[Show abstract][Hide abstract] ABSTRACT: Aims:
Posttranslational modification, such as phosphorylation, plays an essential role in regulating activation of endothelial NO synthase (eNOS). In the present study, we aim to determine whether eNOS could be phosphorylated and regulated by a novel serine/threonine-protein kinase Pim1 in vascular endothelial cells (ECs).
Methods and results:
Using immunoprecipitation and protein kinase assays, we demonstrated that Pim1 specifically interacts with eNOS, which leads to a marked phosphorylation of eNOS at Ser-633 and increased production of nitric oxide (NO). Intriguingly, in response to VEGF stimulation, eNOS phosphorylation at Ser-633 exhibits two distinct phases: transient phosphorylation occurring between 0 and 60 min and sustained phosphorylation occurring between 2 and 24 h, which are mediated by the protein kinase A (PKA) and Pim1, respectively. Inhibiting Pim1 by either pharmacological inhibitor SMI-4a or the dominant negative form of Pim1 markedly attenuates VEGF-induced tube formation, while Pim1 overexpression significantly increases EC tube formation and migration in a NO dependent manner. Importantly, Pim1 expression and eNOS phosphorylation at Ser-633 were substantially decreased in high glucose treated-ECs and in the aorta of db/db diabetic mice. Increased Pim1 expression ameliorates impaired vascular angiogenesis in diabetic mice, as determined by an ex vivo aortic ring assay.
Our findings demonstrate Pim1 as a novel kinase that is responsible for the phosphorylation of eNOS at Ser-633 and enhances endothelial cell sprouting of aortic rings from diabetic mice, suggesting that Pim1 could potentially serve as a novel therapeutic target for revascularization strategies.
Full-text · Article · Nov 2015 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: Previous study showed that mitochondrial ND6 (mitND6) gene missense mutation resulted in NADH dehydrogenase deficiency and was associated with tumor metastasis in several mouse tumor cell lines. In the present study, we investigated the possible role of mitND6 gene nonsense and missense mutations in the metastasis of human lung adenocarcinoma.
The presence of mitND6 gene mutations was screened by DNA sequencing of tumor tissues from 87 primary lung adenocarcinoma patients and the correlation of the mutations with the clinical features was analyzed. In addition, we constructed cytoplasmic hybrid cells with denucleared primary lung adenocarcinoma cell as the mitochondria donor and mitochondria depleted lung adenocarcinoma A549 cell as the nuclear donor. Using these cells, we studied the effects of mitND6 gene nonsense and missense mutations on cell migration and invasion through wounding healing and matrigel-coated transwell assay. The effects of mitND6 gene mutations on NADH dehydrogenase activity and ROS production were analyzed by spectrophotometry and flow cytometry.
mitND6 gene nonsense and missense mutations were detected in 11 of 87 lung adenocarcinoma specimens and was correlated with the clinical features including age, pathological grade, tumor stage, lymph node metastasis and survival rate. Moreover, A549 cell containing mitND6 gene nonsense and missense mutation exhibited significantly lower activity of NADH dehydrogenase, higher level of ROS, higher capacity of cell migration and invasion, and higher pAKT and pERK1/ERK2 expression level than cells with the wild type mitND6 gene. In addition, NADH dehydrogenase inhibitor rotenone was found to significantly promote the migration and invasion of A549 cells.
Our data suggest that mitND6 gene nonsense and missense mutation might promote cell migration and invasion in lung adenocarcinoma, probably by NADH dehydrogenase deficiency induced over-production of ROS.
[Show abstract][Hide abstract] ABSTRACT: Background:
Here we investigated Brahma-related gene 1 (BRG1) expression in aortic smooth muscle cells (SMCs) and its role in the regulation of the pathological changes in aortic SMCs of thoracic arotic dissection (TAD).
BRG1, matrix metalloproteinase 2 (MMP2), and MMP9 mRNA and protein expression in human aortic specimens were examined by qPCR and western blot, respectively. The percentage of apoptotic and contractile SMCs in aortic specimens were determined by TUNEL assay and alpha-SMA immunohistochemical staining, respectively. The role of BRG1 in MMP2 and MMP9 expression, cell apoptosis, and phenotype transition in aortic SMCs were investigated using a human aortic SMC line via adenovirus mediated gene transfer. MMPs mRNA and protein levels were analyzed by qPCR and western blot, respectively. The percentage of apoptotic and contractile cells were determined through flow cytometry analysis.
