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1.  The Phosphodiesterase-4 (PDE4) Inhibitor Rolipram Decreases Ethanol Seeking and Consumption in Alcohol-preferring Fawn-Hooded Rats 
Alcohol dependence is a complex psychiatric disorder demanding development of novel pharmacotherapies. Since the cyclic AMP (cAMP) signaling cascade has been implicated in mediating behavioral responses to alcohol, key components in this cascade may serve as potential treatment targets. Phosphodiesterase 4 (PDE4), an enzyme that specifically catalyzes the hydrolysis of cAMP, represents as a key point in regulating intracellular cAMP levels. Thus, it was of interest to determine whether PDE4 was involved in the regulation of alcohol use and abuse.
Male Fawn-Hooded (FH/Wjd) rats were tested for 5% (v/v) ethanol and 10% (w/v) sucrose operant oral self-administration following treatment with the selective PDE4 inhibitor rolipram (0.0125, 0.025, or 0.05 mg/kg, s.c.); rolipram at higher doses (0.05, 0.1, and 0.2 mg/kg, s.c.) was tested to determine its impact on the intake of ethanol, sucrose, or water using the two-bottle choice drinking paradigm. Subsequent open-field testing was performed to evaluate the influence of higher doses of rolipram on locomotor activity.
Acute administration of rolipram dose-dependently reduced operant self-administration of 5% ethanol, but had no effect on 10% sucrose responding. Time-course assessment revealed significant decreases in ethanol consumption after rolipram (0.1, 0.2 mg/kg) treatment in continuous- and intermittent-access to ethanol at 5% or 10%, respectively. Moreover, chronic rolipram treatment time-dependently decreased 5% ethanol consumption and preference during treatment days and after the termination of rolipram administration. Rolipram at the highest doses (0.1 and 0.2 mg/kg) did decrease locomotor activity, but the effect lasted only 10 and 20 min, respectively, which did not likely alter long-term ethanol drinking.
These results suggest that PDE4 plays a role in alcohol seeking and consumption behavior. Drugs interfering with PDE4 may be a potential pharmacotherapy for alcohol dependence.
PMCID: PMC4335658  PMID: 22671516
Cyclic AMP Signaling; Phosphodiesterase-4 (PDE4); Rolipram; FH/Wjd Rat; Ethanol Intake
2.  Comparison of single versus repeated methamphetamine injection induced behavioral sensitization in mice 
Neuroscience letters  2013;560:103-106.
Repeated exposure to drugs of abuse produces a persistent behavioral sensitization to stimulants, which is often used to study drug-associated behavioral plasticity. Interestingly, even a single exposure to some drugs of abuse is sufficient to elicit long-lasting behavioral sensitization. However, few studies have directly compared the magnitude of sensitization between single versus repeated drug treatments. This study examined the magnitude and duration of single methamphetamine (METH) injection-induced behavioral sensitization and compared it to the more typical repeated drug injection-induced sensitization in mice. Different groups of mice were injected with METH (0.5, 1.0, 2.0 mg/kg, i.p.) only once or daily for 7 consecutive days. A challenge dose of METH (1.0 mg/kg, i.p.) was tested 7 days later. The time-course of a single METH injection-induced behavioral sensitization was assessed where METH (2.0 mg/kg, i.p.) was injected and a challenge dose of METH (1.0 mg/kg, i.p.) was tested after different drug-free periods. Single METH injection produced similar magnitude of behavioral sensitization as compared to repeated injection. Such a sensitized locomotor response peaked 8 days after METH injection and lasted for at least 21 days. This long lasting behavioral alteration induced by single METH injection suggests the value of future studies to explore the underlying neural mechanisms, particularly in comparison to those underlying repeated METH-induced sensitization.
PMCID: PMC4017776  PMID: 24361545
Methamphetamine; Behavioural sensitization; Single-dose injection; Repeated-dose injection
3.  Rapid Identification of α-Glucosidase Inhibitors from Phlomis tuberosa by Sepbox Chromatography and Thin-Layer Chromatography Bioautography 
PLoS ONE  2015;10(2):e0116922.
