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1.  Elevated levels of plasma D-dimer predict a worse outcome in patients with nasopharyngeal carcinoma 
BMC Cancer  2014;14(1):583.
Background
Hemostatic alterations occur during the development of cancer. Plasma D-dimer is a hypercoagulability and fibrinolytic system marker that is increased in patients with various solid tumours. The aim of this study was to evaluate the hemostatic status of nasopharyngeal carcinoma (NPC) patients by assessing plasma D-dimer levels to investigate its value as a prognostic marker.
Methods
We retrospectively analysed 717 patients with nasopharyngeal carcinoma, and we applied Cox regression and log-rank tests to assess the association of D-dimer levels with disease-free survival (DFS), distant metastasis-free survival (DMFS), and overall survival (OS). D-dimer levels were measured using a quantitative D-dimer latex agglutination assay.
Results
Using the 3rd quartile values (0.8 μg/L) as the optimal cut-offs, we found that patients with high D-dimer levels have a shorter 3-year DFS, (79%, 95%CI (73.1–84.9)) vs. (69%, 95%CI (59.2–78.8)), DMFS (87%, 95%CI (83.1–90.9)) vs. (77%, 95%CI (69.2–84.8)), and overall survival (82%, 95%CI (76.1–87.9)) vs. (76%, 95%CI (66.2–85.8)). Multivariate analysis revealed that pre-treatment D-dimer levels and EBV DNA were significant independent factors for DFS, DMFS, and OS in NPC patients. Subgroup analyses indicated that the plasma D-dimer levels could effectively stratify patient prognosis for early cancer, advanced stage cancer, and patients with EBV DNA ≥4000 copies/ml.
Conclusions
High D-dimer levels were associated with poor disease-free survival, distant metastasis-free survival, overall survival, and increased risk of mortality in NPC patients. Prospective trials are required to assess the prognostic value of D-dimer levels.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-583) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-583
PMCID: PMC4242497  PMID: 25109220
Nasopharyngeal carcinoma; D-dimer; Survival
2.  The sequence and de novo assembly of the giant panda genome 
Li, Ruiqiang | Fan, Wei | Tian, Geng | Zhu, Hongmei | He, Lin | Cai, Jing | Huang, Quanfei | Cai, Qingle | Li, Bo | Bai, Yinqi | Zhang, Zhihe | Zhang, Yaping | Wang, Wen | Li, Jun | Wei, Fuwen | Li, Heng | Jian, Min | Li, Jianwen | Zhang, Zhaolei | Nielsen, Rasmus | Li, Dawei | Gu, Wanjun | Yang, Zhentao | Xuan, Zhaoling | Ryder, Oliver A. | Leung, Frederick Chi-Ching | Zhou, Yan | Cao, Jianjun | Sun, Xiao | Fu, Yonggui | Fang, Xiaodong | Guo, Xiaosen | Wang, Bo | Hou, Rong | Shen, Fujun | Mu, Bo | Ni, Peixiang | Lin, Runmao | Qian, Wubin | Wang, Guodong | Yu, Chang | Nie, Wenhui | Wang, Jinhuan | Wu, Zhigang | Liang, Huiqing | Min, Jiumeng | Wu, Qi | Cheng, Shifeng | Ruan, Jue | Wang, Mingwei | Shi, Zhongbin | Wen, Ming | Liu, Binghang | Ren, Xiaoli | Zheng, Huisong | Dong, Dong | Cook, Kathleen | Shan, Gao | Zhang, Hao | Kosiol, Carolin | Xie, Xueying | Lu, Zuhong | Zheng, Hancheng | Li, Yingrui | Steiner, Cynthia C. | Lam, Tommy Tsan-Yuk | Lin, Siyuan | Zhang, Qinghui | Li, Guoqing | Tian, Jing | Gong, Timing | Liu, Hongde | Zhang, Dejin | Fang, Lin | Ye, Chen | Zhang, Juanbin | Hu, Wenbo | Xu, Anlong | Ren, Yuanyuan | Zhang, Guojie | Bruford, Michael W. | Li, Qibin | Ma, Lijia | Guo, Yiran | An, Na | Hu, Yujie | Zheng, Yang | Shi, Yongyong | Li, Zhiqiang | Liu, Qing | Chen, Yanling | Zhao, Jing | Qu, Ning | Zhao, Shancen | Tian, Feng | Wang, Xiaoling | Wang, Haiyin | Xu, Lizhi | Liu, Xiao | Vinar, Tomas | Wang, Yajun | Lam, Tak-Wah | Yiu, Siu-Ming | Liu, Shiping | Zhang, Hemin | Li, Desheng | Huang, Yan | Wang, Xia | Yang, Guohua | Jiang, Zhi | Wang, Junyi | Qin, Nan | Li, Li | Li, Jingxiang | Bolund, Lars | Kristiansen, Karsten | Wong, Gane Ka-Shu | Olson, Maynard | Zhang, Xiuqing | Li, Songgang | Yang, Huanming | Wang, Jian | Wang, Jun
Nature  2009;463(7279):311-317.
Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes.
doi:10.1038/nature08696
PMCID: PMC3951497  PMID: 20010809
3.  Lewis y Regulate Cell Cycle Related Factors in Ovarian Carcinoma Cell RMG-I in Vitro via ERK and Akt Signaling Pathways 
Objective
To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells.
