Nucleophosmin acetylation and construction and expression of its modified sites mutants in breast cancer / 解剖学报

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.

Pathological Changes and Cause of Death Associated with the Global Novel Coronavirus Disease (COVID-19).

The coronavirus disease 2019 (COVID-19) has been a global epidemic for more than three years, causing more than 6.9 million deaths. COVID-19 has the clinical characteristics of strong infectivity and long incubation period, and can cause multi-system damage, mainly lung damage, clinical symptoms of acute respiratory distress syndrome (ARDS) and systemic multiple organ damage. The SARS-CoV-2 virus is still constantly mutating. At present, there is no global consensus on the pathological changes of COVID-19 associated deaths and even no consensus on the criteria for determining the cause of death. The investigation of the basic pathological changes and progression of the disease is helpful to guide the clinical treatment and the development of therapeutic drugs. This paper reviews the autopsy reports and related literature published worldwide from February 2020 to June 2023, with a clear number of autopsy cases and corresponding pathological changes of vital organs as the inclusion criteria. A total of 1 111 autopsy cases from 65 papers in 18 countries are included. Pathological manifestations and causes of death are classified and statistically analyzed, common pathological changes of COVID-19 are summarized, and analytical conclusions are drawn, suggesting that COVID-19 infection can cause life-threatening pathological changes in vital organs. On the basis of different health levels of infected groups, the direct cause of death is mainly severe lung damage and secondary systemic multiple organ failure.

High protein acetylation/succinylation levels and their correlation with high histone 2AX expression level in breast cancer / 解剖学报

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.

Study on promoter methylation of PCDHA13 gene and breast cancer development / 医学研究生学报

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.