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author:("Wang, mianmin")
1.  A Large Scale, Ion Current based Proteomics Investigation of Bronchoalveolar Lavage Fluid in Chronic Obstructive Pulmonary Disease Patients 
Journal of proteome research  2013;13(2):627-639.
Proteomic analysis of bronchoalveolarBlavageBfluid (BALF) in chronic obstructive pulmonary disease (COPD) patients may provide new biomarkers and deeper understanding of the disease mechanisms but remains challenging. Here we describe an ionBcurrentBbased strategy for comparative analysis of BALF proteomes from patients with moderate and stable COPD vs. healthy controls. The strategy includes an efficient preparation procedure providing quantitative recovery and a nanoBLC/MS analysis with a long, heated column. Under optimized conditions, high efficiency and reproducibility were achieved for each step, enabling a “20Bplex” comparison of clinical subjects (n=10/group). Without depletion/fractionation, a total of 423 unique protein groups were quantified under stringent criteria with at least two quantifiable peptides. SeventyBsix proteins were determined as significantlyBaltered in COPD, which represent a diversity of biological processes such as alcohol metabolic process, gluconeogenesis/glycolysis, inflammatory response, proteolysis, and oxidation reduction. Interestingly, altered alcohol metabolism responding to oxidant stress is a novel observation in COPD. The prominently elevated key enzymes involved in alcohol metabolism (e.g. ADH1B, ALDH2&ALDH3A1) may provide a reasonable explanation for a bewildering observation in COPD patients known for decades: the underestimation of the blood alcohol concentrations through breath tests. These discoveries could provide new insights for identifying novel biomarkers and pathological mediators in clinical studies.
PMCID: PMC4073647  PMID: 24188068
Biomarker Discovery; Bronchoalveolar Lavage Fluid; Chronic Obstructive Pulmonary Disease; Peptide Extracted Ion Current
2.  Targeting Oxidative Stress in Embryonal Rhabdomyosarcoma 
Cancer cell  2013;24(6):710-724.
Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histological subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis show that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcoma (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.
PMCID: PMC3904731  PMID: 24332040
3.  Mediastinal Lymph Node Dissection versus Mediastinal Lymph Node Sampling for Early Stage Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis 
PLoS ONE  2014;9(10):e109979.
This systematic review and meta-analysis aimed to evaluate the overall survival, local recurrence, distant metastasis, and complications of mediastinal lymph node dissection (MLND) versus mediastinal lymph node sampling (MLNS) in stage I–IIIA non-small cell lung cancer (NSCLC) patients.
A systematic search of published literature was conducted using the main databases (MEDLINE, PubMed, EMBASE, and Cochrane databases) to identify relevant randomized controlled trials that compared MLND vs. MLNS in NSCLC patients. Methodological quality of included randomized controlled trials was assessed according to the criteria from the Cochrane Handbook for Systematic Review of Interventions (Version 5.1.0). Meta-analysis was performed using The Cochrane Collaboration’s Review Manager 5.3. The results of the meta-analysis were expressed as hazard ratio (HR) or risk ratio (RR), with their corresponding 95% confidence interval (CI).
We included results reported from six randomized controlled trials, with a total of 1,791 patients included in the primary meta-analysis. Compared to MLNS in NSCLC patients, there was no statistically significant difference in MLND on overall survival (HR = 0.77, 95% CI 0.55 to 1.08; P = 0.13). In addition, the results indicated that local recurrence rate (RR = 0.93, 95% CI 0.68 to 1.28; P = 0.67), distant metastasis rate (RR = 0.88, 95% CI 0.74 to 1.04; P = 0.15), and total complications rate (RR = 1.10, 95% CI 0.67 to 1.79; P = 0.72) were similar, no significant difference found between the two groups.
Results for overall survival, local recurrence rate, and distant metastasis rate were similar between MLND and MLNS in early stage NSCLC patients. There was no evidence that MLND increased complications compared with MLNS. Whether or not MLND is superior to MLNS for stage II–IIIA remains to be determined.
PMCID: PMC4190366  PMID: 25296033
4.  A simple model clarifies the complicated relationships of complex networks 
Scientific Reports  2014;4:6197.