Alpha-glucosidase inhibitors currently form an important basis for developing novel drugs for diabetes treatment. In our preliminary tests, the ethyl acetate fraction of Phlomis tuberosa extracts showed significant α-glucosidase inhibitory activity (IC₅₀ = 100 μg/mL). In the present study, a combined method using Sepbox chromatography and thin-layer chromatography (TLC) bioautography was developed to probe α-glucosidase inhibitors further. The ethyl acetate fraction of P. tuberosa extracts was separated into 150 individual subfractions within 20 h using Sepbox chromatography. Then, under the guidance of TLC bioautography, 20 compounds were successfully isolated from these fractions, including four new diterpenoids [14-hydroxyabieta-8,11,13-triene-11-carbaldehyde-18-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone (1), 14-hydroxyabieta-8,11,13-triene-17-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone (2), 14,16-dihydroxyabieta-8,11,13-triene-15,17-dioic acid (3), and phlomisol (15,16-eposy-8,13(16),14-labdatrien-19-ol) (4)], and 16 known compounds. Activity estimation indicated that 15 compounds showed more potent α-glucosidase inhibitory effects (with IC50 values in the range 0.067–1.203 mM) than the positive control, acarbose (IC50 = 3.72 ± 0.113 mM). This is the first report of separation of α-glucosidase inhibitors from P. tuberosa.
PMCID: PMC4319760  PMID: 25658100
4.  Gene coexpression networks reveal key drivers of phenotypic divergence in porcine muscle 
BMC Genomics  2015;16(1):50.
Domestication of the wild pig has led to obese and lean phenotype breeds, and evolutionary genome research has sought to identify the regulatory mechanisms underlying this phenotypic diversity. However, revealing the molecular mechanisms underlying muscle phenotype variation based on differentially expressed genes has proved to be difficult. To characterize the mechanisms regulating muscle phenotype variation under artificial selection, we aimed to provide an integrated view of genome organization by weighted gene coexpression network analysis.
Our analysis was based on 20 publicly available next-generation sequencing datasets of lean and obese pig muscle generated from 10 developmental stages. The evolution of the constructed coexpression modules was examined using the genome resequencing data of 37 domestic pigs and 11 wild boars. Our results showed the regulation of muscle development might be more complex than had been previously acknowledged, and is regulated by the coordinated action of muscle, nerve and immunity related genes. Breed-specific modules that regulated muscle phenotype divergence were identified, and hundreds of hub genes with major roles in muscle development were determined to be responsible for key functional distinctions between breeds. Our evolutionary analysis showed that the role of changes in the coding sequence under positive selection in muscle phenotype divergence was minor.
Muscle phenotype divergence was found to be regulated by the divergence of coexpression network modules under artificial selection, and not by changes in the coding sequence of genes. Our results present multiple lines of evidence suggesting links between modules and muscle phenotypes, and provide insights into the molecular bases of genome organization in muscle development and phenotype variation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1238-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4328970  PMID: 25651817
Muscle; Modules; Weighted gene coexpression network analysis; Phenotype variation; Artificial selection
5.  Impact on growth and invasion of gastric cancer cell lines by silencing NEDD9 
OncoTargets and therapy  2015;8:223-231.
Gastric adenocarcinoma is a predominant disease with latent attribute, high malignancy, and poor prognosis in People’s Republic of China. Gastric cancer is the most common malignant tumor of the digestive tract. It has been suggested that abnormal expression of NEDD9 was associated with stage progression and metabolism of carcinomas. Some authors demonstrated that both messenger RNA (mRNA) and protein of NEDD9 were highly expressed in gastric cancer, and paired paracancerous atypical hyperplasia tissues were correlated with lymph node metastasis, tumor depth, and tumor-lymph node-metastasis (TNM) staging. In this study, we found that NEDD9 small interfering RNA (siRNA) can induce apoptosis and suppress proliferation, migration, and invasion of BGC823 cell lines. These findings suggested that NEDD9 siRNA might serve as a tumor suppressor by targeting NEDD9 in gastric cancer cell. It has been suggested that abnormal expression of NEDD9 was associated with carcinogenesis, and in the first part of the study, we found that NEDD9 was highly expressed in gastric cancer tissues; and it too was correlated with lymph node metastasis, tumor depth, and TNM staging. In this project, experiments were carried out to silence NEDD9 in BGC823 cell lines using NEDD9 siRNA, and the biological activity of BGC823 cells was observed after RNA interference.
