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Objective To determine the acetylation level of nucleophosmin(NPM)in female breast cancer and to discuss its function through mutation of modified lysine sites.To construct positive and negative NPM mutants on its acetylated lysine sites and to express them in breast cancer cells.Methods Acetylation level and acetylated lysine sites of NPM in three breast cancer tissues and para-carcinoma tissues were detected by acetylome technology;NPM mutants were constructed by site-directed mutagenesis PCR,specific PCR products were digested by DpnI and transformed into Escherichia coli(E.coli)to obtain specific plasmids for mutants;The accuracy of mutants were verified by double restriction enzyme digestion and sequencing;The mutants were expressed in BT-549 cells by transient transfection and verified by RT-PCR method.Protein expression and acetylation level of NPM were validated by Western blotting;Function of NPM acetylation was analyzed by proteomic detection and bioinformatic analysis.Results The 27th and 32nd lysine of NPM were highly acetylated in breast cancer tissues,which were 2.76 and 2.22 times higher than those in adjacent normal tissues,respectively;The NPM mutants showed the same molecular weight as that of wild type NPM and contained expected mutation sites;Corresponding NPM mRNA levels of BT-549 cells transfected with NPM mutants were significantly increased.With the increase of wild type NPM expression level,NPM acetylation level increased,while decreased after 27th lysine underwent negative mutation.NPM acetylation can significantly change the expression levels of 101 proteins in BT-549 cells,which are enriched in regulation of cellular macromolecule biosynthesis,DNA-template transcription,RNA biosynthesis and RNA metabolism process.Conclusion NPM is highly acetylated in breast cancer and can play a key role in cellular macromolecule biosynthesis,DNA-templated transcription,RNA biosynthesis and RNA metabolism process.
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Objective To explore the protein acetylation/succinylation and histone 2AX (H2AX) expression levels in breast cancer, as well as their correlation. Methods By Western blotting and RT-PCR methods to detect the protein modification and H2AX expression levels in 11 breast cancer tissues and cells, as well as to explore the common regulation way of protein acetylation and succinylation by treatment of histone deacetylase inhibitors ; To study the relationship between H2AX expression level and protein modification level through the construction and over-expression of indicated plasmids. Results Compared with the adjacent normal tissues, there existed an increase protein acetylation/succinylation levels in breast cancer tissues, and the protein acetylation and succinylation were both regulated by histone deacetylase (HDAC) members. The H2AX mRNA and protein expression levels were increased both in breast cancer cell and tissues, its expression level and the expression and modification level of represented protein nucleophosmin 1(NPM1) showed a positive correlation. Conclusion The breast cancer possesses a characteristic of high protein acetylation/succinylation levels and high H2AX expression level, the H2AX expression level and the modification level of partial proteins in breast cancer have a positive correlation.
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Objective The mechanisms of PCDHA13 promoter methylation in breast cancer have not yet been elucidated at present. This study was to investigate the role of PCDHA13 gene promoter methylation in the development of breast cancer.Methods The methylation state of PCDHA13 gene promoter in human breast cancer tissues was detected by MassARRAY mass spectrum methylation sequencing. 100μmol/L 5-Aza was prepared with culture medium. The ZR-75-1 cells with 60% cell confluence were added to the final concentration of 5 μmol/L(low concentration group) and 10 μmol/L(high concentration group) 5-Aza, and the control group was only added culture medium. Detection of methylation status of PCDHA13 gene promoter in human breast cancer cells by bisulfite sequencing and methylation-specific PCR, and analysis of methylation status and mRNA expression of PCDHA13 gene by semi-quantitative RT-PCR. Western blot, MTT and DAPI staining were used to detect the effect of 5-Aza treatment on proliferation and apoptosis of breast cancer ZR-75-1 cells.Results The methylation degree of PCDHA13 gene promoter in the 1, 4-6, 9, 10 and 11 CpG loci in breast cancer tissues was significantly higher than that in normal breast group \[(0.2639±0.1575) vs (0.1612±0.1706), (0.2509±0.1377) vs (0.1688±0.0992), (0.4204±0.2087) vs (0.2621±0.1731), (0.3761±0.1407) vs (0.2824±0.1486), (0.3922±0.1294) vs (0.3072±0.1496)\], and the difference was statistically significant (P0.05). The expression of PCDHA13 mRNA of ZR-75-1 cells was loss in control group, but the expression of PCDHA13 mRNA was reversed after treated with 5-Aza, and the expression of PCDHA13 mRNA was significantly higher in high concentration group than that in low concentration group(P>0.05). After treated with 5-Aza for 24, 48 and 72 hours, the growth inhibition rates were lower in low concentration group than that in high concentration group (P>0.05). The morphology of the nuclei was basically normal and there was no apoptosis occurred in ZR-75-1 cells. But after treated with 5-Aza, some ZR-75-1 cells showed nuclear condensation, chromatin agglutination and heavy coloration.Conclusion This study showed that the low expression or loss of mRNA is associated with hypermethylation of the PCDHA13 gene promoter in breast carcinoma. The PCHDA13 gene expression can be reversed by 5-Aza in ZR-75-1 cells. The re-expression of PCHAD13 not only inhibit the proliferation of cells, but also promote apoptosis. Abnormal methylation of PCDHA13 may become a potential tumor marker for breast cancer.
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The intra-arterial thrombolysis is one of the most effective approaches in salvaging brain ischemic penumbra and treating acute cerebral infarction.However,because of the limita- tions of short time windows for treatment,intracerebral hemorrhage after thrombolysis and reocclusion,more clinical studies are needed before this technique is widely developed and applied.
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<p><b>OBJECTIVE</b>To evaluate the effects of community intervention on risk factors of cerebrovascular disease.</p><p><b>METHODS</b>With cluster sampling, 2,586 and 2,723 women and men aged 35 to 74 were selected randomly as intervention cohort and control cohort, respectively, from about 300 thousand community population in Beijing, Shanghai and Changsha, during 1997 to 2000. Their blood pressure, levels of blood lipid, body mass index (BMI) were measured and status of smoking and alcohol consumption were surveyed as indices of evaluation. Health education for smoking quit, alcohol consumption restriction and reinforced treatment for diabetic and hypertensive patients were implemented in the intervention communities. And, 2,544 and 2,533 persons in the two cohorts responded three years after intervention, and then all the indices mentioned above were measure again for them.</p><p><b>RESULTS</b>Average reduction in systolic blood pressure by 3.21 mm Hg and serum level of total cholesterol by 0.58 mmol/L was achieved in intervention cohort after intervention, but with 0.48 mmol/L increase in triglyceride. Average blood levels of both high density lipoprotein-cholesterol (HDL-C) and glucose increased in the two cohorts after intervention, but with 0.30 mmol/L net increase of blood glucose in control cohort. There was no significant difference in average increase of HDL-C between the two cohorts. BMI increased by 0.56 in control cohort, and no significant change in intervention cohort. Proportion of smoking decreased by 5.4% in men and 2.4% in women of intervention cohort, and no change in control cohort.</p><p><b>CONCLUSION</b>Level of risk factors for cerebrovascular disease reduced significantly with community intervention.</p>