Real-world networks such as the Internet and WWW have many common traits. Until now, hundreds of models were proposed to characterize these traits for understanding the networks. Because different models used very different mechanisms, it is widely believed that these traits origin from different causes. However, we find that a simple model based on optimisation can produce many traits, including scale-free, small-world, ultra small-world, Delta-distribution, compact, fractal, regular and random networks. Moreover, by revising the proposed model, the community-structure networks are generated. By this model and the revised versions, the complicated relationships of complex networks are illustrated. The model brings a new universal perspective to the understanding of complex networks and provide a universal method to model complex networks from the viewpoint of optimisation.
PMCID: PMC4145283  PMID: 25160506
5.  Dysfunction of Bone Marrow Vascular Niche in Acute Graft-Versus-Host Disease after MHC-Haploidentical Bone Marrow Transplantation 
PLoS ONE  2014;9(8):e104607.
Acute graft-versus-host disease (aGvHD) is the most common complication of allogeneic hematopoietic stem cell transplantation (HSCT), which is often accompanied by impaired hematopoietic reconstitution. Sinusoidal endothelial cells (SECs) constitute bone marrow (BM) vascular niche that plays an important role in supporting self-renewal capacity and maintaining the stability of HSC pool. Here we provide evidences that vascular niche is a target of aGvHD in a major histocompatibility complex (MHC)–haploidentical matched murine HSCT model. The results demonstrated that hematopoietic cells derived from GvHD mice had the capacity to reconstitute hematopoiesis in healthy recipient mice. However, hematopoietic cells from healthy donor mice failed to reconstitute hematopoiesis in GvHD recipient mice, indicating that the BM niche was impaired by aGvHD in this model. We further demonstrated that SECs were markedly reduced in the BM of aGvHD mice. High level of Fas and caspase-3 expression and high rate of apoptosis were identified in SECs, indicating that SECs were destroyed by aGvHD in this murine HSCT model. Furthermore, high Fas ligand expression on engrafted donor CD4+, but not CD8+ T cells, and high level MHC-II but not MHC-I expression on SECs, suggested that SECs apoptosis was mediated by CD4+ donor T cells through the Fas/FasL pathway.
PMCID: PMC4131885  PMID: 25119573
6.  Recurrent Somatic Structural Variations Contribute to Tumorigenesis in Pediatric Osteosarcoma 
Cell reports  2014;7(1):104-112.
Osteosarcoma is a neoplasm of mesenchymal origin with features of osteogenic differentiation. Patients with recurrent or metastatic disease have a very poor prognosis. To define the landscape of somatic mutations in pediatric osteosarcoma, we performed whole-genome sequencing of DNA from 20 osteosarcoma tumor samples and matched normal tissue (obtained from 19 patients) in the discovery cohort as well as 14 samples from 13 patients in the validation cohort. Our results demonstrate that pediatric osteosarcoma is characterized by multiple somatic chromosomal lesions, including structural variations (SVs) and copy number alterations (CNAs). Moreover, single nucleotide variations (SNVs) exhibit a pattern of localized hypermutation called “kataegis” in 50% of the tumors. Despite these regions of kataegis across the osteosarcoma genomes, we detected relatively few recurrent SNVs, and only when SVs were included did we identify the major pathways that are mutated in osteosarcoma. We identified p53 pathway lesions in all 19 patient’s tumors in the discovery cohort, 9 of which were translocations in the first intron of the TP53 gene, leading to gene inactivation. This mechanism of p53 gene inactivation is unique to osteosarcoma among pediatric cancers. In an additional cohort of 32 patients, TP53 gene alterations were identified in 29 of those tumors. Beyond TP53, the RB1, ATRX and DLG2 genes showed recurrent somatic alterations (SNVs and/or SVs) in 29–53% of the tumors. These data highlight the power of whole-genome sequencing in identifying recurrent somatic alterations in cancer genomes that may be missed using other methods.