The target analysis of NEDD9 siRNA was forecast using online tools. In order to determine a more efficient NEDD9 siRNA, three pairs of NEDD9 siRNA primer were designed, synthesized, and then transfected into BGC823 cells. NEDD9-2 siRNA was finally adopted by detecting the quantitative real-time polymerase chain reaction (qRT-PCR). Cells were collected for detecting mRNA by qRT-PCR or protein by western blot analysis. Cell apoptosis was detected using flow cytometry, and the transwell invasion system was used for cell migration and invasion assays. The effect of NEDD9 siRNA in silencing the target gene in BGC823 cells was then assessed. Also, the impact of NEDD9-2 siRNA on cell proliferation, apoptosis, migration, and invasion were detected in BGC823 cell lines.
The relative quantity of expression of mRNA and protein showed a decrease in all cells transfected with siNEDD9-2 at different concentrations. The cell proliferation inhibition assay showed that the inhibition rate was significantly increased in all transfected cells compared with control groups. Cell apoptosis assay showed that the number of living cells were significantly reduced compared with control groups, and cell migration and invasion assay showed that siNEDD9 could inhibit BGC823 cell migration and invasion in vitro.
NEDD9 siRNA could inhibit expression of NEDD9 and induce apoptosis, suppress proliferation, migration, and invasion of BGC823 cells, acting as a tumor suppressor in carcinogenesis of gastric cancer. These findings suggested that NEDD9 siRNA plays an important role in the proliferation, apoptosis, and invasion of BGC823 cells.
PMCID: PMC4309781  PMID: 25653543
NEDD9 siRNA; apoptosis
6.  Heterogeneous intrinsic excitability of murine spiral ganglion neurons is determined by Kv1 and HCN channels 
Neuroscience  2013;257:96-110.
The spiral ganglion conveys afferent auditory information predominantly through a single class of type I neurons that receive signals from inner hair cell (IHC) sensory receptors. These auditory primary afferents, like in other systems (Puopolo and Belluzzi, 1998, Gascon and Moqrich, 2010, Leao et al., 2012) possess a marked diversity in their electrophysiological features (Taberner and Liberman, 2005). Consistent with these observations, when the auditory primary afferents were assessed in neuronal explants separated from their peripheral and central targets it was found that individual neurons were markedly heterogeneous in their endogenous electrophysiological features. One aspect of this heterogeneity, obvious throughout the ganglion, was their wide range of excitability as assessed by voltage threshold measurements (Liu and Davis, 2007). Thus, while neurons in the base differed significantly from apical and middle neurons in their voltage thresholds, each region showed distinctly wide ranges of values. To determine whether the resting membrane potentials (RMP) of these neurons correlate with the threshold distribution and to identify the ion channel regulatory elements underlying heterogeneous neuronal excitability in the ganglion, patch-clamp recordings were made from postnatal day (P5-8) murine spiral ganglion neurons in vitro. We found that RMP mirrored the tonotopic threshold distribution, and contributed an additional level of heterogeneity in each cochlear location. Pharmacological experiments further indicated that threshold and RMP was coupled through the Kv1 current, which had a dual impact on both electrophysiological parameters. Whereas, hyperpolarization-activated cationic channels (HCN) decoupled these two processes by primarily affecting RMP without altering threshold level. Thus, beyond mechanical and synaptic specializations, ion channel regulation of intrinsic membrane properties imbues spiral ganglion neurons with different excitability levels, a feature that contributes to primary auditory afferent diversity.
PMCID: PMC3897933  PMID: 24200924
spiral ganglion neuron; intrinsic excitability; Kv1; Ih; auditory
7.  Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves 
Plant Biotechnology Journal  2013;12(2):231-239.
High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.
PMCID: PMC4285938  PMID: 24151938
triacylglycerol; Nicotiana tabacum; leaf; WRI1; DGAT1; oleosin
8.  Detection of molecular alterations in methamphetamine-activated Fos-expressing neurons from a single rat dorsal striatum using fluorescence-activated cell sorting (FACS) 
Journal of neurochemistry  2013;128(1):10.1111/jnc.12381.
Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a FACS-based method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. However, this method requires pooling samples from many rats. We now describe a modified FACS-based method to characterize molecular alterations in Fos-expressing dorsal striatal neurons from a single rat using a multiplex pre-amplification strategy. Fos and NeuN (a neuronal marker) immunohistochemistry indicate that 6–7% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p) while less than 1% of neurons were activated by saline injections. We used FACS to separate NeuN-labeled neurons into Fos-positive and Fos-negative neurons and assessed mRNA expression using RT-qPCR from as little as 5 Fos-positive neurons. Methamphetamine induced 3–20-fold increases of immediate early genes arc, homer-2, c-fos, fosB and its isoforms (ΔfosB and a novel isoform ΔfosB-2) in Fos-positive but not Fos-negative neurons. IEG mRNA induction was 10-fold lower or absent when assessed in unsorted samples from single dorsal striatum homogenates. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models.
PMCID: PMC3867527  PMID: 23895375
flow cytometry; immediate early genes; gene expression
9.  Endocannabinoid Signaling in the Etiology and Treatment of Major Depressive Illness 
Current pharmaceutical design  2014;20(23):3795-3811.
The purpose of this review is to examine human and preclinical data that are relevant to the following hypotheses. The first hypothesis is that deficient CB1R-mediated signaling results in symptoms that mimic those seen in depression. The second hypothesis is that activation of CB1R-mediated signaling results in behavioral, endocrine and other effects that are similar to those produced by currently used antidepressants. The third hypothesis is that conventional antidepressant therapies act through enhanced CB1R mediated signaling. Together the available data indicate that activators of CB1R signaling, particularly inhibitors of fatty acid amide hydrolase, should be considered for clinical trials for the treatment of depression.
PMCID: PMC4002665  PMID: 24180398
CB1 receptor; 2-arachidonoylglycerol; fatty acid amide hydrolase; URB597; tetrahydrocannabinol; genetics; circulation
10.  Clinical observation of the therapeutic effects of pegylated recombinant human granulocyte colony-stimulating factor in patients with concurrent chemoradiotherapy-induced grade IV neutropenia 
The aim of the present study was to investigate the efficacy and side-effects of preventive treatment with pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) on concurrent chemoradiotherapy-induced grade IV neutropenia and to provide a rational basis for its clinical application. A total of 114 patients with concurrent chemoradiotherapy-induced grade IV neutropenia were enrolled. A randomized approach was used to divide the patients into an experimental group and a control group. The experimental group included three subgroups, namely a P-50 group, P-100 group and P + R group. The P-50 group had 42 cases, which were given a single 50-μg/kg subcutaneous injection of PEG-rhG-CSF. The P-100 group had 30 cases, which received a single 100-μg/kg subcutaneous injection of PEG-rhG-CSF. The P + R group comprised 22 cases, which were given a single 50-μg/kg subcutaneous injection of PEG-rhG-CSF and rhG-CSF 5 μg/kg/day; when the absolute neutrophil count (ANC) was ≥2.0×109/l, the administration of rhG-CSF was stopped. The control group (RC group) comprised 20 patients, who received rhG-CSF 5 μg/kg/day by subcutaneous injection until the ANC was ≥2.0×109/l. Changes in the neutrophil proliferation rate and ANC values over time, the neutropenic symptom remission time and incidence of adverse drug reactions were analyzed statistically in each group of patients. In the experimental group, the neutrophil proliferation rate and ANC values were significantly higher than those in the control group; the clinical effects began 12–24 h after treatment in the experimental group, and indicated that the treatment improved neutropenia in ~48 h after treatment. There was no significant difference in the neutrophil proliferation rate and ANC values between the P-50 and P+R groups. In the experimental group, the remission time of neutropenia-induced fever and muscle pain after administration was significantly shorter than that in the control group, with a statistically significant difference (P<0.05). The adverse drug reaction rates showed no significant difference between the experimental group and the control group. PEG-rhG-CSF had good efficacy and safety in the treatment of concurrent chemotherapy-induced grade IV neutropenia. For the treatment of concurrent chemotherapy-induced grade IV neutropenia, a single subcutaneous injection of 50 μg/kg PEG-rhG-CSF is the recommended dose. The effects begin at 12–24 h; if the ANC values are not significantly improved during this time, no supplementary administration of rhG-CSF is necessary.
PMCID: PMC4316958  PMID: 25667625
pegylated recombinant human granulocyte colony-stimulating factor; concurrent chemoradiotherapy; grade IV neutropenia
11.  Influence factors for successful corneal donation among Chinese adults: data from Nanjing between 2001 and 2012 
To investigate the factors that may influence the successful corneal donation among adults in China.
