Feedback Loop Between Mitochondrial Fission/Fussion And Notch Signaling Regulates Survivin-mediated Survival Of TNBC Cells

Mitochondria are highly dynamic organelles,which are in constant process of fission and fusion.Mitochondrial fission and fusion not only have an important effect on maintaining mitochondrial morphology,calcium homeostasis,ROS production and so on,but also affect cell metabolism,proliferation and migration.Studies have shown that triple negative breast cancer(TNBC)has a worse prognosis than non triple negative breast cancer.The reason may be associated with enhanced tumor cell survival ability and the lack of effective therapeutic targets.But the mechanism is not yet fully clear.In this study,mitochondrial fission and fusion was chosen as a breakthrough point to explore the effect of mitochondrial dynamics on the survival of triple negative breast cancer cells and the mechanism.ObjectiveIn this study,we chose triple negative breast cancer as the research object,Drp1 and Mfn1 as the breakthrough point(the molecules of which mediated mitochondrial fission and fusion respectively),to explore:(1),the distribution state of mitochondrial fusion and fission in triple negative breast cancer and its correlation with prognosis;(2),the regulatory role of mitochondrial fission and fusion involved in triple negative breast cancer cell growth;(3),the mechanism of mitochondrial fission and fusion involved in triple negative breast cancer cell growth.Materials and Methods1.Transmission electron microscope(TEM)was used to observe the distribution state of mitochondrial fission and fusion in triple negative breast cancer patients,and statistical analysis was used to assess prognosis.2.Immunohistochemical staining(IHC),Western blot and qRT-PCR were used to detect the levels of Drp1 and Mfn1 protein and mRNA in the tissue samples and cells of triple negative breast cancer.3.Transfection was used to construct Drp1 mediated mitochondrial fission and Mfn1 mediated mitochondrial fusion triple negative breast cancer cell models.4.MTS assay was used to detect the cell viability of the cell models of Drp1/Mfn1 mediated mitochondrial fission/fusion.5.Situ tumor formation in nude mice was used to detect the ability of Drp1/Mfn1 mediated mitochondrial fission/fusion cell models.6.Annexin V-FITC/PI/flow cytometry and TUNEL staining were used to detect cell apoptosis in Drp1/Mfn1 mediated mitochondrial fission/fusion cells.7.Eud assay and IHC Ki67 staining were used to detect the cell proliferation of Drp1/Mfn1 mediated mitochondrial fission/fusion cells.8.Western blot,IHC were used to detect Drp1,Mfn1,Notch1,NICD1 protein levels,and mitochondrial morphology staining was used to explore the interaction of mitochondrial fission/ fusion and Notch signal.9.Annexin V-FITC/PI/flow cytometry and Edu were used to detect the effect of the interaction between mitochondrial fission/fusion and Notch signaling on cell apoptosis and proliferation.10.Western blot was used to detect the expression of survivin regulated by the interaction between mitochondrial fission/fusion and Notch signal.11.MST experiment demonstrated that survivin mediated the effect of mitochondrial fission/fusion and Notch signal on cell viability.Results1.Mitochondrial fission increased in triple negative breast cancer and the expression of Drp1 was negatively correlated with prognosis;mitochondrial fusion decreased and the expression of Mfn1 was positively correlated with prognosis in TNBC cells.2.Drp1 mediated mitochondrial fission promoted cell survival,proliferation and inhibited apoptosis in TNBC cells;Mfn1 mediated mitochondrial fusion inhibited cell survival,proliferation and promoted apoptosis.3.Drp1 mediated mitochondrial fission promoted Notch1 expression and NICD1 activation;Mfn1mediated mitochondrial fusion inhibited Notch1 expression and NICD1 activation.4.Activation of Notch signal promoted mitochondrial fission and inhibited mitochondrial fusion.5.The interaction of mitochondrial fission and Notch signaling inhibited cell apoptosis and promoted cell proliferation,and positively regulated the expression of survivin.6.Survivin mediated the effect of the interaction of mitochondrial fission and Notch signaling on cell viability of triple negative breast cancer cells.ConclusionIn this study,the positive feedback regulation of mitochondrial fission and Notch signaling pathway was identified.And it’s regulating effect on promoting cell survival and inhibiting cell apoptosis in triple negative breast cancer cells was mediated by survivin.