The expression level of BRG1 in the aortic walls (adventitia-removed) was significantly higher in the TAD than the normal group. BRG1 expression was positively correlated to expression of MMP2 and MMP9 and SMC apoptosis, but was negatively correlated to the percentage of contractile aortic SMCs in TAD specimens. In human aortic SMC line, BRG1 transfection led to significant upregulation of MMP2 and MMP9 expression and a concomitant increase in SMC apoptosis as well as a decrease in the percentage of contractile phenotype of cells.
BRG1 is significantly upregulated in the aortic SMCs of TAD, and its overexpression might promote the development of TAD by increasing MMP2 and MMP9 expression, inducing SMC apoptosis and the transition from contractile to synthetic phenotype.
Preview · Article · Oct 2014 · BMC Cardiovascular Disorders
[Show abstract][Hide abstract] ABSTRACT: Background:
Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Hippo kinase from Drosophila and it is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart is not known.
Methods and results:
To identify novel cardiac proteins that may regulate Mst1 activity in the heart under pathophysiological conditions, a yeast two-hybrid screening of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait was performed. As a result, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) was identified as an Mst1-interacting protein. The interaction of PCMT1 with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiomyocytes, in which PCMT1 interacted with the kinase domain of Mst1, but not with its C-terminal regulatory domain. Overexpression of PCMT1 did not affect the Mst1 expression, but significantly attenuated the Mst1 activation and its apoptotic effects in response to the hypoxia/reoxygenation induced injury in cardiomyocytes. Indeed, upregulation of PCMT1 by CGP3466B, a compound related to the anti-Parkinson's drug R-(-)-deprenyl with potent antiapoptotic effects, inhibited the hypoxia/reoxygenation induced Mst1 activation and cardiomyocyte apoptosis.
These findings implicate PCMT1 as a novel inhibitor of Mst1 activation in cardiomyocytes and suggest that targeting PCMT1 may prevent myocardial apoptosis through inhibition of Mst1.
No preview · Article · May 2013 · International journal of cardiology
[Show abstract][Hide abstract] ABSTRACT: Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.
[Show abstract][Hide abstract] ABSTRACT: Recent data from human and animal studies have shown an upregulated expression of advanced glycosylation end product-specific receptor (RAGE) in human atherosclerotic plaques 1 and in retina, messangial, and aortic vessels, suggesting an important role of RAGE in the pathogenesis of atherothrombotic diseases. In the past few years, the relationship between RAGE polymorphisms (-429T/C, -374T/A, and G82S) and coronary heart disease (CHD) has been reported in various ethnic groups; however, these studies have yielded contradictory results.
PubMed, ISI web of science, EMBASE and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. Data were abstracted independently by two reviewers. A meta-analysis was performed to examine the association between RAGE polymorphisms and susceptibility to CHD. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated.
A total of 17 studies including 4343 patients and 5402 controls were involved in this meta-analysis. Overall, no significant results were observed for -429T/C (OR = 1.01, 95% CI: 0.92-1.12, P = 0.78), -374T/A (OR = 1.11, 95% CI: 0.98-1.26, P = 0.09) and G82S (OR = 1.12, 95% CI: 0.86-1.45, P = 0.41) polymorphism. In the stratified analyses according to ethnicity, sample size, CHD endpoint and Hardy-Weinberg status, no evidence of any gene-disease association was obtained.
This meta-analysis demonstrates that there is no association between the RAGE -429T/C, -374T/A and G82S polymorphisms and CHD.