This retrospective study was conducted in 2012. The eligible participants were all the adults registered in Nanjing Red Cross Eye Bank to donate their corneas after death during the period of 2001 and 2012. Multivariate logistic regression models were applied to investigate the influence factors for successful donation, the outcome events.
Totally, 210 of 328 (64.0%) registered potential donors successfully donated their corneas after death. The mean (SD) age at registration was 64.7 (12.5) for all participants, with 65.5 (10.1) and 63.2 (15.8) for successful and unsuccessful donors, respectively. With multivariate logistic regression analysis, five factors, the willingness of donation, age, education level, residence area, and cause of death were identified to be associated with successful corneal donation.
The willingness of donation and some socio-demographic factors might substantially affect their successful donation after death for people who registered to donate corneas.
PMCID: PMC4270993  PMID: 25540751
cornea donation; influence factors; China
12.  Two functional loci in the promoter of EPAS1 gene involved in high-altitude adaptation of Tibetans 
Scientific Reports  2014;4:7465.
EPAS1 involves in the hypoxic response and is suggested to be responsible for the genetic adaptation of high-altitude hypoxia in Tibetans. However, the detailed molecular mechanism remains unknown. In this study, a single nucleotide polymorphism rs56721780:G>C and an insertion/deletion (indel) polymorphism −742 indel in the promoter region showed divergence between Tibetans and non-Tibetan lowlanders. rs56721780:G>C regulated the transcription of EPAS1 by IKAROS family zinc finger 1 (IKZF1), which was identified as a new transcriptional repressor for EPAS1 gene. It demonstrated that the C allele of rs56721780:G>C decreased the binding of IKZF1, leading to the attenuated transcriptional repression of EPAS1 gene. The insertion at −742 indel provided a new binding site for Sp1 and was related to the activation of EPAS1 promoter. Further functional analysis revealed that lysyl oxidase (LOX) gene, which was reported to be responsible for extracellular matrix protein cross-linking of amnion previously, was a direct target of EPAS1. The CC genotype at rs56721780:G>C and the insertion genotype at −742 indel were found associated with higher EPAS1 and LOX expression levels in amnion, as well as higher birth weight of Tibetan newborns, suggesting that EPAS1 gene might play important roles in the development of amnion, fetus growth and high-altitude adaptation of Tibetans.
PMCID: PMC4264014  PMID: 25501874
13.  An Outbreak of Type Π Vaccine-Derived Poliovirus in Sichuan Province, China: Emergence and Circulation in an Under-Immunized Population 
PLoS ONE  2014;9(12):e113880.
During August 2011–February 2012, an outbreak of type Π circulating vaccine-derived poliovirus (cVDPVs) occurred in Sichuan Province, China.
A field investigation of the outbreak was conducted to characterize outbreak isolates and to guide emergency response. Sequence analysis of poliovirus capsid protein VP1 was performed to determine the viral propagation, and a coverage survey was carried out for risk assessment.
One clinical compatible polio case and three VDPV cases were determined in Ngawa County, Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province. Case patients were unimmunized children, 0.8–1 years old. Genetic sequencing showed that the isolates diverged from the VP1 region of the type Π Sabin strain by 5–12 nucleotides (nt) and shared the same 5 nt VP1 substitutions, which indicate single lineage of cVDPVs. Of the 7 acute flaccid paralysis cases (all>6 months) reported in Ngawa Prefecture in 2011, 4 (57.1%) cases (including 2 polio cases) did not receive oral attenuated poliovirus vaccine. Supplementary immunization activities (SIAs) were conducted in February–May, 2012, and the strain has not been isolated since.
High coverage of routine immunization should be maintained among children until WPV transmission is globally eradicated. Risk assessments should be conducted regularly to pinpoint high risk areas or subpopulations, with SIAs developed if necessary.
