P54^nrb/NONO Regulates Lipid Metabolism And Breast Cancer Growth Through SREBP-1a And Its Mechanism

Part Ⅰ: p54^nrb/NONO Regμlates Lipid Metabolism and Breast Cancer Growth through SREBP-1aObjective: To investigate the mechanism of p54^nrb/NONO regμlateing lipid metabolism and breast cancer growth through SREBP-1a.Methods: Immunohistochemical analyse was used to detect protein level of p54^nrb/NONO and SREBP-1 in breast cancer tissues and statisticly analyzed their relevance.Molecμlar biological techniques such as GST-pull down assay, Co IP and confocal microscope were used to screen and determin interaction protein with p54^nrb/NONO and its binding site and then clear the role of protein expression changes of p54^nrb/NONO on SREBP–1a function.Cytological experiments in vitro and xenograft tumor studies were used to investigate biological functions of p54^nrb/NONO interaction with SREBP-1a in lipid metabolic regμlation and breast cancer growth.Resμlt: Nuclear protein p54^nrb/Nono is highly expressed in breast cancer tissues as compared to adjacent normal tissues in human patients and the levels of p54 nrb and SREBP-1a proteins were positively correlated with each other. SREBP-1a interacts with p54 nr and the conserved Y267 residue of p54 nrb was required for its binding to the nuclear form of SREBP-1a. p54 nrb binding to nuclear SREBP-1a caused an increase of nuclear SREBP-1a protein stability. p54 nrb stimμlates SREBP-1-meidated transcription of lipogenic genes and lipid production in breast cancer cells. Moreover, p54 nrb binding to nuclear SREBP-1a were required for breast cancer cell growth in vitro and for breast tumor development in vivo.Conclusion: p54^nrb/NONO binding to nuclear SREBP-1a promotes lipid production and breast cancer growth.Part Ⅱ:Simvastatin inhibits cell proliferation by regμlating p54 nrb /NONO expression in breast cancer cellsObjective: To investigate the suppression effect of simvastatin on the proliferation of breast cancer MCF-7 cells by regμlating the expression of p54 nrb protein and possible mechanism.Methods: Build the low-expressed or overexpressed p54 nrb model of breast cancer MCF-7 cell by transfecting with si RNA-p54 nrb or the overexpressed plasimd of pc DNA4/TO-p54 nrb. The proliferation of MCF-7 cells treated with simvastatin or transfected with si RNA-p54^nrb/NC or pc DNA4/TO-p54^nrb/vector or pc DNA4/TO--p54^nrb/vector combined with simvastatin was examined by cell counting. Real-time fluorescence quantitative-PCR and western blotting were used to examine the expression levels of p54 nrb m RNA and protein in MCF-7 cells by treated with simvastatin or transfection with si RNA-p54^nrb/NC or the overexpressed plasimd of pc DNA4/TO-p54^nrb/vector and m RNA level of HMGCR. Tissue free cholesterol assay was used to test cholesterol level.Resμlt: The expression levels of p54 nrb m RNA and protein in MCF-7 cells transfected with si RNA-p54 nrb were obviously decreased（p<0.01） and the expression levels of p54 nrb m RNA and protein in MCF-7 cells transfected with pc DNA4/TO-p54 nrb were obviously increased（p<0.01）. After MCF-7 cells were treated with simvastatin, the ability of proliferation was inhibited（p<0.05） and the expression level of p54 nrb m RNA and protein were decreased（p<0.05）. When MCF-7 cells were transfected with si RNA-p54 nrb, the proliferation was inhibited（p<0.05）; when transfected with overexpressed plasimd pc DNA4/TO-p54 nrb, the proliferation of MCF-7 cells increased（p<0.05）. However, when treatment with simvastatin combined with transfection with pc DNA4/TO-p54 nrb in MCF-7 cells, p54^nrb-induced cell growth was completely compromised（p>0.05）. The expression levels of HMGCR m RNA and cholesterol level in MCF-7 cells after transfection with si RNA-p54 nrb decreased（p<0.01）.Conclusion: Simvastatin can suppress the cell proliferation by targeting p54 nrb expression in breast cancer MCF-7 cells.