Vascular Transcriptome Profiling Reveals Aging-Related Genes in Angiotensin Ⅱ-Induced Hypertensive Mouse Aortas / 中国医学科学杂志(英文版)

Objective Angiotensin Ⅱ (Ang Ⅱ)-induced vascular damage is a major risk of hypertension. However, the underlying molecular mechanism of AngⅡ-induced vascular damage is still unclear. In this study, we explored the novel mechanism associated with Ang II-induced hypertension. Methods We treated 8- to 12-week-old C57BL/6J male mice with saline and Ang Ⅱ(0.72 mg/kg·d) for 28 days, respectively. Then the RNA of the media from the collected mice aortas was extracted for transcriptome sequencing. Principal component analysis was applied to show a clear separation of different samples and the distribution of differentially expressed genes was manifested by Volcano plot. Functional annotations including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed to reveal the molecular mechanism of Ang Ⅱ-induced hypertension. Finally, the differentially expressed genes were validated by using quantitative real-time PCR. Results The result revealed that a total of 773 genes, including 599 up-regulated genes and 174 down-regulated genes, were differentially expressed in the aorta of Ang Ⅱ-induced hypertension mice model. Functional analysis of differentially expressed genes manifested that various cellular processes may be involved in the Ang Ⅱ-induced hypertension, including some pathways associated with hypertension such as extracellular matrix, inflammation and immune response. Interestingly, we also found that the differentially expressed genes were enriched in vascular aging pathway, and further validated that the expression levels of insulin-like growth factor 1 and adiponectin were significantly increased (P<0.05). Conclusion We identify that vascular aging is involved in Ang Ⅱ-induced hypertension, and insulin-like growth factor 1 and adiponectin may be important candidate genes leading to vascular aging.

Up-regulation of Fas ligand expression by sirtuin 1 in both flow-restricted vessels and serum-stimulated vascular smooth muscle cells / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms.</p><p><b>METHODS</b>SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell (VSMC) lysate. Smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) C57BL/6 mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (ligation groups) or the ligation-excluded operation (sham groups). The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 (SIRT1H363Y), and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis.</p><p><b>RESULTS</b>SIRT1 was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation (P<0.001) and VSMCs treated with serum (P<0.05 at the transcriptional level, P<0.001 at the protein level). No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL (P<0.001). The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (P<0.001), while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 (P<0.001).</p><p><b>CONCLUSIONS</b>Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT1.</p>

Lysine-specific demethylase 1 represses THP-1 monocyte-to-macrophage differentiation / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the role of lysine-specific demethylase 1 (LSD1) in the process of THP-1 monocyte-to-macrophage differentiation.</p><p><b>METHODS</b>Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting were performed to analyze the expression of LSD1 and interleukin-6 (IL-6) in THP-1 monocytes and THP-1-derived macrophages. Chromatin immunoprecipitation (ChIP) assay was applied to detect the occupancy of LSD1 and H3K4 methylation at IL-6 promoter during THP-1 monocyte-to-macrophage differentiation. IL-6 mRNA level and H3K4 methylation at IL-6 promoter were analyzed using qRT-PCR and ChIP assay in LSD1-knockdown THP-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 0, 4, 8, 12, and 24 hours. Fluorescence activated flow cytometry was performed to reveal the percentage of macrophages differentiated from THP-1 monocytes.</p><p><b>RESULTS</b>The expression of LSD1 reduced during THP-1 monocyte-to-macrophage differentiation (P<0.01). LSD1 occupancy decreased and H3K4 methylation increased at IL-6 promoter during the differentiation. With knockdown of LSD1, H3K4 methylation at IL-6 promoter was found increased after TPA treatment at different times points (all P<0.05, except 24 hours). The percentage of macrophages increased significantly in the THP-1 cells with LSD1 knockdown (P<0.05).</p><p><b>CONCLUSIONS</b>LSD1 is repressed during the monocyte-to-macrophage differentiation of THP-1 cells. Suppression of LSD1-mediated H3K4 demethylation may be required for THP-1 monocyte-to-macrophage differentiation.</p>