Methods
mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot.
Results
Lewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression.
Conclusion
Lewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation.
doi:10.3390/ijms13010828
PMCID: PMC3269723  PMID: 22312289
Lewis(y) antigen; cell cycle; cyclin; cyclin-dependent kinases; cyclin-dependent kinase inhibitors
4.  BDNF–ERK–CREB signalling mediates the role of miR-132 in the regulation of the effects of oleanolic acid in male mice 
Background
Although previous study has demonstrated that brain-derived neurotrophic factor (BDNF) is involved in the antidepressant-like effect of oleanolic acid, there is little information regarding the details of the molecular mechanism involved in this effect.
Methods
We used a chronic unpredictable mild stress (CUMS) model to test the antidepressant-like effect of oleanolic acid on depressant-like behaviour, miR-132 expression and synaptic protein expression in the male mouse hippocampus. Furthermore, we explored the possible signalling pathways associated with miR-132 expression that mediate the effect of oleanolic acid on neuronal proliferation.
Results
The results demonstrated that a 3-week treatment with oleanolic acid ameliorated CUMS-induced anhedonic and anxiogenic behaviours. Furthermore, we found that oleanolic acid led to the BDNF-related phosphorylation and activation of extracellular signal-regulated kinases (ERK) and cyclic adenosine monophosphate response element binding protein (CREB), which was associated with the upregulation of miR-132 and hippocampal neuronal proliferation. Moreover, experiments with an miR-132 antagomir revealed that targeting miR-132 led to inhibition of neuronal proliferation and the postsynaptic density protein 95, but did not affect presynaptic protein synapsin I.
Limitations
Several other stimuli can also induce CREB phosphorylation in the hippocampus. Thus, regulation of miR-132 may not be restricted to neurotrophic signalling.
Conclusion
Our results show that oleanolic acid induces the upregulation of miR-132, which serves as an important regulator of neurotrophic actions, mainly through the activation of the hippocampal BDNF–ERK–CREB signalling pathways.
doi:10.1503/jpn.130169
PMCID: PMC4160364  PMID: 25079084
5.  High Expression of Lewis y Antigen and CD44 Is Correlated with Resistance to Chemotherapy in Epithelial Ovarian Cancers 
PLoS ONE  2013;8(2):e57250.
Objectives
To measure Lewis y antigen and CD44 antigen expression in epithelial ovarian carcinoma and to correlate the levels of these antigens with clinical response to chemotherapy.
Methods
The study cases included 34 cases of ovarian carcinoma with resistance to chemotherapeutic drugs, 6 partially drug-sensitive cases, and 52 drug-sensitive cases (92 total).
Results
The rates of expression of Lewis y antigen and CD44 antigen were significantly greater in the drug-resistant group than that in the partially-sensitive or sensitive groups. Surgical stage, residual tumor size and expression of CD44 and Lewis y antigen in ovarian carcinoma tissues were independent risk factors for chemotherapeutic drug resistance.
Conclusions
Over-expression of Lewis y and CD44 antigen are strong risk factors for chemotherapeutic drug resistance in ovarian carcinoma patients.
doi:10.1371/journal.pone.0057250
PMCID: PMC3585297  PMID: 23468946
6.  Combined administration of anisodamine and neostigmine produces anti-shock effects: involvement of α7 nicotinic acetylcholine receptors 
Acta Pharmacologica Sinica  2012;33(6):761-766.
Aim:
To evaluate the anti-effects of anisodamine and neostigmine in animal models of endotoxic and hemorrhagic shock.
Methods:
Kunming mice were injected with lipopolysaccharide (LPS 30 mg/kg, ip) to induce endotoxic shock. Anisodamine (12.5, 25, and 50 mg/kg, ip) and neostigmine (12.5, 25, and 50 μg/kg, ip) were administered immediately after LPS injection. Survival rate was monitored, and the serum levels of TNF-α and IL-1β were analyzed using ELISA assays. The effects of anisodamine and neostigmine were also examined in α7 nicotinic acetylcholine receptor (α7 nAChR) knockout mice with endotoxic shock and in Beagle dogs with hemorrhagic shock.
Results:
In mice with experimental endotoxemia, combined administration of anisodamine and neostigmine significantly increased the survival rate and decreased the serum levels of inflammatory cytokines, as compared to those produced by either drug alone. The anti-shock effect of combined anisodamine and neostigmine was abolished in α7 nAChR knockout mice. On the other hand, intravenous injection of the combined anisodamine and neostigmine, or the selective α7 nAChR agonist PNU282987 exerted similar anti-shock effects in dogs with hemorrhagic shock.
Conclusion:
The results demonstrate that combined administration of anisodamine and neostigmine produces significant anti-shock effects, which involves activation of α7 nAChRs.
doi:10.1038/aps.2012.26
PMCID: PMC4010367  PMID: 22580739
anisodamine; neostigmine; PNU282987; α7 nicotinic acetylcholine receptor; endotoxic shock; hemorrhagic shock
7.  The Stimulation of IGF-1R Expression by Lewis(y) Antigen Provides a Powerful Development Mechanism of Epithelial Ovarian Carcinoma 
Objective
This study aimed to measure and correlate the expression of insulin-like growth factor receptor-1 (IGF-1R) and the Lewis(y) antigen in ovarian cancer cell lines and tissue samples.