[Show abstract][Hide abstract] ABSTRACT: Pericardial fibrocalcification (PF) is a prominent feature of human pericardial pathology, including constrictive pericarditis and, to a lesser extent, degenerated autologous pericardial substitutes. However, the role of pericardial interstitial cells (PICs) in the pathogenesis of PF has yet to be established. Using a combination of histology and immunohistochemistry, we showed that the critical cellular event in PF in situ was the transdifferentiation of PICs into myofibroblasts/osteoblasts and that the percentage of myofibroblasts/osteoblasts correlated positively with the severity of PF. In vitro studies demonstrated that PICs, similar to mesenchymal stem cells, had the potential to differentiate along adipogenic, osteogenic, chondrogenic or myogenic lineages. However, PICs exhibited a more limited self-renewal capacity and a lower expression of Oct4 (POU5F1) and Kruppel-like transcription factor Klf4, underwent earlier senescence and spontaneously transdifferentiated into myofibroblasts/osteoblasts. Quantitative-real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) confirmed that the mRNA levels of α-smooth muscle actin (α-SMA), alkaline phosphatase (ALP), core-binding factor α1/runt-related transcription factor2 (Cbfa1/Runx2), transforming growth factor (TGF)-β1 and bone morphogenetic protein (BMP)-2 were upregulated as the passage number increased. The mRNA level of platelet-derived growth factor (PDGF)-AA was also significantly upregulated with higher levels at passage 3. Ectopic expression of Oct4 and Klf4 enhanced the colony formation of PICs and selectively impaired induction of genes involved in transdifferentiation into myofibroblasts/osteoblasts (α-SMA, ALP, Cbfa1/Runx2, PDGF-AA and BMP-2). These data, while offering new insights into the biology of PICs, reinforce the central role of these cells in cell-mediated PF and may assist in future strategies to treat fibrocalcific pericardial diseases.
No preview · Article · Dec 2012 · Journal of Molecular and Cellular Cardiology
[Show abstract][Hide abstract] ABSTRACT: Lung cancer is a major worldwide health problem. The aim of this study is to establish a novel Chinese human lung adenocarcinoma cell line and examine its biological characteristics.
Lung adenocarcinoma specimens were freshly resected during surgery. The tissues were incubated in vitro and the cell line was named Ch-Huang-1. The biological characteristics of the cells were investigated by light microscopy, chromosome analysis, and transplantation experiment.
Light microscopy revealed that cells from the primary tumor, Ch-Huang-1 cell line, and transplanted tumor possessed the characteristics of a malignant glandular epithelial tumor. The cell growth curve, doubling time, and mitotic index were also observed in vitro. Nuclear chromosome analysis revealed that the tumor was a subtriploid with a mode of 35-44 per cell. Tumor nodes were observed under the skin of nude mice by heterogenic transplantation.
The characteristics of the established cell line suggest that it is a newly established human adenocarcinoma cell line.
No preview · Article · May 2012 · Zhongguo fei ai za zhi = Chinese journal of lung cancer
[Show abstract][Hide abstract] ABSTRACT: It is well-known that angiopoietin-2 (Ang-2) plays an important role in the formation of the blood vascular system. Angiopoietin is involved in many diseases about angiogenesis such as tumor, so may have great prospects for the treatment of these diseases. The aim of this study is to evaluate the influence of inhibiting Ang-2 via adeno-associated virus induced RNA interference (RNAi) on the biological characteristics of bronchogenic adenocarcinoma.
AAV-Ang-2shRNA driven by H1 promoter was constructed to transfect A549 cell line. Normal and AAV-Null cell line were utilized in the control groups. The influence of RNAi on Ang-2 expression as well as the growth rate, tumorigenic efficiency, proliferation rate, apoptosis, and microvessel density of A549 cell line were analyzed.
In vitro experiment indicated that the Ang-2 expression level (P<0.001) and growth rate (P<0.001) of A549 cell line 48 h transfected with AAV-Ang-2shRNA were significantly lower than those in the normal and AAV-Null cell lines. Cell cycle analysis showed the proliferation index (PI) of normal, AAV-Null, and AAV-Ang-2shRNA transfected A549 cell line were 0.51± 0.43, 0.48 ± 0.29, and 0.26 ± 0.31, respectively, which indicated the PI of AAV-Ang-2shRNA transfected cell line was significantly lower, compared with the normal and AAV-Null cell lines. In vivo experiment exhibited that AAV-Ang-2shRNA transfected cell line possessed a lower mass and volume of tumor relative to two control groups. In addition, the apoptosis index (AI) of AAV-Ang-2shRNA transfected, normal, and AAV-Null cell lines were (5.98 ± 3.11)%, (7.51 ± 4.42)% and (17.06 ± 7.43)% respectively, which manifested that AAV-Ang-2shRNA transfected cell line possessed a higher AI (P=0.005, P=0.007). A lower percentage of PCNA-positive cell was observed in AAV-Ang-2shRNA transfected cell line (92.75 ± 9.7)% as well, compared with the normal (85.8 ± 11.8)% and AAV-Null (69.8 ± 16.5)% cell lines.