PMCID: PMC4263476  PMID: 25503964
14.  Computed tomography angiography manifestations of collateral circulations in Budd-Chiari syndrome 
The aim of this study was to assess the computed tomography angiography (CTA) manifestations of collateral circulations in patients with Budd-Chiari syndrome (BCS). Eighty patients with BCS were examined by CT scan. Using the CTA images of the relevant blood vessels, including the affected hepatic veins (HVs) and inferior venae cavae (IVCs), the collateral circulations were reconstructed. In addition to obstructed HVs and IVCs, collateral circulations were found in each of the patients. The collateral circulations were classified as intrahepatic, extrahepatic and portosystemic pathways. Intrahepatic collateral pathways were further classified as the following six types: HV-accessory HV (n=51, 63.8%), HV-HV (n=6, 7.5%), HV-accessory HV plus HV (n=6, 7.5%), IVC-HV/accessory HV-HV-right atrium (n=5, 6.3%), HV-umbilical vein (n=4, 5.0%) and HV-inferior phrenic vein (n=8, 10.0%). Extrahepatic collateral pathways included IVC-lumbar-ascending lumbar-hemiazygos/azygos vein (n=80, 100.0%), IVC-left renal-ascending lumbar-hemiazygos vein (n=75, 93.8%), IVC-left renal-inferior phrenic vein (n=49, 61.3%), IVC-renal -peri-renal -superficial epigastric vein (n=26, 32.5%) and superficial epigastric vein (n=12, 15.0%) types. The CTA characteristics of each type of collateral circulation were demonstrated. In conclusion, the present study revealed that CTA is able to show the intra- and extrahepatic collateral circulations of patients with BCS, which may be useful for therapeutic planning.
PMCID: PMC4280983  PMID: 25574205
Budd-Chiari syndrome; computed tomography angiography; collateral circulation
15.  The role of death receptor 3 in the biological behavior of hepatocellular carcinoma cells 
Molecular Medicine Reports  2014;11(2):797-804.
Death receptor 3 (DR3) belongs to the tumor necrosis factor (TNF) receptor superfamily, primarily found in lymphoid tissues. Reports have determined that DR3 may also be distributed in numerous types of tumors. Therefore, it is thought that DR3 may have an important role in the process of tumorigenesis. The aim of the present study was to observe the effect of silencing DR3 expression on hepatocarcinoma cell growth, apoptosis and invasion in order to elucidate the role of DR3 in tumor development. The hepatocarcinoma cell lines (HepG2, Huh7, SMMC7721 and Bel-7402) and normal human liver cells (HL-7702) were transfected with three stealth RNA interference (RNAi) sequences that target the DR3 gene. Reverse transcription quantitative polymerase chain reaction was used to detect the expression levels of DR3 in hepatocarcinoma cell lines and normal liver HL-7702 cells. MTT assay and flow cytometry (FCM) were used to determine the rates of cell proliferation and apoptosis, respectively. Following silencing of the DR3 gene, western blot analysis was used to determine the protein expression of P53, Fas, Caspase8, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Caspase3. DR3 messenger RNA (mRNA) expression in hepatocarcinoma cell lines was significantly increased compared with that in the normal liver cell line. Three targeted DR3 gene small interfering RNAs significantly inhibited DR3 gene expression in Bel-7402 cells at the nucleic acid level. AF02670.1_stealth_883 and cocktail demonstrated the most efficient inhibition of DR3 gene expression at 48 and 72 h following transfection, with mRNA inhibition rates of 89.46 and 92.75%, and 90.53 and 94.25% (P<0.01), respectively. Cell viability was significantly reduced by AF02670.1_stealth_883 and RNAi cocktail at 24, 48 and 72 h following transfection. The inhibition rates of cell proliferation were 50.76 and 61.76% (P<0.05) at 72 h following transfection. FCM revealed that AF02670.1_stealth_883 and RNAi cocktail also induced apoptosis in Bel-7402 cells at 72 h following transfection. Reduction of NF-κB and P53 levels was observed (P<0.05) in Bel-7402 cells following DR3 silencing, whereas levels of Fas, Caspase3 and Caspase8 were markedly elevated (P<0.05). DR3 expression levels in hepatocellular carcinoma cells were significantly higher than those in normal cells. DR3 silencing effectively inhibited proliferation and invasion of hepatocellular carcinoma cells in vitro. However, silencing of the DR3 gene affect levels of apoptosis antigen-3 ligand in cells, therefore indicating that it may be involved with other pathways that regulate apoptosis in HCCs. In conclusion, the results of the present study indicated that DR3 may be a promising therapeutic target molecule for further study of hepatocellular carcinoma gene therapy.