PMCID: PMC4096827  PMID: 24703847
7.  Thymic Damage, Impaired Negative Selection, and Development of Chronic Graft-versus-Host Disease Caused by Donor CD4+ and CD8+ T Cells 
Prevention of chronic graft-versus-host disease (cGVHD) remains a major challenge in allogeneic hematopoietic cell transplantation (HCT), due to limited understanding of cGVHD pathogenesis and lack of appropriate animal models. Here, we report that, in classical acute GVHD models with C57BL/6 donors and MHC-mismatched BALB/c recipients and with C3H.SW donors and MHC-matched C57BL/6 recipients, GVHD recipients surviving for more than 60 days after HCT developed cGVHD characterized by cutaneous fibrosis, tissue damage in the salivary gland and the presence of serum autoantibodies. Donor CD8+ T cells were more potent than CD4+ T cells for inducing cGVHD. The recipient thymus and de novo-generated, donor-derived CD4+ T cells were required for induction of cGVHD by donor CD8+ T cells but not by donor CD4+ T cells. Donor CD8+ T cells preferentially damaged recipient medullary thymic epithelial cells and impaired negative selection, resulting in production of autoreactive CD4+ T cells that perpetuated damage to the thymus and augmented the development of cGVHD. Short-term anti-CD4 monoclonal antibody treatment early after HCT enabled recovery from thymic damage and prevented cGVHD. These results demonstrate that donor CD8+ T cells cause cGVHD solely through thymic-dependent mechanisms, while CD4+ T cells can cause cGVHD through either thymic-dependent or independent mechanisms.
PMCID: PMC3746979  PMID: 23709681
8.  A Genome-Wide RNAi Screen Identifies FOXO4 as a Metastasis-Suppressor through Counteracting PI3K/AKT Signal Pathway in Prostate Cancer 
PLoS ONE  2014;9(7):e101411.
Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness.
PMCID: PMC4077825  PMID: 24983969
9.  Class I ADP-Ribosylation Factors Are Involved in Enterovirus 71 Replication 
PLoS ONE  2014;9(6):e99768.
Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.
PMCID: PMC4049829  PMID: 24911624
10.  Inhibition of Enterovirus 71 Replication by 7-Hydroxyflavone and Diisopropyl-Flavon7-yl Phosphate 
PLoS ONE  2014;9(3):e92565.
Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease, which has been continuously prevalent in Asia in recent years. In children, severe cases can lead to death, and no prophylactic or therapeutic measures against EV71 infection are available. The 3C proteases of EV71 play an important role in viral replication and are an ideal drug target. In previous work, we resolved the crystal structure for EV71 3Cpro. In this report, we took advantage of the automated docking program AutoDock 4.0 to simulate EV71 3Cpro-ligand conformation. 7-hydroxyflavone (HF) and its phosphate ester(FIP) were predicted to bind with EV71 3Cpro.In an in vitro protease inhibition assay, FIP inhibited EV71 3Cpro protease activity. Both flavones were highly active against EV71, protecting cells from EV71 infection. Replication of viral RNA and formation of EV71 plaque were all strongly inhibited in cells. These results indicated that HF and FIP may serve as potential protective agents in the treatment of patients with chronic EV71 infection.
PMCID: PMC3963929  PMID: 24664133
11.  Anti-Enterovirus 71 Effects of Chrysin and Its Phosphate Ester 
PLoS ONE  2014;9(3):e89668.
Enterovirus 71 (EV71) can cause severe disease and even lead to death in children, and an effective antiviral drug is currently unavailable. The anti-EV71 effect of chrysin (5,7-dihydroxyflavone), a natural flavonoid commonly found in many plants, was tested in this report. By using the predicting program Autodock 4.0 and an in vitro protease inhibition assay, we found that chrysin could suppress viral 3Cpro activity. Replication of viral RNA and production of viral capsid protein and the infectious virion were strongly inhibited by chrysin, without noticeable cytotoxicity. Cytopathic effects on cells were also prevented. Diisopropyl chrysin-7-yl phosphate (CPI), the phosphate ester for chrysin, was generated through a simplified Atheron-Todd reaction to achieve stronger anti-viral activity. CPI was also able to bind with and inhibit viral 3Cpro activity in vitro. As expected, CPI demonstrated more potent antiviral activity against EV71.
PMCID: PMC3943725  PMID: 24598537
12.  Analysis of Differential Gene Expression and Novel Transcript Units of Ovine Muscle Transcriptomes 
PLoS ONE  2014;9(2):e89817.