AAV-mediated expression of shRNA significantly reduces concentration of Ang-2 in A549 cell line, lowers proliferation and growth rate and induce .apoptosis of A549 cell line.
No preview · Article · Jul 2011 · Zhongguo fei ai za zhi = Chinese journal of lung cancer
[Show abstract][Hide abstract] ABSTRACT: The human paraoxonase (PON) is calcium dependent HDL associated ester hydrolase which has attracted considerable attention as a candidate gene for coronary heart disease based on its enzyme function as a key factor in lipoprotein catabolism pathways. Many studies have examined the association between polymorphisms in the PON gene and risk of coronary heart disease (CHD), but the results have been inconsistent.
We conducted a meta-analysis of 88 studies on 4 PON polymorphisms [Q192R, L55M, and T(-107)C in the PON1 and the S311C in the PON2] published before August 2010, including a total of 24,702 CHD cases and 38,232 controls. We also systematically explored potential sources of heterogeneity.
In a combined analysis, the summary per-allele odds ratio for CHD of the 192R was 1.11 (95% CI: 1.05-1.17). However, when the analyses were restricted to 10 larger studies (n>500 cases), the summary per-allele odds ratio was 0.96 (95% CI: 0.90-1.02). Our analyses detected a possibility of publication bias with an overestimate of the true association by smaller studies. A meta-analysis of studies on the 55M, (-107)T, and 311C variant showed no significant overall association with CHD, yielding a per-allele odds ratio of 0.94 (95% CI: 0.88-1.00), 1.02 (95% CI: 0.91-1.15) and 1.02 (95% CI: 0.90-1.16) respectively.
This meta-analysis suggested an overall weak association between the R192 polymorphism and CHD risk.
[Show abstract][Hide abstract] ABSTRACT: T-cell tolerance is an important mechanism for tumor escape, but the molecular pathways involved in T-cell tolerance remain poorly understood. It remains unknown whether the inhibitory immunoreceptor programmed death-1 (PD-1) plays a role in conditions of human non-small cell lung cancer (NSCLC). In this study, we detected PD-1 expression on CD8+ T cells from healthy control peripheral blood mononuclear cells (PBMCs) and the PBMCs of NSCLC patients as well as NSCLC tissues. Results showed that tumor-infiltrating CD8+ T cells had increased PD-1 expression and impaired immune function, including reducing cytokine production capability and impairing capacity to proliferate. Blockade of the PD-1/PD-L1 pathway by the PD-L1-specific antibody partially restored cytokine production and cell proliferation. These data provide direct evidence that the PD-1/PD-L1 pathway is involved in CD8+ T-cell dysfunction in NSCLC patients. Moreover, blocking this pathway provides a potential therapy target in lung cancer.
[Show abstract][Hide abstract] ABSTRACT: To investigate genetic mechanisms of hepatocarcinogenesis and identify potential anticancer targets in hepatocellular carcinoma (HCC), we analyzed microarray gene expression profiles between 33 HCCs and their corresponding noncancerous liver tissues. Functional analysis of differentially-expressed genes in HCC indicated that cell cycle dysregulation plays an important role in hepatocarcinogenesis. Based on 14 differentially-expressed genes involved in cell cycle in HCC, we applied Structural Equation Modeling (SEM) to establish a potential genetic network which could assist understanding of HCC molecular mechanisms. siRNA-mediated knock-down of two significantly up-regulated genes, minichromosome maintenance protein 2 (MCM2) and cyclin B1 (CCNB1), in HCC cells (SMMC-7721 and QGY-7703) induced G2/M-phase arrest, apoptosis and antiproliferation in HCC. Some up-regulated cell cycle-related genes in HCC were down-regulated following specific depletion of MCM2 or/and CCNB1 in HCC cells, which might well validate and complement the reconstructed cell cycle network. This study may contribute to further disclose hepatocarcinogenesis mechanism through systematically analyzed the HCC-related-cell-cycle pathway. This study also shows that MCM2 and CCNB1 could be promising prognostic and therapeutic targets for HCC.