Methods
Reverse transcriptase PCR (RT-PCR), Western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence double-labeling techniques were applied to detect and measure the expression of Lewis(y) and IGF-1R.
Results
In α1,2-fucosyltransferase (α1,2-FT)-transfected cells, IGF-1R expression was significantly upregulated compared with cells that do not overexpress α1,2-FT (P < 0.05). The amount of Lewis(y) expressed on IGF-1R increased 1.81-fold in α1,2-FT-overexpressing cells (P < 0.05), but the ratio of Lewis(y) expressed on IGF-1R to total IGF-1R was unaltered between two cells (P > 0.05). In malignant epithelial ovarian tumors, the positivity rates of Lewis(y) and IGF-1R detection were 88.3% and 93.33%, respectively, which is higher than the positivity rates in marginal (60.00% and 63.33%, all P < 0.05), benign (33.00% and 53.33%, all P < 0.01), and normal (0% and 40%, all P < 0.01) ovarian samples. No correlations were detected in positivity rates of Lewis(y) or IGF-1R expression with respect to clinicopathological parameters in ovarian cancers (all P > 0.05). Both IGF-1R and Lewis(y) were highly expressed in ovarian cancer tissues, and their expression levels were positively correlated (P < 0.05).
Conclusion
Overexpression of Lewis(y) results in overexpression of IGF-1R. Both IGF-1R and Lewis(y) are associated with the occurrence and development of ovarian cancers.
doi:10.3390/ijms12106781
PMCID: PMC3211010  PMID: 22072919
epithelial ovarian tumor; Insulin-like growth factor receptor-1; Lewis(y) antigen; immunohistochemistry; immunofluorescence double labeling method
8.  Tumor suppression by miR-31 in esophageal carcinoma is p21-dependent 
Genes & Cancer  2014;5(11-12):436-444.
microRNA regulation network is important for the cancer genetic heterogeneity. Relative to the increasing numbers of microRNA's targets identified, upstream regulatory mechanisms that control functional microRNAs are less well-documented. Here, we investigated the function of miR-31, a pleiotropically-acting microRNA, in esophageal squamous cell cancer (ESCC). We demonstrated that miR-31 only exerted tumor-suppressive effects in TE-7 ESCC cells, but not in TE-1 ESCC cells, although both of these cell lines harbor inactive p53. Interestingly, TE-1 cells highly expressed p21, while p21 levels were virtually undetectable in TE-7 cells, suggesting a p21-dependent mechanism of miR-31-mediated tumor suppression. Accordingly, knockdown of p21 in TE-1 cells reversed the tumor suppressive actions of miR-31. In patient ESCC specimens, real-time RT-PCR analysis revealed that expression of E2F2 and STK40, two known miR-31 target oncogenes, was negatively correlated with the expression of miR-31 in a p21-dependent manner, supporting the conclusion that miR-31 only downregulates its target oncogenes when p21 levels are low. Collectively, these data suggest a novel mechanism through which the tumor-suppressive effect of miR-31 is p21-dependent. In addition, we speculate that delivery of miR-31 could provide therapeutic benefit in the personalized management of a subgroup of ESCC patients with p21-deficient tumors.
PMCID: PMC4279440  PMID: 25568668
microRNA; miR-31; p21; esophageal squamous cell cancer; personalized medicine
9.  MicroRNA-21 Promotes Cell Growth and Migration by Targeting Programmed Cell Death 4 Gene in Kazakh's Esophageal Squamous Cell Carcinoma 
Disease Markers  2014;2014:232837.
Esophageal cancer (EC) is the eighth most common cancer worldwide and the sixth most common cause of cancer death. There are two main types of EC—squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). Although some advances in the exploration of its possible etiological mechanism were made recently including behaviors and environmental risk factors as well as gene alterations, the molecular mechanism underlying ESCC carcinogenesis and progression remains poorly understood. It has been reported that miR-21 was upregulated in most malignant cancers, the proposed mechanism of which was through suppressing expression of programmed cell death 4 (PDCD4). In present study, it is firstly reported that miR-21 was upregulated in Kazakh's ESCC and that miR-21 played a negative role in regulating PDCD4 using in situ hybridization (ISH) and luciferase reporter approach. Morever, in model of ESCC xenografted nude mice, miR-21 maybe used as an effective target in the treatment. The present results demonstrated that miR-21 may be a potential therapeutic target in management of ESCC.
doi:10.1155/2014/232837
PMCID: PMC4221975  PMID: 25400316
10.  Genotypes and Transmitted Drug Resistance among Treatment-Naive HIV-1-Infected Patients in a Northwestern Province, China: Trends from 2003 to 2013 
PLoS ONE  2014;9(10):e109821.
Background
Transmitted drug resistance (TDR) reduces the efficacy of initial antiretroviral treatment and has become a public health concern. Little information is available regarding the genetic diversity of HIV-1 and the prevalence of TDR among treatment-naïve patients in a northwestern province of China since the implementation of national free antiretroviral therapy (ART).
Methods
Blood samples from 372 HIV-1 treatment-naive patients were collected between 2003 and 2013 in Shaanxi province. Viral RNA was extracted for nested PCR, and phylogenetic reconstruction and recombination analyses were performed to characterize patterns of the HIV-1 subtypes. Genotypic drug resistance testing was performed using an in-house assay to determine trends in the prevalence of HIV-1 transmitted drug resistance.