PMCID: PMC4262488  PMID: 25370568
hepatocellular carcinoma; death receptor 3; RNA interference; apoptosis; caspase3; apoptosis antigen-3 ligand
16.  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
17.  Cyclin-Dependent Kinase 5 in the Ventral Tegmental Area Regulates Depression-Related Behaviors 
The Journal of Neuroscience  2014;34(18):6352-6366.
Dopamine neurons in the ventral tegmental area (VTA) govern reward and motivation and dysregulated dopaminergic transmission may account for anhedonia and other symptoms of depression. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that regulates a broad range of brain functions through phosphorylation of a myriad of substrates, including tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis. We investigated whether and how Cdk5 activity in VTA dopamine neurons regulated depression-related behaviors in mice. Using the Cre/LoxP system to selectively delete Cdk5 in the VTA or in midbrain dopamine neurons in Cdk5loxP/loxP mice, we showed that Cdk5 loss of function in the VTA induced anxiety- and depressive-like behaviors that were associated with decreases in TH phosphorylation at Ser31 and Ser40 in the VTA and dopamine release in its target region, the nucleus accumbens. The decreased phosphorylation of TH at Ser31 was a direct effect of Cdk5 deletion, whereas decreased phosphorylation of TH at Ser40 was likely caused by impaired cAMP/protein kinase A (PKA) signaling, because Cdk5 deletion decreased cAMP and phosphorylated cAMP response element-binding protein (p-CREB) levels in the VTA. Using Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology, we showed that selectively increasing cAMP levels in VTA dopamine neurons increased phosphorylation of TH at Ser40 and CREB at Ser133 and reversed behavioral deficits induced by Cdk5 deletion. The results suggest that Cdk5 in the VTA regulates cAMP/PKA signaling, dopaminergic neurotransmission, and depression-related behaviors.
PMCID: PMC4004818  PMID: 24790206
cAMP; Cdk5; depression; dopamine; DREADD; tyrosine hydroxylase
18.  Protection of Tong-Sai-Mai Decoction against Apoptosis Induced by H2O2 in PC12 Cells: Mechanisms via Bcl-2-Mitochondria-ROS-INOS Pathway 
Tong-Sai-Mai decoction (TSM) is a Chinese materia medica polyherbal formulation that has been applied in treating brain ischemia for hundreds of years. Because it could repress the oxidative stress in in vivo studies, now we focus on the in vitro studies to investigate the mechanism by targeting the oxidative stress dependent signaling. The relation between the neurogenesis and the reactive oxygen species (ROS) production remains largely unexamined. PC12 cells are excitable cell types widely used as in vitro model for neuronal cells. Most marker genes that are related to neurotoxicity, apoptosis, and cell cycles are expressed at high levels in these cells. The aim of the present study is to explore the cytoprotection of TSM against hydrogen peroxide- (H2O2-) induced apoptosis and the molecular mechanisms underlying PC12 cells. Our findings revealed that TSM cotreatment with H2O2 restores the expression of bcl-2, inducible nitric oxide synthase (INOS), and mitochondria membrane potential. Meanwhile, it reduces intracellular [Ca2+] concentration, lactate dehydrogenase (LDH) release, and the expression of caspase-3 and bax. The results of the present study suggested that the cytoprotective effects of the TSM might be mediated, at least in part, by the bcl-2-mitochondria-ROS-INOS pathway. Due to its nontoxic characteristics, TSM could be further developed to treat the neurodegenerative diseases which are closely associated with the oxidative stress.
PMCID: PMC4227446  PMID: 25404948
19.  Prediction of disease-related genes based on weighted tissue-specific networks by using DNA methylation 
BMC Medical Genomics  2014;7(Suppl 2):S4.
Predicting disease-related genes is one of the most important tasks in bioinformatics and systems biology. With the advances in high-throughput techniques, a large number of protein-protein interactions are available, which make it possible to identify disease-related genes at the network level. However, network-based identification of disease-related genes is still a challenge as the considerable false-positives are still existed in the current available protein interaction networks (PIN).
Considering the fact that the majority of genetic disorders tend to manifest only in a single or a few tissues, we constructed tissue-specific networks (TSN) by integrating PIN and tissue-specific data. We further weighed the constructed tissue-specific network (WTSN) by using DNA methylation as it plays an irreplaceable role in the development of complex diseases. A PageRank-based method was developed to identify disease-related genes from the constructed networks. To validate the effectiveness of the proposed method, we constructed PIN, weighted PIN (WPIN), TSN, WTSN for colon cancer and leukemia, respectively. The experimental results on colon cancer and leukemia show that the combination of tissue-specific data and DNA methylation can help to identify disease-related genes more accurately. Moreover, the PageRank-based method was effective to predict disease-related genes on the case studies of colon cancer and leukemia.