In this study, we characterized differentially expressed genes (DEGs) between the muscle transcriptomes of Small-tailed Han sheep and Dorper sheep and predicted novel transcript units using high-throughput RNA sequencing technology. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that 1,300 DEGs were involved in cellular processes, metabolic pathways, and the actin cytoskeleton pathway. Importantly, we identified 34 DEGs related to muscle cell development and differentiation. Additionally, we were able to optimize the gene structure and predict the untranslated regions (UTRs) for some of the DEGs. Among the 123,678 novel predicted transcript units (TUs), 15,015 units were predicted protein sequences. The reliability of the sequencing data was verified through qRT-PCR analysis of 12 genes. These results will provide useful information for functional genetic research in the future.
PMCID: PMC3935930  PMID: 24587058
13.  Homoharringtonine and omacetaxine for myeloid hematological malignancies 
Homoharringtonine (HHT), a plant alkaloid with antitumor properties originally identified nearly 40 years ago, has a unique mechanism of action by preventing the initial elongation step of protein synthesis. HHT has been used widely in China for the treatment of chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Omacetaxine, a semisynthetic form of HHT, with excellent bioavailability by the subcutaneous route, has recently been approved by FDA of the United States for the treatment of CML refractory to tyrosine kinase inhibitors. This review summarized preclinical and clinical development of HHT and omacetaxine for myeloid hematological malignancies.
PMCID: PMC3884015  PMID: 24387717
Homoharringtonine; Omacetaxine; Chronic myeloid leukemia; Acute myeloid leukemia; Myelodysplastic syndrome
14.  Characteristics and Mechanisms of Cardiopulmonary Injury Caused by Mine Blasts in Shoals: A Randomized Controlled Study in a Rabbit Model 
PLoS ONE  2013;8(12):e81310.
Because the characteristics of blast waves in water are different from those in air and because kinetic energy is liberated by a pressure wave at the water-air interface, thoracic injuries from mine blasts in shoals may be serious. The aim of the present study was to investigate the characteristics and mechanisms of cardiopulmonary injury caused by mine blasts in shoals.
To study the characteristics of cardiopulmonary injury, 56 animals were divided randomly into three experimental groups (12 animals in the sham group, 22 animals in the land group and 22 animals in the shoal group). To examine the biomechanics of injury, 20 animals were divided randomly into the land group and the shoal group. In the experimental model, the water surface was at the level of the rabbit's xiphoid process, and paper electric detonators (600 mg RDX) were used to simulate mines. Electrocardiography and echocardiography were conducted, and arterial blood gases, serum levels of cardiac troponin I and creatine kinase-MB and other physiologic parameters were measured over a 12-hour period after detonation. Pressures in the thorax and abdomen and the acceleration of the thorax were measured.
The results indicate that severe cardiopulmonary injury and dysfunction occur following exposure to mine blasts in shoals. Therefore, the mechanisms of cardiopulmonary injury may result from shear waves that produce strain at the water-air interface. Another mechanism of injury includes the propagation of the shock wave from the planta to the thorax, which causes a much higher peak overpressure in the abdomen than in the thorax; as a result, the abdominal organs and diaphragm are thrust into the thorax, damaging the lungs and heart.
PMCID: PMC3864783  PMID: 24358110
15.  Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas 
Nature genetics  2013;45(6):602-612.
The commonest pediatric brain tumors are low-grade gliomas (LGGs). We utilized whole genome sequencing to discover multiple novel genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24/39 (62%) tumors. Intragenic duplications of the FGFR1 tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes containing TKD-duplicated FGFR1 into brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. TKD-duplicated FGFR1 induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs/LGGNTs.
PMCID: PMC3727232  PMID: 23583981
16.  An inv(16)(p13.3q24.3)-encoded CBFA2T3-GLIS2 fusion protein defines an aggressive subtype of pediatric acute megakaryoblastic leukemia 
Cancer cell  2012;22(5):683-697.
To define the mutation spectrum in non-Down syndrome acute megkaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL leukemia samples. Our analysis identified a cryptic chromosome 16 inversion [inv(16)(p13.3q24.3)] in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling, and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.
PMCID: PMC3547667  PMID: 23153540
18.  FISH+CD34+CD38- cells detected in newly diagnosed acute myeloid leukemia patients can predict the clinical outcome 
In acute myeloid leukemia (AML), the leukemia initiating cells (LICs) or leukemia stem cells (LSCs) is found within the CD34+CD38- cell compartment. The LICs subpopulation survives chemotherapy and is most probable the cause of minimal residual disease (MRD), which in turn is thought to cause relapse. The aim of this study was to determine the prognostic value of the percentage of LICs in blasts at diagnosis.