Results
Multiple genotypes were identified among the patients in Shaanxi, including B (25.0%), C (0.3%), G (0.3%), and CRF01_AE (39.2%), CRF07_BC (32.7%), CRF08_BC (0.8%), CRF55_01B (1.1%), and URFs (0.6%). The subtypes were associated with the transmission routes (χ2 = 77.113, p<0.01). In this study, a low baseline CD4+ T cell count and a high viral load were found among CRF01_AE-infected patients compared with patients who were infected with non-CRF01_AE (p<0.01) through sexual transmission; however, the CRF01_AE subtype was not associated with a low baseline CD4+ T cell count or a high viral load in Chinese patients infected through blood transmission (p = 0.249). The overall TDR rate in this population was 4.4% between 2003 and 2013. A univariate logistic regression model revealed that a low CD4 T cell count (≤100 cells/µL) was associated with the development of drug-resistant strains.
Conclusion
Our work revealed diverse HIV-1 subtype distributions in Shaanxi province. We identified a low and stable TDR time trend among ART-naive patients. These findings enhance our understanding of HIV-1 genetic diversity and provide some guidelines for the improvement and implementation of a comprehensive public health strategy of HIV-1 TDR prevention.
doi:10.1371/journal.pone.0109821
PMCID: PMC4198111  PMID: 25333965
11.  Non-Genetic Direct Reprogramming and Biomimetic Platforms in a Preliminary Study for Adipose-Derived Stem Cells into Corneal Endothelia-Like Cells 
PLoS ONE  2014;9(10):e109856.
Cell fate and function can be regulated and reprogrammed by intrinsic genetic program, extrinsic factors and niche microenvironment. Direct reprogramming has shown many advantages in the field of cellular reprogramming. Here we tried the possibility to generate corneal endothelia (CE) -like cells from human adipose-derived stem cells (ADSCs) by the non-genetic direct reprogramming of recombinant cell-penetrating proteins Oct4/Klf4/Sox2 (PTD-OKS) and small molecules (purmorphamine, RG108 and other reprogramming chemical reagents), as well as biomimetic platforms of simulate microgravity (SMG) bioreactor. Co-cultured with corneal cells and decellularized corneal ECM, Reprogrammed ADSCs revealed spherical growth and positively expressing Nanog for RT-PCR analysis and CD34 for immunofluorescence staining after 7 days-treatment of both purmorphamine and PTD-OKS (P-OKS) and in SMG culture. ADSCs changed to CEC polygonal morphology from spindle shape after the sequential non-genetic direct reprogramming and biomimetic platforms. At the same time, induced cells converted to weakly express CD31, AQP-1 and ZO-1. These findings demonstrated that the treatments were able to promote the stem-cell reprogramming for human ADSCs. Our study also indicates for the first time that SMG rotary cell culture system can be used as a non-genetic means to promote direct reprogramming. Our methods of reprogramming provide an alternative strategy for engineering patient-specific multipotent cells for cellular plasticity research and future autologous CEC replacement therapy that avoids complications associated with the use of human pluripotent stem cells.
doi:10.1371/journal.pone.0109856
PMCID: PMC4198143  PMID: 25333522
12.  Infrageneric Phylogeny and Temporal Divergence of Sorghum (Andropogoneae, Poaceae) Based on Low-Copy Nuclear and Plastid Sequences 
PLoS ONE  2014;9(8):e104933.
The infrageneric phylogeny and temporal divergence of Sorghum were explored in the present study. Sequence data of two low-copy nuclear (LCN) genes, phosphoenolpyruvate carboxylase 4 (Pepc4) and granule-bound starch synthase I (GBSSI), from 79 accessions of Sorghum plus Cleistachne sorghoides together with those from outgroups were used for maximum likelihood (ML) and Bayesian inference (BI) analyses. Bayesian dating based on three plastid DNA markers (ndhA intron, rpl32-trnL, and rps16 intron) was used to estimate the ages of major diversification events in Sorghum. The monophyly of Sorghum plus Cleistachne sorghoides (with the latter nested within Sorghum) was strongly supported by the Pepc4 data using BI analysis, and the monophyly of Sorghum was strongly supported by GBSSI data using both ML and BI analyses. Sorghum was divided into three clades in the Pepc4, GBSSI, and plastid phylograms: the subg. Sorghum lineage; the subg. Parasorghum and Stiposorghum lineage; and the subg. Chaetosorghum and Heterosorghum lineage. Two LCN homoeologous loci of Cleistachne sorghoides were first discovered in the same accession. Sorghum arundinaceum, S. bicolor, S. x drummondii, S. propinquum, and S. virgatum were closely related to S. x almum in the Pepc4, GBSSI, and plastid phylograms, suggesting that they may be potential genome donors to S. almum. Multiple LCN and plastid allelic variants have been identified in S. halepense of subg. Sorghum. The crown ages of Sorghum plus Cleistachne sorghoides and subg. Sorghum are estimated to be 12.7 million years ago (Mya) and 8.6 Mya, respectively. Molecular results support the recognition of three distinct subgenera in Sorghum: subg. Chaetosorghum with two sections, each with a single species, subg. Parasorghum with 17 species, and subg. Sorghum with nine species and we also provide a new nomenclatural combination, Sorghum sorghoides.
doi:10.1371/journal.pone.0104933
PMCID: PMC4133246  PMID: 25122516
13.  Association between CYP1A2 and CYP1B1 Polymorphisms and Colorectal Cancer Risk: A Meta-Analysis 
PLoS ONE  2014;9(8):e100487.
