| Objective: Breast cancer is one of the leading causes of cancer death among women worldwide.The main therapeutic approaches to treat breast cancer include surgery,radiotherapy and chemotherapy.However,it has some chances to recurrence and metastasis.Cancer stem cells(CSCs),including breast cancer stem cells(BCSCs),have been shown to play important roles in cancer's recurrence and metastasis.BCSCs are also relatively resistant to chemotherapy and radiotherapy compared with their non-tumorigenic progeny.Therefore,it is necessary to discover and identify the novel and specific molecular targets or related signaling pathways to inhibit the mammary stem/progenitor cell population and suppress carcinogenesis and tumor metastasis.N-myc and STAT interactor(NMI)is such a protein that involved in a variety of signaling mechanisms and interacts with different transcription factors.It is associated with DNA damage,cell cycle control and epithelial-mesenchymal transition.However the role of NMI in cancer stem cell remains poorly understood.In this study,we try to explore the function of NMI in BCSCs and find out the mechanisms.Methods: 1.Mammosphere formation assay was used to detect the expression level of NMI in breast cancer stem cell enriched populations.Breast cancer stem cell surface markers(CD44/CD24)were used to detect the effect of NMI to breast cancer stem cells by flow cotymetry.q RT-PCR and Western blot were used to evaluate the expression level of NMI and stem cell associated proteins Nanog,Oct4 and Sox2.TheNMI m RNA levels in CD44+CD24-and CD44-CD24+ cell populations were analyzed using GEO database(GSE15192).2.Mammosphere formation ability,flow cytometry,western blot and q RT–PCR were used to detected the influence of NMI in breast cancer stem cells by knockdown/overexpression of NMI.3.Dual Luciferase Assays of h TERT promoter were used in MCF7 cells transfected with NMI knockdown plasmids.Western blot was used to detect the effect of NMI on the level of h TERT protein after NMI knockdown.Western blot,flow cytometry,mammosphere formation,transwell and immunofluorescence were used to detect the effect of NMI if it can be rescued by h TERT.4.Animal experiment was performed by subcutaneous injection of tumor cells to construct nude mice tumor model.The Tumor formation ability of NMI stable knockdown or overexpression cell lines and their control cell lines were detected.Intravenous injection was performed to construct breast cancer lung metastasis model.5.GEO database and immunohistochemistry were used to analyze the relationship between NMI and h TERT.The relationship between NMI/h TERT and clinic pathological stage were analyzed.6.Immunoprecipitation and mass spectrometry were used to find the transcription factor cooperated with NMI on h TERT promoter and verified by Western blot.Results: 1.NMI is downregulated in breast cancer stem cell(BCSC)-enriched populations.2.NMI knockdown promotes the expression of CSC-related markers NANOG,OCT4,SOX2,mammosphere formation ability and CD44+CD24-cells population.3.NMI overexpression inhibits the expression of CSC-related markers Nanog,Oct4,Sox2,mammosphere formation ability and CD44+CD24-cells population.4.NMI inhibits BCSCs traits by down-regulating h TERT expression.5.NMI suppresses EMT in breast cancer cells and tumorigenicity in mouse model with xenografts of human breast cancer by down-regulating h TERT.6.NMI inversely correlates with h TERT in breast cancer samples.7.YY1 interacts with NMI to mediate the down-regulation of h TERT.Conclusion: Our findings show that NMI was lower expressed in tumor tissue compared with normal tissue and NMI inhibits cancer stem-like cell traits in breast cancer by downregulation of h TERT. |
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