1 in adjacent normal tissues and cancer tissues of breast cancer patients.2.The results of the first part have found that the m RNA and protein levels of NEMP1 are dramatically elevated in breast cancer tissues.And the expression of NEMP1 is also significantly up-regulated in the tamoxifen-resistant breast cancer cell line MCF7/TAMR.Therefore,the second part is mainly to explore whether NEMP1 is involved in the regulation of tamoxifen resistance in breast cancer cell lines.3.The results of the previous two sections show that NEMP1 is highly associated with breast cancer and can contribute to developing tamoxifen resistance in MCF7 cells.Studies have shown that nuclear receptor coactivator 1 is also considered to be a key regulator of breast cancer and is positively associated with tamoxifen resistance in breast cancer.In summary,based on the hypothesis that NEMP1 and NCOA1 are two potential regulators of breast cancer development,we want to reveal the specific molecular mechanisms of these two genes in the development of breast cancer and tamoxifen resistance.Most importantly,the aim of this study was to find potential therapeutic targets and feasible treatment strategies for breast cancer patients who have developed tamoxifen resistance.Methods1.We first treated MCF7 cells,an estrogen receptor-positive and tamoxifensensitive breast cancer cell line,with 4-hydroxytamoxifen(active metabolite of tamoxifen)to obtain tamoxifen-resistant MCF7 cell line,which was named MCF7/TAMR cells.The proliferative capacity of MCF7 and MCF7/TAMR cells was detected by MTT assay after 96 hours of treatment with different doses of4-hydroxytamoxifen.Next,Western blot and real-time quantitative PCR were used to detect the protein and m RNA levels of NEMP1 in tamoxifen-sensitive MCF7 cells and tamoxifen-resistant MCF7/TAMR cells.In addition,we also detected the expression of NEMP1 protein in normal tissues and cancer tissues of breast cancer patients by immunohistochemistry.Moreover,we examined the m RNA levels of NEMP1 in 53 breast cancer tissues and 18 matched adjacent normal tissues by realtime quantitative PCR.2.The MCF7/TAMR stable cell lines which NEMP1 was knockdown and NEMP1 overexpressing MCF7 stable cell lines were first obtained using the lentiviral packaging system,and the efficiency of NEMP1 knockdown and overexpression was detected by western blotting.Next,the cell number status of NEMP1 knockdown MCF7/TAMR cells and NEMP1 overexpressing MCF7 cells in tamoxifen treatment and untreated was examined using cell total assay,MTT assay and colony formation assay,respectively.3.Firstly,the NEMP1 knockdown MCF7/TAMR stable cell line was obtained by using the lentiviral packaging system.And then the m RNA levels of HER2,EGFR,MYC,CCND1,PTEN,NCOA1,BCL2,and SRC which are important regulatory factor involved in the development of breast cancer were detected by real-time quantitative PCR in control cells and NEMP1 knocking down MCF7/TAMR cells.In addition,the expression level of NCOA1 in NEMP1 knocking down MCF7/TAMR cells and the control group and was detected by western blotting.Results1.(1)The results showed that when the concentration of 4-hydroxytamoxifen was gradually increased,the proliferative ability of MCF7 cells was significantly inhibited and showed a dosage-dependent effect.When the concentration of4-hydroxytamoxifen was 0.2 ?M,0.5 ?M and 1 ?M,the proliferation ability of MCF7/TAMR cells was enhanced,and the number of cells was significantly higher than that of MCF7 cells.When the concentration of 4-hydroxytamoxifen was 2 ?M,the proliferation of MCF7/TAMR cells was slightly inhibited compared with that without 4-hydroxytamoxifen treatment,but the number of cells was still more than that of MCF7 cells which were significantly inhibited by 4-hydroxytamoxifen.(2)The results of western blot and real-time quantitative PCR showed that, compared with MCF7 cells,the protein and m RNA levels of NEMP1 were significantly increased in MCF7/TAMR cells.(3)The results of immunohistochemistry showed that NEMP1 expression was significantly elevated in breast cancer tissues compared with adjacent normal tissues.The results of real-time quantitative PCR showed that the m RNA levels of NEMP1 were significantly elevated in tumor tissues compared to para-cancerous normal tissues of berast cancer patients.2.(1)The results of western blotting showed that the protein level of NEMP1 was significantly down-regulated in NEMP1 knocking down MCF7/TAMR cells compared with the control group.(2)There were no significant differences in the growth,proliferation,and colony forming ability of the control group between the tamoxifen-treated or untreated groups.However,after NEMP1 knockdown,the growth,proliferation,and colony forming ability of cells in the tamoxifen-treated group was significantly inhibited compared with the untreated group.(3)The results of western blotting showed that the protein level of NEMP1 was significantly up-regulated in MCF7 cells stably overexpressing NEMP1-CDS compared with the control group.(4)In the control cells,tamoxifen treatment significantly inhibited cell growth,proliferation,and colony forming ability compared to untreated group.However,in NEMP1 overexpressing cells,there was no significant difference in cell growth,proliferation,and colony forming ability between tamoxifen-treated and untreated groups.3.(1)Compared with the control cells,only the m RNA level of NCOA1 was decreased in NEMP1 knockdown MCF7/TAMR cells.While the m RNA levels of other regulatory factors HER2,EGFR,MYC,CCND1,PTEN,BCL2 and SRC were compared between the control cells and NEMP1 knockdown MCF7/TAMR cells.(2)The results of western blotting showed that the protein level of NCOA1 was also significantly decreased in NEMP1 knockdown MCF7/TAMR cells compared with control cells.Conclusions1.MCF7/TAMR cells indeed develop tamoxifen resistance compared with MCF7 cells which are sensitive to tamoxifen treatment.Moreover,the protein and m RNA levels of NEMP1 were significantly elevated in MCF7/TAMR cells.In addition,we also found that the protein and m RNA levels NEMP1 were significantly elevated in breast cancer tissues compared with adjacent normal tissues,indicating that NEMP1 is closely related to breast cancer and breast cancer drug resistance.2.Using the lentiviral system,we successfully obtained the NEMP1 knockdown MCF7/TAMR cell line and the NEMP1 overexpressing MCF7 cell line.In NEMP1 knockdown MCF7/TAMR cell line,we found that the decrease of NEMP1 inhibited tamoxifen resistance in MCF7/TAMR cells by cell number counting,MTT assay,and colony formation assays.Furthermore,in the NEMP1 overexpressing MCF7 cell line,we found that NEMP1 overexpression reduced the sensitivity of MCF7 cells to tamoxifen and caused tamoxifen resistance in MCF7 cells.3.In NEMP1 knockdown MCF7/TAMR cells,we found that only the m RNA levels of NCOA1 were decreased,while the m RNA levels of other regulatory factors such as HER2,EGFR,MYC,CCND1,PTEN,BCL2,and SRC were compared between the two groups.In addition,we also found that the protein level of NCOA1 was also significantly decreased in NEMP1 knockdown MCF7/TAMR cells.These experimental results indicate that NEMP1 regulates tamoxifen resistance in MCF7/TAMR cells by regulating the expression of NCOA1. |
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