Background
The previous published data on the association between CYP1A2*F (rs762551), CYP1B1 Leu432Val (rs1056836), Asn453Ser (rs180040), and Arg48Gly (rs10012) polymorphisms and colorectal cancer risk remained controversial.
Methodology/Principal Findings
The purpose of this study is to evaluate the role of CYP1A2*F, CYP1B1 Leu432Val, Asn453Ser, and Arg48Gly genotypes in colorectal cancer susceptibility. We performed a meta-analysis on all the eligible studies that provided 5,817 cases and 6,544 controls for CYP1A2*F (from 13 studies), 9219 cases and 10406 controls for CYP1B1 Leu432Val (from 12 studies), 6840 cases and 7761 controls for CYP1B1 Asn453Ser (from 8 studies), and 4302 cases and 4791 controls for CYP1B1Arg48Gly (from 6 studies). Overall, no significant association was found between CYP1A2*F, CYP1B1 Leu432Val, Asn453Ser, and Arg48Gly and colorectal cancer risk when all the eligible studies were pooled into the meta-analysis. And in the subgroup by ethnicity and source of controls, no evidence of significant association was observed in any subgroup analysis.
Conclusions/Significance
In summary, this meta-analysis indicates that CYP1A2*F, CYP1B1 Leu432Val, Asn453Ser, and Arg48Gly polymorphisms do not support an association with colorectal cancer, and further studies are needed to investigate the association. In addition, our work also points out the importance of new studies for CYP1A2*F polymorphism in Asians, because high heterogeneity was found (dominant model: I2 = 81.3%; heterozygote model: I2 = 79.0).
doi:10.1371/journal.pone.0100487
PMCID: PMC4130485  PMID: 25115775
14.  Association Between Variants of PRDM1 and NDP52 and Crohn’s Disease, Based on Exome Sequencing and Functional Studies 
Gastroenterology  2013;145(2):339-347.
Background & Aims
Genome-wide association studies (GWASs) have identified 140 Crohn’s disease (CD) susceptibility loci. For most loci, the variants that cause disease are not known and the genes affected by these variants have not been identified. We aimed to identify variants that cause CD through detailed sequencing, genetic association, expression, and functional studies.
Methods
We sequenced whole exomes of 42 unrelated subjects with Crohn’s disease (CD) and 5 healthy individuals (controls), and then filtered single-nucleotide variants by incorporating association results from meta-analyses of CD GWASs and in silico mutation effect prediction algorithms. We then genotyped 9348 patients with CD, 2868 with ulcerative colitis, and 14,567 controls, and associated variants analyzed in functional studies using materials from patients and controls and in vitro model systems.
Results
We identified rare missense mutations in PR domain-containing1 (PRDM1) and associated these with CD. These increased proliferation of T cells and secretion of cytokines upon activation, and increased expression of the adhesion molecule L-selectin. A common CD risk allele, identified in GWASs, correlated with reduced expression of PRDM1 in ileal biopsies and peripheral blood mononuclear cells (combined P=1.6×0−8). We identified an association between CD and a common missense variant, Val248Ala, in nuclear domain 10 protein 52 (NDP52) (P=4.83×10−9). We found that this variant impairs the regulatory functions of NDP52 to inhibit NFκB activation of genes that regulate inflammation and affect stability of proteins in toll-like receptor pathways.
Conclusions
We have extended GWAS results and provide evidence that variants in PRDM1 and NDP52 determine susceptibility to CD. PRDM1 maps adjacent to a CD interval identified in GWASs and encodes a transcription factor expressed by T and B cells. NDP52 is an adaptor protein that functions in selective autophagy of intracellular bacteria and signaling molecules, supporting the role for autophagy in pathogenesis of CD.
doi:10.1053/j.gastro.2013.04.040
PMCID: PMC3753067  PMID: 23624108
inflammatory bowel disease; whole-exome sequencing; complex disease
15.  GABAB Receptor Gene Transfer into the Nucleus Tractus Solitarii Induces Chronic Blood Pressure Elevation in Normotensive Rats 
Background
Increasing evidence indicates that GABAergic neurons in the nucleus of the solitary tract (NTS) play a significant role in the arterial baroreceptor reflex and in the control of cardiovascular homeostasis. However, the role of these neurons in the development of hypertension is not yet fully clear.
Methods and Results
In the present study, we first confirmed that GABA B receptor (GBR) expression is enhanced in the NTS of SHR as compared with WKY rats using real time RT-PCR and Western Blots. To study the functional consequence of upregulated GBR expression, GBR was overexpressed in the NTS by bilateral microinjection of the AAV2-GBR1 viral vector into the NTS of WKY rats. Immunofluorescence staining and Western blots demonstrated that microinjection of AAV2-GBR1 into the NTS of WKY rats resulted in a significant increase in GBR1 expression in the NTS neurons. Overexpression of GBR in the NTS induced a chronic elevation in blood pressure and heart rate in the normotensive WKY rats. In an acute study, the pressor response to baclofen microinjected into the NTS was enhanced in SHR as compared with WKY rats.
