| ObjectiveTo investigate the effects of fructose-1,6-bisphosphatase 1(FBP1)on mitophagy and apoptosis as well as their underlying mechanisms in human breast cancer MCF-7 and MDA-MB-231 cells.Methods1.The mRNA and protein levels of FBP1 and autophagy related markers,such as p62 and LC3,in luminal(MCF-7、BT-474、T47D、ZR-75-1)and basal-like(MDA-MB-231、BT-549、MDA-MB-435、MDA-MB-468)breast cancer cells were detected by(qRT-PCR)and western blot,respectively.Expression correlations between FBP1 and autophagy related genes was obtained and analyzed by The Cancer Genome Atlas(TCGA)Breast Cancer database.2.The specific small interfering RNA against FBP1 and recombinant plasmids carrying FBP1 gene were transfected by liposome into MCF-7 and MDA-MB-231 cells,respectively.Then,to verify the silencing and overexpressing efficiency of FBP1 gene and the effects of the above treatment on the autophagy level,the expression of FBP1,p62,LC3 and Beclin 1 was detected by western blot.After carbonyl cyanide m-chlorophenyl hydrazine(CCCP)treatment,cellular mitochondrial membrane potential(MMP)was assessed by a fluorescent probe JC-1.The morphology of mitochondria was observed by transmission electron microscope(TEM).The intracellular distribution and expression of translocase of outer membrane 20(TOM20)were examined by immunofluorescence staining.The levels of reactive oxygen species(ROS)and the percentages of apoptotic cells were calculated by flow cytometry.The expression of apoptosis associated protein,such as PARP,Caspase 3,Bcl-2 and Bax,was detected by western blot.3.The expression plasmids containing FBP1 gene and HIF-1A gene were co-transfected with lipofectine into human embryonic kidney 293 T cells.The protein-protein interaction between FBP1 and HIF-1α was verified by co-immunoprecipitation(co-IP)assay.The correlation between expression of FBP1 gene and HIF-1A gene was analyzed by TCGA breast cancer database.Then,the specific small interfering RNA against FBP1 and recombinant plasmids carrying FBP1 gene were transfected by liposome into MCF-7 and MDA-MB-231 cells,respectively.The effects of FBP1 gene expression on the transcriptional activity of hypoxia response elements(HREs)was examined by luciferase report assay.The mRNA and protein levels of HIF-1α,BNIP3L/NIX and BNIP3 were detected by qRT-PCR and western blot.The changes of co-action between Bcl-2 and Beclin 1,BNIP3L/NIX as well as BNIP3 were detected by co-IP assay.4.FBP1 expression in breast cancer tissue with different pathological pattern and different tumor stage was analyzed by The Human Protein Atlas database and TCGA database.The correlation between FBP1 expression and survival rate of patients with breast cancer was analyzed by Kaplan-Meier Plotter database.Results1.According to the results of qRT-PCR and western blot,we found that both RNA and protein levels of FBP1 in luminal cells were significantly higher than those in basal-like cells.Besides,the expression of LC3-II was significantly up-regulated and the expression of p62 was significantly down-regulated in basal-like cells compared to those in luminal cells.The results of RNAseq from TCGA breast cancer database also suggested that the expression of FBP1 gene was negatively correlated with the expression of autophagy-promoting gene,such as microtubule-associated protein 1 light chain 3 beta(MAP1LC3B),BCL2/adenovirus E1 B 19 kDa interacting protein 3(BNIP3),and phosphatidylinositol 3-kinase catalytic subunit type 3(PIK3C3)genes,but positively correlated with the expression of RUN and cysteine-rich domain containing Beclin 1 interacting protein(RUBCN)gene.2.The results of western blot indicated that after overexpressing FBP1 gene in MDA-MB-231 cells,the expression of FBP1 and p62 was conspicuously up-regulated and the expression of LC3-II and Beclin 1 was obviously down-regulated.After CCCP treatment,the MMP of cells with FBP1 expression was down-regulated.Under TEM observation,cells with high level of FBP1 expression were characterized by plenty of severe swelling mitochondria.The results of immunofluorescence staining with the antibody against TOM20 revealed quantities of mitochondria conglomerated and united around the perinuclear region.Correspondingly,the mean fluorescence intensity of TOM20 was also elevated in FBP1-expressing MDA-MB-231 cells.On the basis of results from flow cytometry,we noticed that FBP1 could increase the ROS level and the percentages of apoptotic cells,which was identical to the results of the western blot that FBP1 increased the expression of pro-apoptotic proteins,such as cleaved-PARP,cleaved-Caspase 3 and Bax,and decreased the expression of anti-apoptotic protein,such as Bcl-2.All of the above results had been verified in FBP1-kowndown MCF-7 cells and arrived at the same conclusion.3.The results of co-IP assay pointed out that FBP1 could interact with HIF-1α.After an in-depth analysis of the results from TCGA breast cancer database,we confirmed that there was a negative correlation of the gene expression between FBP1 and HIF-1A.The results of luciferase report assay revealed that ectopic FBP1 expression suppressed HREs activity at the level of transcription.Both the results of qRT-PCR and western blot indicated that the mRNA and protein levels of HIF-1α together with its target genes,such as BNIP3L/NIX and BNIP3,were significantly down-regulated after FBP1 overexpression in MDA-MB-231 cells.The results of co-IP suggested that FBP1 encourages Bcl-2 to dissociate from BNIP3 and BNIP3L/NIX and combine with Beclin 1.All of the above results had been verified in FBP1-kowndown MCF-7 cells and arrived at same conclusion.4.The results of immunohistochemistry from The Human Protein Atlas and TCGA breast cancer database showed that FBP1 had negative week expression in infiltrating ductal carcinoma and positive strong expression in infiltrating lobular carcinoma.More importantly,the results downloaded from Kaplan-Meier Plotter database also implied that patients with lower levels of FBP1 expression might give rise to poorer disease-free survival(DFS)and disease-specific survival(DSS),which was also been verified by the result that FBP1 expression was down-regulated with the increase of tumor progression in breast cancer patients.Conclusions1.FBP1 expression is inversely correlated with autophagic level in breast cancer.2.FBP1 inhibits mitophagy and promotes the generation of ROS,which eventually leads to cell apoptosis.3.The direct interaction between FBP1 and HIF-1α blocks the activation of HIF-1α/BNIP3 pathway,which promotes the interaction between Beclin 1 and Bcl-2 in the process of inhibiting mitophagy.4.Loss of FBP1 indicates poor prognosis in breast cancer... |
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