| Objective:To study the correlations of SIRT3,IDH2,FAS expressions and their clinical significance in breast cancer tissues and cells.Methods:Forty-five postoperative tissue specimens of primary breast cancer and matched normal breast tissue specimens at affiliated hospital of Guilin Medical University were used to examine the expression levels of SIRT3,IDH2 and FAS by immunohistochemistry Envision method.Then the expressions and relationships of SIRT3,IDH2 and FAS were determined by semi quantitative integration method and chi-square tests.And Pearson method was used to analyze the correlations among three proteins expressions.Quantitative real-time PCR and western blot were used to measure the expression levels of SIRT3,IDH2 and FAS m RNAs and proteins in breast cancer cells and normal breast epithelial cells.ELISA was used to examine SIRT3 protein expression,IDH2 activity,FAS activity and ACC activity in breast cancer cells and normal breast epithelial cells.The correlations among SIRT3 protein expression,IDH2 activity and FAS activity in breast cancer cells were analyzed by Pearson method.The interaction between SIRT3 and IDH2 in breast cancer cells was performed by immune-co-precipitation experiment.Immunoprecipitation combined with LC-MS/MS method was directly used to prove the interaction.And the co-localization of SIRT3 and IDH2 in breast cancer cells was detected by double-color immunofluorescence combined with laser confocal microscopy.Results:The positive rates of SIRT3,IDH2 and FAS in breast cancer tissues were significantly higher than those in normal breast tissues[75.56%(34/45)vs42.22%(19/45),χ~2=10.326,P=0.001;84.44%(38/45)vs 26.67%(12/45),χ~2=30.420,P<0.001;86.67%(39/45)vs 17.78%(8/45),χ~2=42.796,P<0.001].The ratios of SIRT3,IDH2 and FAS high expression in breast cancer tissues were also significantly higher than those in matched normal breast tissues[55.56%(25/45)vs 20.00%(9/45),χ~2=12.101,P=0.001;66.67%(30/45)vs8.89%(4/45),χ~2=31.954,P<0.001;71.11%(32/45)vs 6.67%(3/45),χ~2=39.319,P<0.001].Pearson correlation analysis showed that SIRT3 expression intensity was positively correlated with that of DIH2(r~2=0.7352,P=0.05)and FAS(r~2=0.8108,P=0.05).And IDH2 expression intensity was positively correlated with the that of FAS in breast cancer tissues(r~2=0.6530,P=0.05).QRT-PCR and western blot results showed that the expressions of SIRT3,IDH2 and FAS m RNAs and proteins in breast cancer cells were higher than that in normal breast epithelial cells.The results of ELISA and related quantitative reagent kit assays suggested that SIRT3 expression,IDH2 activity and FAS activity were all increased,and the contents of citric acid in mitochondria and cytoplasm,and intracellular free fatty acid were all increased,compared with normal breast epithelial cells.Pearson correlation analysis revealed that SIRT3 protein expression was positively correlated with IDH2 activity(r~2=0.7332,P=0.0295),FAS activity(r~2=0.7270,P=0.0310),and IDH2 activity was positively correlated with FAS activity(r~2=0.9256,P=0.0021).Immune-co-precipitation and LC-MS/MS assay suggested that SIRT3 and IDH2 could bind to each other in breast cancer cells.The interactive combination of these two proteins were clearly seen by double-color immunofluorescence and laser confocal images.And the place where they interact was in the cytoplasm.Conclusion:High expressions of SIRT3,IDH2 and FAS in breast cancer tissues and up-regulation of SIRT3 protein,increase of IDH2 activity and FAS activity in breast cancer cells may jointly promote the occurrence and development of breast cancer.SIRT3 and IDH2 may interact in the cytoplasm of breast cancer cells.The modificatory effect of SIRT3 on IDH2 may be the key to regulate the metabolism of citric acid,which affects FAS to synthesize fatty acids.It will be expected to provide theoretical basis and molecular targets for the treatment of breast cancer. |