Conclusion
GBR1 expression is enhanced in the NTS of SHR versus WKY rats and overexpression of this gene in the NTS results in chronic elevation of blood pressure and heart rate in normotensive rats.
PMCID: PMC4113594  PMID: 23803332
GABAB receptor; blood pressure; hypertension
16.  Down-Regulation of TRPM8 in Pulmonary Arteries of Pulmonary Hypertensive Rats 
Background
Pulmonary hypertension (PH) is characterized by profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Multiple transient receptor potential melastatin-related (TRPM) subtypes have been identified in vascular tissue. However, the changes in the expression and function of TRPM channels in pulmonary hypertension have not been characterized in detail.
Methods
We examined the expression of TRPM channels and characterized the functions of the altered TRPM channels in two widely used rat models of chronic hypoxia (CH)- and monocrotaline (MCT)-induced PH.
Results
CH-exposed and MCT-treated rats developed severe PH and right ventricular hypertrophy, with a significant decrease in TRPM8 mRNA and protein expression in pulmonary arteries (PAs). The downregulation of TRPM8 was associated with significant reduction in menthol-induced cation-influx. Time-profiles showed that TRPM8 down-regulation occurred prior to the increase of right ventricular systolic pressure (RVSP) and right ventricular mass index (RVMI) in CH-exposed rats, but these changes were delayed in MCT–treated rats. The TRPM8 agonist menthol induced vasorelaxation in phenylephrine-precontracted PAs, and the vasorelaxing effects were significantly attenuated in PAs of both PH rat models, consistent with decreased TRPM8 expression.
Conclusion
Downregulation of TRPM8 may contribute to the enhanced vasoreactivity in PH.
doi:10.1159/000350107
PMCID: PMC4034698  PMID: 23817166
Transient receptor potential melastatin; Pulmonary hypertension; Calcium signaling; Monocrotaline; Chronic hypoxia; Menthol
17.  Distinct patterns of ALDH1A1 expression predict metastasis and poor outcome of colorectal carcinoma 
Purpose: Aldehyde dehydrogenase 1A1 (ALDH1A1) has been proposed as a candidate biomarker for colorectal carcinoma (CRC). However, the heterogeneity of its expression makes it difficult to predict the outcome of CRC. The aim of this study was to evaluate the diagnostic and prognostic value of this molecule in CRC. Methods and Results: In this study, we examined ALDH1A1 expression by immunohistochemistry including 406 cases of primary CRC with corresponding adjacent mucosa, with confirmation of real-time PCR and Western blotting. We found that the expression patterns of ALDH1A1 were heterogeneous in the CRC and corresponding adjacent tissues. We defined the ratio of ALDH1A1 level in adjacent mucosa to that in tumor tissues as RA/C and found that the capabilities of tumor invasion and metastasis in the tumors with RA/C < 1 were significantly higher than those with RA/C ≥ 1. Follow-up data showed the worse prognoses in the CRC patients with RA/C < 1. For understanding the underlying mechanism, the localization of β-catenin was detected in the CRC tissues with different patterns of ALDH1A1 expression from 221 patients and β-catenin was found preferentially expressed in cell nuclei of the tumors with RA/C < 1 and ALDH1A1high expression of HT29 cell line, indicating that nuclear translocation of β-catenin might contribute to the increased potentials of invasion and metastasis. Conclusion: Our results indicate that RA/C is a novel biomarker to reflect the distinct expression patterns of ALDH1A1 for predicting metastasis and prognosis of CRC.
PMCID: PMC4097282  PMID: 25031716
Colorectal carcinoma; metastasis; prognosis; aldehyde dehydrogenase 1A1; β-catenin
18.  Association of ERCC1 and ERCC2 polymorphisms with colorectal cancer risk in a Chinese population 
Scientific Reports  2014;4:4112.
The ERCC1 and ERCC2 genes are important in repairing DNA damage and genomic instability, and are involved in the nucleotide excision repair pathway. We hypothesized that single nucleotide polymorphisms (SNPs) in ERCC1 and ERCC2 are associated with the risk of colorectal cancer in a Chinese population. To test this hypothesis, we genotyped four functional SNPs (ERCC1 Asn118Asn, C8092A, ERCC2 Asp312Asn, and Lys751Gln) in a case-control study with 213 colorectal cancer cases and 240 cancer-free controls. We found that the ERCC1 C8092A polymorphism AA and CA/AA variant genotypes were associated with a significantly increased risk of colorectal cancer, compared with the CC genotype (OR = 2.50, 95% CI = 1.10–5.70 for AA versus CC, and OR = 1.58, 95% CI = 1.08–2.30 for CA/AA versus CC). Furthermore, the effect appeared to be more prominent among men, smokers, drinkers, and patients with rectal cancer. However, no other SNPs were observed for any significant association with colorectal cancer risk. These results suggest that the ERCC1 C8092A polymorphism may contribute to colorectal cancer susceptibility in the Chinese population. Further large and functional studies are needed to confirm our findings.
doi:10.1038/srep04112
PMCID: PMC3925949  PMID: 24531312
19.  The Low Chamber Pancreatic Cancer Cells Had Stem-Like Characteristics in Modified Transwell System: Is It a Novel Method to Identify and Enrich Cancer Stem-Like Cells? 