Design and methods
The percentage of LICs in the blast population was determined at diagnosis using a unique Flow-FISH analysis, which applies fluorescent in situ hybridization (FISH) analysis on flow cytometry sorted cells to distinguish LICs within the CD34+CD38- cell compartment. Fourty-five AML patients with FISH-detectable cytogenetic abnormalities treated with standardized treatment program were retrospectively included in the study. Correlations with overall survival (OS), events-free survival (EFS) and cumulative incidence of relapse (CIR) were evaluated with univariate and multivariate analysis.
The percentage of LICs is highly variable in patients with acute myeloid leukemia, ranged from 0.01% to 52.8% (median, 2.1%). High LIC load (≥1%) negatively affected overall survival (2-year OS: 72.57% vs. 16.75%; P = 0.0037) and events-free survival (2-year EFS: 67.23% vs. 16.33%; P = 0.0018), which was due to an increased cumulative incidence of relapse (2-year CIR: 56.7% vs. 18.0%; P = 0.021). By multivariate analysis, high LIC load retained prognostic significance for OS and EFS.
In the present study, we established the Flow-FISH protocol as a useful method to distinguish normal and leukemic cells within the CD34+CD38- cell subpopulation. The high percentage of LICs at diagnosis was significantly correlated with increased risk of poor clinical outcome.
PMCID: PMC4028871  PMID: 24517186
Acute myeloid leukemia; Leukemia initiating cells; Minimal residual disease
19.  Computational methods for detecting copy number variations in cancer genome using next generation sequencing: principles and challenges 
Oncotarget  2013;4(11):1868-1881.
Accurate detection of somatic copy number variations (CNVs) is an essential part of cancer genome analysis, and plays an important role in oncotarget identifications. Next generation sequencing (NGS) holds the promise to revolutionize somatic CNV detection. In this review, we provide an overview of current analytic tools used for CNV detection in NGS-based cancer studies. We summarize the NGS data types used for CNV detection, decipher the principles for data preprocessing, segmentation, and interpretation, and discuss the challenges in somatic CNV detection. This review aims to provide a guide to the analytic tools used in NGS-based cancer CNV studies, and to discuss the important factors that researchers need to consider when analyzing NGS data for somatic CNV detections.
PMCID: PMC3875755  PMID: 24240121
copy number variation; next generation sequencing; cancer genome analysis; somatic mutations
20.  6p22.3 amplification as a biomarker and potential therapeutic target of advanced stage bladder cancer 
Oncotarget  2013;4(11):2124-2134.
Genetic and epigenetic alterations have been identified as to contribute directly or indirectly to the generation of transitional cell carcinoma of the urinary bladder (TCC-UB). In a comparative fashion much less is known about copy number alterations in TCC-UB, but it appears that amplification of chromosome 6p22 is one of the most frequent changes. Using fluorescence in situ hybridization (FISH) analyses, we evaluated chromosomal 6p22 amplification in a large cohort of bladder cancer patients with complete surgical staging and outcome data. We have also used shRNA knockdown candidate oncogenes in the cell based study. We found that amplification of chromosome 6p22.3 is significantly associated with the muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) (22%) in contrast to superficial TCC-UB (9%) (p=7.2-04). The rate of 6p22.3 amplification in pN>1 patients (32%) is more than twice that in pN0 (16%) patients (p=0.05). Interestingly, we found that 6p22.3 amplification is as twice as high (p=0.0201) in African American (AA) than European American (EA) TCC-UB patients. Moreover, we showed that the expression of some candidate genes (E2F3, CDKAL1 and Sox4) in the 6p22.3 region is highly correlated with the chromosomal amplification. In particular, knockdown of E2F3 inhibits cell proliferation in a 6p22.3-dependent manner, whereas knockdown of CDKAL1 and Sox4 has no effect on cell proliferation. Using gene expression profiling, we further identified some common as well as distinctive subset targets of the E2F3 family members. In summary, our data indicate that E2F3 is a key regulator of cell proliferation in a subset of bladder cancer and the 6p22.3 amplicon is a biomarker of aggressive phenotype in this tumor type.
PMCID: PMC3875774  PMID: 24231253
bladder cancer; chromosome 6p22; FISH; outcome; survival
21.  Hypoxia-inducible C-to-U coding RNA editing downregulates SDHB in monocytes 
PeerJ  2013;1:e152.