Effect of LMO2 overexpression in hemangioblast on its proliferation and hematopoietic differentiation / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate function of the Lim-only protein(LMO2) in hemangioblast generated from murine embryonic stem cells differentiation to hematopoietic cells.</p><p><b>METHODS</b>The hemangioblast-specific expression vector with lmo2 or green fluorescence protein gene was constructed, respectively. The murine embryonic stem cells were transfected by the hemangioblast-specific expression vectors. The neomycin-resistance ES cell clones were obtained after having been screened by G418. The cell clones were spontaneously differentiated into embryo bodies(EB) containing hemangioblast.Expression of the hematopoietic genes was investigated by real-time reverse transcription-ploymerase chain reaction during EB differentiation.For the EB cells, blast-cloning forming cells analysis and blood-colony forming unit analysis were then performed, respectively. The numbers of the blasts were counted during hematopoietic differentiation.</p><p><b>RESULTS</b>The hemangioblast-specific expression vector with lmo2 or green fluorescence protein was transfected into ES cells.The neomycin-resistance ES cells generated EBs from 2.5 days to 10 days.Real time reverse transcription-ploymerase chain reaction analysis indicated that overexpression of lmo2 increased the expression of hematopoietic genes(gata1, tal1, Β-h1, and Β-major globin) during EB formation.Blast-cloning forming cells analysis showed that the numbers of the blasts generated by ES/lmo2 was 2-or 3-fold than those in the controls.The total numbers of the blood-colony forming unit or the numbers of the erythrocyte colony-forming unit generated by ES/lmo2 were 2.5 times or 3 times, respectively, when compared with the controls.</p><p><b>CONCLUSION</b>LMO2 enhances the proliferation and differentiation of hemangioblasts.</p>

Prokaryotic expression and purification of mitochondrial transcription complex proteins / 中国医学科学院学报

<p><b>OBJECTIVE</b>To obtain human mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M), and mitochondrial transcription factor B2 (TFB2M) that were expressed efficiently in E. coli BE21 and to purify the target proteins.</p><p><b>METHODS</b>TFAM, TFB1M, and TFB2M segments were designed and synthesized. After having been sequenced, the reconstructed expression vectors were constructed by enzyme digestion and by cloning into an expression vector pET42a. Then the reconstructed vectors were transformed into E. coli BL21. Recombinant glutathione S transferase (GST) fusion proteins were expressed via the induction of IsoPropyl beta-D-ThioGalactoside (IPTG) and purified by glutathione Sepharose 4B.</p><p><b>RESULTS</b>The expression plasmids of pET42a-TFAM, pET42a-TFB1M, and pET42a-TFB1M were successfully constructed. The sequences of the cloned gene segments were identical with GenBank reported. The protein bands with relative molecular masses of 56 000, 67 000, and 69 000 appeared on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after the expressed GST-TFAM, GST-TFB1M, and GST-TFB2M fusion proteins were separated by SDS-PAGE. The expressed fusion proteins were purified to high purity.</p><p><b>CONCLUSION</b>The recombinant plasmids pET42a-TFAM, pET42a-TFB1M, and pET42a-TFB2M were successfully constructed, and the GST-fused target proteins were prepared.</p>

Inhibition of SIRT1 increases EZH2 protein level and enhances the repression of EZH2 on target gene expression / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To study the regulatory rolesof SIRT1 on EZH2 expression and the further effects on EZH 2’ s repression of target gene expression.</p><p><b>METHODS</b>The stable SIRT1 RNAi and Control RNAi HeLa cells were established by infection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation(ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells.</p><p><b>RESULTS</b>Western blot results showed that EZH2 protein level increased upon SIRT1 depletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter regionof EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases.</p><p><b>CONCLUSIONS</b>Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expression.</p>

Gaussia luciferase reporter assay for assessment of gene delivery systems in vivo / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To develop an alternative method for assessment of gene delivery systems in vivo.</p><p><b>METHODS</b>Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase (Gluc) expression cassette. After implantation of these cells into recipient mice, the expression of Gluc was detected in whole blood or plasma collected.</p><p><b>RESULTS</b>As little as 10 muL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer. And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.</p><p><b>CONCLUSIONS</b>Gluc may be useful as an in vivo reporter for gene therapy researches, and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.</p>