BioMed Research International  2014;2014:760303.
Cancer stem cells (CSCs) or cancer-initiating cells (CICs) play an important role in tumor initiation, progression, metastasis, chemoresistance, and recurrence. It is important to construct an effective method to identify and isolate CSCs for biotherapy of cancer. During the past years, many researchers had paid more attention to it; however, this method was still on seeking. Therefore, compared to the former methods that were used to isolate the cancer stem cell, in the present study, we tried to use modified transwell system to isolate and enrich CSCs from human pancreatic cancer cell lines (Panc-1). Our results clearly showed that the lower chamber cells in modified transwell system were easily forming spheres; furthermore, these spheres expressed high levels of stem cell markers (CD133/CD44/CD24/Oct-4/ESA) and exhibited chemoresistance, underwent epithelial-to-mesenchymal transition (EMT), and possessed the properties of self-renewal in vitro and tumorigenicity in vivo. Therefore, we speculated that modified transwell assay system, as a rapid and effective method, can be used to isolate and enrich CSCs.
doi:10.1155/2014/760303
PMCID: PMC3934619  PMID: 24689055
20.  miR-200b as a prognostic factor in breast cancer targets multiple members of RAB family 
Background
miR-200b has been reported to be a tumor suppressor and a promising therapeutic target in cancer. miR-200b has been associated with epithelial-mesenchymal transition and chemo-resistance in cancer. The aim of this study is to investigate the expression of miR-200b, its prognostic roles and its potential targets in breast cancer.
Methods
qRT-PCR was used to detect miR-200b expression in breast cancer tissues and cell lines. In situ hybridization of miR-200b on tissue microarray including 134 breast cancer samples was used to evaluate its prognostic role. Novel targets of miR-200b in breast cancer were predicted and confirmed by luciferase reporter assay and western bloting. Immunohistochemical staining was used for protein detection. The biological effects of miR-200b in breast cancer cells were further confirmed by ectopic expression of its mimics followed by MTT assay and invasion test.
Results
miR-200b was downregulated in breast cancer tissues and cell lines and its low-expression correlated with poor outcome in breast cancer patients. Members of RAB family, RAB21, RAB23, RAB18 and RAB3B were predicted to be the targets of miR-200b. The luciferase reporter assay was performed to certificate this prediction. The expressions of RAB21, RAB23, RAB18 and RAB3B were suppressed by transfection of miR-200b in breast cancer cells. Over-expression of miR-200b or knock-down of RAB21, RAB23, RAB18 and RAB3B inhibited breast cancer cell proliferation and invasion in vitro.
Conclusions
Our study provides evidence that miR-200b is a prognostic factor in breast cancer targeting multiple members of RAB family. MiR-200b could be a potential therapeutic target in breast cancer.
doi:10.1186/1479-5876-12-17
PMCID: PMC3898994  PMID: 24447584
miR-200b; RAB family; Breast cancer; Prognosis
21.  Disturbing miR-182 and -381 Inhibits BRD7 Transcription and Glioma Growth by Directly Targeting LRRC4 
PLoS ONE  2014;9(1):e84146.
Inactivated LRRC4 has been clinically detected in gliomas, and promoter hypermethylation has been implicated as the mechanism of inactivation in some of those tumors. Our previous researches indicated that LRRC4 is a target gene of miR-381, the interaction of miR-381 and LRRC4 is involved in glioma growth. In this study, we demonstrate that LRRC4 is a target gene of the other microRNA, miR-182. We found that the high expression of miR-182 and miR-381 in gliomas are involved in pathological malignant progression. The silencing of miR-182 and miR-381 inhibited the proliferation in vitro and growth of glioma cell with in vivo magnetic resonance imaging by intracranial transplanted tumor model in rats. We also demonstrated that BRD7, a transcriptional cofactor for p53, is highly expressed and negatively correlated with LRRC4 expression in gliomas. Disturbing miR-182 and miR-381 affected transcriptional regulation of the BRD7 gene. This finding was verified by ectopic overexpression of LRRC4 or restoration of endogenous LRRC4 expression by treatment with the DNA demethylating agent 5-Aza-dC. Taken together, miR-182 and miR-381 may be a useful therapeutic target for treatment of glioma.
doi:10.1371/journal.pone.0084146
PMCID: PMC3880275  PMID: 24404152
22.  A Large Cohort Study Reveals the Association of Elevated Peripheral Blood Lymphocyte-to-Monocyte Ratio with Favorable Prognosis in Nasopharyngeal Carcinoma 
PLoS ONE  2013;8(12):e83069.
Background
Nasopharyngeal carcinoma (NPC) is an endemic neoplasm in southern China. Although NPC sufferers are sensitive to radiotherapy, 20–30% of patients finally progress with recurrence and metastases. Elevated lymphocyte-to-monocyte ratio (LMR) has been reported to be associated with favorable prognosis in some hematology malignancies, but has not been studied in NPC. The aim of this study was to evaluate whether LMR could predict the prognosis of NPC patients.
Methods
A retrospective cohort of 1,547 non-metastatic NPC patients was recruited between January 2005 and June 2008. The counts for peripheral lymphocyte and monocyte were retrieved, and the LMR was calculated. Receiver operating characteristic curve analysis, univariate and multivariate COX proportional hazards analyses were applied to evaluate the associations of LMR with overall survival (OS), disease-free survival (DFS), distant metastasis-free survival (DMFS) and loco-regional recurrence-free survival (LRRFS), respectively.