Background. RNA editing is a post-transcriptional regulatory mechanism that can alter the coding sequences of certain genes in response to physiological demands. We previously identified C-to-U RNA editing (C136U, R46X) which inactivates a small fraction of succinate dehydrogenase (SDH; mitochondrial complex II) subunit B gene (SDHB) mRNAs in normal steady-state peripheral blood mononuclear cells (PBMCs). SDH is a heterotetrameric tumor suppressor complex which when mutated causes paraganglioma tumors that are characterized by constitutive activation of hypoxia inducible pathways. Here, we studied regulation, extent and cell type origin of SDHB RNA editing.
Methods. We used short-term cultured PBMCs obtained from random healthy platelet donors, performed monocyte enrichment by cold aggregation, employed a novel allele-specific quantitative PCR method, flow cytometry, immunologic cell separation, gene expression microarray, database analysis and high-throughput RNA sequencing.
Results. While the editing rate is low in uncultured monocyte-enriched PBMCs (average rate 2.0%, range 0.4%–6.3%, n = 42), it is markedly upregulated upon exposure to 1% oxygen tension (average rate 18.2%, range 2.8%–49.4%, n = 14) and during normoxic macrophage differentiation in the presence of serum (average rate 10.1%, range 2.7%–18.8%, n = 17). The normoxic induction of SDHB RNA editing was associated with the development of dense adherent aggregates of monocytes in culture. CD14-positive monocyte isolation increased the percentages of C136U transcripts by 1.25-fold in normoxic cultures (n = 5) and 1.68-fold in hypoxic cultures (n = 4). CD14-negative lymphocytes showed no evidence of SDHB editing. The SDHB genomic DNA remained wild-type during increased RNA editing. Microarray analysis showed expression changes in wound healing and immune response pathway genes as the editing rate increased in normoxic cultures. High-throughput sequencing of SDHB and SDHD transcripts confirmed the induction of C136U RNA editing in normoxic cultures but showed no additional verifiable coding edits. Analysis of SDHB RNA sequence data from 16 normal human tissues from the Illumina Body Map and from 45 samples representing 23 different cell types from the ENCODE projects confirmed the occurrence of site-specific C136U editing in whole blood (1.7%) and two primary CD14+ monocyte samples (1.9% and 2.6%). In contrast, the other cell types showed an average of 0.2% and 0.1% C136U editing rates in the two databases, respectively.
Conclusions. These findings demonstrate that C-to-U coding RNA editing of certain genes is dynamically induced by physiologically relevant environmental factors and suggest that epigenetic downregulation of SDHB by site-specific RNA editing plays a role in hypoxia adaptation in monocytes.
PMCID: PMC3775634  PMID: 24058882
Epigenetic; Environment; RNA editing; Cytidine deaminase; Monocyte; Macrophage; Mitochondrion; Hypoxia; Complex II
22.  Central nervous system involvement in adult patients with diffuse large B-cell lymphoma: Influence of rituximab 
Oncology Letters  2012;4(3):541-545.
CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone)-like chemotherapy, in combination with rituximab (R-CHOP-like), improves outcome in patients with diffuse large B-cell lymphoma (DLBCL). We aimed to investigate the impact of rituximab on central nervous system (CNS) disease in adult patients. We studied 315 patients (aged 18–60 years old) from six hospitals between July 2003 and May 2008. All patients received CHOP-like (n=165) or R-CHOP-like (n=150) regimen every 3 weeks. With a median follow-up of 3.69 years, 10 patients (3.17%) developed CNS disease. The cumulative risk of CNS occurrence was not significantly different between the two treatment groups (P=0.871). We conclude that the addition of rituximab did not reduce the risk of CNS disease in adult patients with DLBCL.
PMCID: PMC3439009  PMID: 22970053
central nervous system; diffuse large B-cell lymphoma; rituximab
23.  Characterization and Comparative Analyses of Muscle Transcriptomes in Dorper and Small-Tailed Han Sheep Using RNA-Seq Technique 
PLoS ONE  2013;8(8):e72686.