Change of the chromosome conformation of human alpha-globin gene locus in transgenic mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish chromosome conformation capture (3C) strategy and to use this method for exploring the effect of chromosome conformation on human alpha-globin gene expression in the human alpha-globin transgenic mouse.</p><p><b>METHODS</b>Homozygous human alpha-globin transgenic male mouse was crossed with KM female mouse. The 14.5-day post-coitum (dpc) embryos were used for the isolation of fetal liver and fetal brain cells. Homogeneous single-cell suspension was treated with formaldehyde to crosslink the chromatin conformation in the nuclear. The cross-linked chromatin compound was digested with Nco I and then ligated with T4 DNA ligase. The ligated compound was reversely cross-linked and then the ligated genomic DNA was purified for PCR analysis. The primers were designed along the two sides of cut and ligated sites. Semi-quantitative PCR was used to analyze the chromosome conformation of the whole human alpha-globin gene locus in fetal liver and fetal brain cells.</p><p><b>RESULTS</b>When HS40 fragment was used as the fixed fragment, in fetal brain cells, the ligation frequencies of HS40 fragment with other fragments were decreased as the linear distances to HS40 fragment were increasing; while in fetal liver cells, two active genes (alpha1 and alpha2) fragments showed higher ligation frequencies with HS40 fragment than other fragments. However, the fragment containing an inactive gene (xi) displayed the comparable low ligation frequency as that in fetal brain. When alpha2 fragment was used as the fixed fragment, similarly, in fetal brain cells the ligation frequencies of alpha2 fragment with other ones were decreased as the linear distances increasing; when in fetal liver cells, it showed higher ligation frequencies with two upstream regulatory elements (HS 40 and 33). However, it showed a little bit lower ligation frequency with another two upstream regulatory elements (HS10 and 8) than those in fetal brain.</p><p><b>CONCLUSION</b>In fetal liver cells, the distant regulatory elements are in close proximity to the downstream of the expressed globin genes through looping out, the interval region; however, in fetal brain, they were not in vicinity to the expressed globin genes.</p>

Research progression of deacetylase (SIRT1) / 中国医学科学院学报

The silent information regulator protein 2 (Sir2) and its homologues play an important role in the regulation of cellular physiological processes such as survival, apoptosis, and aging. SIRT1, the mammalian Sir 2 homologue, has been shown to deacetylate a wide range of non-histone substrates and histone substrates. It has been constantly reported that SIRT1 may be associated with the occurrence of metabolic syndrome, genomic homeostasis, tumors, and neurodegenerative diseases. Calorie restriction may mitigate many major diseases in rodent models by SIRT1-mediated deacetylase activity and prolong the life expectancies in these animals. Therefore, SIRT1 may be emphasized as a new therapy target for many different diseases.

Establishment of BAC mediated transgenic mice containing 97 kb beta-globin gene cluster / 中国医学科学院学报

<p><b>OBJECTIVE</b>To delete IL-11 receptor alpha chain gene from the Bacterial Artificial Chromosome (BAC) chimeric DNA by RecA protein mediated homologous recombination method and establish the transgenic mice model containing whole beta-globin gene cluster.</p><p><b>METHODS</b>Two 500 bp homologous sequences (A and B) located at the upstream and downstream of IL-11 receptor alpha chain gene respectively were cloned into the Hind III and Xba I sites of pBV vector, then the 1 kb A + B fragment was recovered from the building vector and inserted into the Sal I site of the shuttle vector pSV-RecA. After transforming the shuttle vector into the competent DH10B E. Coli containing BAC DNA, the IL-11 receptor alpha chain gene was finally deleted from the BAC DNA through chloramphenicol positive selection and fusaic acid negative selection. The new BAC clone was characterized by Pulse Field Gel Electrophoresis (PFGE). Then, we microinjected the linearized and purified BAC DNA into the mouse fertilized eggs and prepared the transgenic mice.</p><p><b>RESULTS</b>By RecA protein mediated homologous recombination method, we deleted the IL-11 receptor alpha chain gene from the BAC DNA containing the complete beta-globin Gene Cluster and established 3 respective transgenic mice lines.</p><p><b>CONCLUSION</b>Human beta-globin gene cluster in the transgenic mice mediated by new BAC expresses in a correct mode and level as compared with previous transgenic mice.</p>