Results
Univariate analysis revealed that higher LMR level (≥5.220) was significantly associated with superior OS, DFS and DMFS (P values <0.001). The higher lymphocyte count (≥2.145×109/L) was significantly associated with better OS (P = 0.002) and DMFS (P = 0.031), respectively, while the lower monocyte count (<0.475×109/L) was associated with better OS (P = 0.012), DFS (P = 0.011) and DMFS (P = 0.003), respectively. Multivariate Cox proportional hazard analysis showed that higher LMR level was a significantly independent predictor for superior OS (hazard ratio or HR  = 0.558, 95% confidence interval or 95% CI  = 0.417–0.748; P<0.001), DFS (HR  = 0.669, 95% CI  = 0.535–0.838; P<0.001) and DMFS (HR = 0.543, 95% CI  = 0.403–0.732; P<0.001), respectively. The advanced T and N stages were also independent indicators for worse OS, DFS, and DMFS, except that T stage showed borderline statistical significance for DFS (P = 0.053) and DMFS (P = 0.080).
Conclusions
The elevated pretreatment peripheral LMR level was a significant favorable factor for NPC prognosis and this easily accessed variable may serve as a potent marker to predict the outcomes of NPC patients.
doi:10.1371/journal.pone.0083069
PMCID: PMC3873908  PMID: 24386144
23.  Protein phosphorylation-acetylation cascade connects growth factor deprivation to autophagy 
Autophagy  2012;8(9):1385-1386.
Different from unicellular organisms, metazoan cells require the presence of extracellular growth factors to utilize environmental nutrients. However, the underlying mechanism was unclear. We have delineated a pathway, in which glycogen synthase kinase 3 (GSK3) in cells deprived of growth factors phosphorylates and activates the acetyltransferase KAT5/TIP60, which in turn stimulates the protein kinase ULK1 to elicit autophagy. Cells with the Kat5/Tip60 gene replaced with Kat5S86A that cannot be phosphorylated by GSK3 are resistant to serum starvation-induced autophagy. Acetylation sites on ULK1 were mapped to K162 and K606, and the acetylation-defective mutant ULK1K162,606R displays reduced kinase activity and fails to rescue autophagy in Ulk1−/− mouse embryonic fibroblasts, indicating that acetylation is vital to the activation of ULK1. The GSK3-KAT5-ULK1 cascade seems to be specific for cells to sense growth factors, as KAT5 phosphorylation is not enhanced under glucose deprivation. Distinct from the glucose starvation-autophagy pathway that is conserved in all eukaryotic organisms, the growth factor deprivation response pathway is perhaps unique to metazoan organisms.
doi:10.4161/auto.20959
PMCID: PMC3442885  PMID: 22717509
GSK3; Tip60; Ulk1; acetylation; autophagy; growth factor; phosphorylation
24.  Direct Interaction of Selenoprotein R with Clusterin and Its Possible Role in Alzheimer’s Disease 
PLoS ONE  2013;8(6):e66384.
Selenoprotein R (SelR) plays an important role in maintaining intracellular redox balance by reducing the R-form of methionine sulfoxide to methionine. As SelR is highly expressed in brain and closely related to Alzheimer′s disease (AD), its biological functions in human brain become a research focus. In this paper, the selenocysteine-coding TGA of SelR gene was mutated to cysteine-coding TGC and used to screen the human fetal brain cDNA library with a yeast two-hybrid system. Our results demonstrated that SelR interacts with clusterin (Clu), a chaperone protein. This protein interaction was further verified by fluorescence resonance energy transfer (FRET), coimmunoprecipitation (co-IP), and pull-down assays. The interacting domain of Clu was determined by co-IP to be a dynamic, molten globule structure spanning amino acids 315 to 381 with an amphipathic-helix. The interacting domain of SelR was investigated by gene manipulation, ligand replacement, protein over-expression, and enzyme activity measurement to be a tetrahedral complex consisting of a zinc ion binding with four Cys residues. Study on the mutual effect of SelR and Clu showed synergic property between the two proteins. Cell transfection with SelR gene increased the expression of Clu, while cell transfection with Clu promoted the enzyme activity of SelR. Co-overexpression of SelR and Clu in N2aSW cells, an AD model cell line, significantly decreased the level of intracellular reactive oxygen species. Furthermore, FRET and co-IP assays demonstrated that Clu interacted with β-amyloid peptide, a pathological protein of AD, which suggested a potential effect of SelR and Aβ with the aid of Clu. The interaction between SelR and Clu provides a novel avenue for further study on the mechanism of SelR in AD prevention.
doi:10.1371/journal.pone.0066384
PMCID: PMC3689823  PMID: 23805218
25.  Genomic Editing of the HIV-1 Coreceptor CCR5 in Adult Hematopoietic Stem and Progenitor Cells Using Zinc Finger Nucleases 
Molecular Therapy  2013;21(6):1259-1269.
The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5Δ32/Δ32) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5Δ32/Δ32 donors, we reasoned that engineered autologous CD34+ hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.
doi:10.1038/mt.2013.65
PMCID: PMC3677314  PMID: 23587921

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