No preview · Article · Jun 2010 · Frontiers in bioscience (Scholar edition)
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to identify and validate novel prognostic biomarkers in human hepatocellular carcinoma (HCC). We analyzed gene expression profiles not only between 33 HCCs and their corresponding noncancerous liver tissues, but also between 25 HCCs and pooled normal liver tissues using cDNA microarrays containing 12800 genes. Functional analysis of differentially expressed genes involved in HCC carcinogenesis and tumor progression revealed that up-regulated and down-regulated genes are mainly associated with cell cycle and immune response, respectively. We detected two regions of cytogenetic changes only in poorly-differentiated HCCs using the expression data. We identified a 9-gene expression signature, which was able to predict differentiation degree and survival of HCC samples. Among the 9 most discriminatory genes, minichromosome maintenance protein 2 (MCM2), a significantly up-regulated gene involved in cell cycle pathway, was selected for further analysis. Overexpression of MCM2 protein related to poor-differentiation in HCC was validated using tissue microarray-based immunohistochemistry containing 96 HCCs. Our studies show that the 9-gene expression signature may serve as promising prognostic biomarkers involved in hepatocarcinogenesis and tumor progression.
No preview · Article · Jun 2010 · Frontiers in bioscience (Elite edition)
[Show abstract][Hide abstract] ABSTRACT: To clarify the role of glutathione S-transferases (GSTs; GSTM1 and GSTT1) status in susceptibility to coronary heart disease (CHD), a meta-analysis of published studies was performed. A total of 19 studies including 8020 cases and 11 501 controls were included in this meta-analysis. In a combined analysis, the relative risks for CHD of the GSTM1 null and GSTT1 null polymorphisms were 1.47 [95% confidence interval (CI): 1.08-2.01] and 1.26 (95% CI: 0.90-1.75), respectively. Three potential sources of heterogeneity including ethnicity, source of control and sample size of study were also assessed. However, no significant association was found in stratified analyses. By pooling data from eight studies (2909 cases and 3745 controls) that considered combinations of GSTT1 and GSTM1 genotypes, a statistically significant increased risk for CHD [odds ratio (OR = 2.38, 95% CI: 1.03-5.48)] was detected for individuals with combined deletion mutations in both genes compared with positive genotypes. Results from the meta-analysis of five studies on GSTs stratified according to smoking status showed an increased risk for individuals with null genotype (OR = 2.21, 95% CI: 1.24-3.92 for GSTM1 and OR = 3.29, 95% CI: 1.49-7.26 for GSTT1) versus non-null genotypes. This meta-analysis suggests that the GSTM1 null genotype may slightly increase the risk of CHD and that interaction between unfavourable GSTs genotypes may exist.
[Show abstract][Hide abstract] ABSTRACT: Objective
The aim of the research was to study the function of human yrdC gene in the gastric carcinoma cells.
Human yrdC gene was isolated from human spleen tissue by RT-PCR. Anti-human yrdC monoclonal antibody was prepared by hybridoma cell technique. Recombinant adenovirus Ad.yrdC carrying yrdC gene was constructed by using the AdEasy adenoviral vector system. Recombinant adenovirus Ad.yrdCshRNA mediated yrdCshRNA was prepared by RNA interference technology. Gastric adenocarcinoma BGC-823 cells of moderate differentiation were transfected and absorbance of the transfected cells was calculated at 490 nm by methyl thiazolyl tetrazolium (MTT) method.
A value of the transfected Ad.yrdC group was significantly greater than that of the non-transfected and transfected Ad.Null groups, and A value of Ad.yrdCshRNA group was significantly lower than that of the non-transfected and transfected Ad.Null groups.
Expression of yrdC gene has a function of promoting the proliferation of gastric carcinoma cells.
No preview · Article · Aug 2009 · The Chinese-German Journal of Clinical Oncology
[Show abstract][Hide abstract] ABSTRACT: Lipoprotein lipase (LPL) plays a key role in the lipid metabolism and transporting. It can catalyze the hydrolysis of chylomicron and very low-density lipoprotein triglyceride. Moreover, the abnormality of LPL associates with many pathophysiological conditions. Herein cDNA microarray and Northern blots analysis were used to study the expression of lipoprotein lipase in lung adenocarcinoma tissues. There were 113 genes of all tested blots in cDNA microarray expressed lowly. LPL gene is expressed lowly at the average ratio 0.26 (Cy5/Cy3) in lung adenocarcinoma tissues over controls. Northern blots confirmed those changes detected from the cDNA microarray and suggested that low expression of LPL may play an important role in the lung adenocarcinoma development.