The sheep is an important domestic animal and model for many types of medically relevant research. An investigation of gene expression in ovine muscle would significantly advance our understanding of muscle growth. RNA-seq is a recently developed analytical approach for transcriptome profiling via high-throughput sequencing. Although RNA-seq has been recently applied to a wide variety of organisms, few RNA-seq studies have been conducted in livestock, particularly in sheep. In this study, two cDNA libraries were constructed from the biceps brachii of one Small-tailed Han sheep (SH) and one Dorper sheep (DP). The Illumina high-throughput sequencing technique and bioinformatics were used to determine transcript abundances and characteristics. For the SH and DP libraries, we obtained a total of 50,264,608 and 52,794,216 high quality reads, respectively. Approximately two-thirds of the reads could be mapped to the sheep genome. In addition, 40,481 and 38,851 potential coding single nucleotide polymorphisms (cSNPs) were observed, respectively, of which a total of 59,139 cSNP coordinates were different between the two samples. Up to 5,116 and 5,265 respective reference genes had undergone 13,827 and 15,684 alternative splicing events. A total of 6,989 reference genes were extended at the 5’, 3’ or both ends, and 123,678 novel transcript units were found. A total of 1,300 significantly differentially expressed genes were identified between the two libraries. These results suggest that there are many differences in the muscle transcriptomes between these two animals. This study addresses a preliminary analysis and offers a foundation for future genomic research in the sheep.
PMCID: PMC3758325  PMID: 24023632
24.  MicroRNA-200a Regulates Grb2 and Suppresses Differentiation of Mouse Embryonic Stem Cells into Endoderm and Mesoderm 
PLoS ONE  2013;8(7):e68990.
The mechanisms by which microRNAs (miRNAs) affect cell fate decisions remain poorly understood. Herein, we report that miR-200a can suppress the differentiation of mouse embryonic stem (ES) cells into endoderm and mesoderm. Interestingly, miR-200a directly targets growth factor receptor-bound protein 2 (Grb2), which is a key adaptor in the Erk signaling pathway. Furthermore, high levels of miR-200a dramatically decrease Grb2 levels and suppress the appearance of mesoderm and endoderm lineages in embryoid body formation, as well as suppressing the activation of Erk. Finally, Grb2 supplementation significantly rescues the miR-200a-induced layer-formation bias and the Erk suppression. Collectively, our results demonstrate that miR-200a plays critical roles in ES cell lineage commitment by directly regulating Grb2 expression and Erk signaling.
PMCID: PMC3715486  PMID: 23874841
25.  Concurrent Infection of Hepatitis B Virus Negatively Affects the Clinical Outcome and Prognosis of Patients with Non-Hodgkin’s Lymphoma after Chemotherapy 
PLoS ONE  2013;8(7):e69400.
Hepatitis B virus (HBV) is hepatotropic and lymphotropic. HBV-infected individuals have an increased risk of developing malignant lymphoma, and the HBV infection rate in lymphoma patients is significantly higher than that in the general population. However, the exact mechanism and correlation between HBV infection and lymphoma onset and progression currently remain unclear. We retrospectively analyzed clinical data from non-Hodgkin’s lymphoma (NHL) patients with different HBV infection statuses. The results showed that the HBV infection rate was significantly higher in patients with B-cell type and late stage of NHL. The chemotherapy efficacy for NHL patients with chronic active HBV infection was significantly lower than that for the patients with chronic inactive HBV infection, the patients with HBV carriers and the patients without HBV infection. In addition, the NHL chemotherapy activated HBV replication and caused significant liver dysfunction, which could further reduce the chemotherapy efficacy. Through Kaplan-Meier survival curve and log-rank analysis, we found that the HBV infection status in NHL patients was significantly correlated with the patients’ progression-free survival (PFS) and overall survival (OS). Compared with the patients without HBV infection (PFS: 95% CI 47.915 to 55.640; OS: 95% CI 81.324 to 86.858), the PFS and OS of the patients with chronic active HBV infection were significantly shorter (PFS: 95% CI 9.424 to 42.589, P < 0.001; OS: 95% CI 42.840 to 82.259, P = 0.006). The study demonstrated that the sustained HBV replication in patients with chronic active HBV infection could be a key factor that influences the prognosis of NHL patients after chemotherapy, and thus may provide information for designing rational clinical treatments for NHL patients with different HBV infection statuses and improve the treatment efficacy and prognosis.
PMCID: PMC3704665  PMID: 23861969

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