Tissue-specific data and DNA methylation are two important factors to the study of human diseases. The same method implemented on the WTSN can achieve better results compared to those being implemented on original PIN, WPIN, or TSN. The PageRank-based method outperforms degree centrality-based method for identifying disease-related genes from WTSN.
PMCID: PMC4243158  PMID: 25350763
20.  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.
PMCID: PMC4221975  PMID: 25400316
21.  Effects of miR-33a-5P on ABCA1/G1-Mediated Cholesterol Efflux under Inflammatory Stress in THP-1 Macrophages 
PLoS ONE  2014;9(10):e109722.
The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages.
PMCID: PMC4201478  PMID: 25329888
22.  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.
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).
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.
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.
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.
PMCID: PMC4198111  PMID: 25333965
23.  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.
PMCID: PMC4198143  PMID: 25333522
24.  Evolutionarily Conserved Repulsive Guidance Role of Slit in the Silkworm Bombyx mori 
PLoS ONE  2014;9(10):e109377.
Axon guidance molecule Slit is critical for the axon repulsion in neural tissues, which is evolutionarily conserved from planarians to humans. However, the function of Slit in the silkworm Bombyx mori was unknown. Here we showed that the structure of Bombyx mori Slit (BmSlit) was different from that in most other species in its C-terminal sequence. BmSlit was localized in the midline glial cell, the neuropil, the tendon cell, the muscle and the silk gland and colocalized with BmRobo1 in the neuropil, the muscle and the silk gland. Knock-down of Bmslit by RNA interference (RNAi) resulted in abnormal development of axons and muscles. Our results suggest that BmSlit has a repulsive role in axon guidance and muscle migration. Moreover, the localization of BmSlit in the silk gland argues for its important function in the development of the silk gland.
PMCID: PMC4186835  PMID: 25285792
25.  Sparstolonin B Attenuates Hypoxia-Induced Apoptosis, Necrosis and Inflammation in Cultured Rat Left Ventricular Tissue Slices 
Ischemia/reperfusion results in tissue damage, a rapid increase in cytokines and chemokines and inflammatory cell infiltration. Herein we investigated the ability of a selective TLR2/4 antagonist, Sparstolonin B (SsnB), to protect rat cultured left ventricular tissue (LV) slices from hypoxic injury by inhibiting the myocardial inflammatory response independent of inflammatory cell infiltration.
Methods and Results
Media Lactate dehydrogenase (LDH) levels were measured to reflect hypoxia-induced cytotoxicity and cell injury with and without SsnB. Incubation with SsnB (15 and 30 μM) significantly reduced by 20 and 40 %, respectively, the amount of LDH released from the hypoxic LV slices. TUNEL staining showed that SsnB significantly attenuated the levels of hypoxia-induced apoptotic cells from 61.5 ±4.0 to 27.0±2.1 (15 μM SsnB) and 23.5±2.2 (30 μM SsnB) cells/unit area. Similarly, the Periodic Acid-Schiff (PAS) staining of ischemic areas in untreated hypoxic LV slices was increased 17 fold from 0.26±0.09 to 4.41±0.43 %, while in hypoxic slices incubated with 15 and 30 μM of SsnB, the PAS positive ischemic areas were increased by only 6.4 fold to 1.66±0.39 % and 3.8 fold to 1.00±0.22 %, respectively. Rt-PCR confirmed that MCP1 and IL-6 expression during hypoxia was elevated by 2 and 4 fold, respectively, while their upregulation was significantly inhibited (i.e., <0.7 fold increase) by SsnB.
The selective TLR2/4 antagonist, Sparstolonin B, can substantially protect LV myocardium via its ability to inhibit injury resulting from hypoxic myocardial-generated inflammation. Accordingly SsnB has potential as a therapeutic agent for the attenuation of myocardial ischemia-reperfusion injury.
PMCID: PMC4164598  PMID: 25117676
Myocardial ischemia injury; LDH; Apoptosis; Necrosis; Inflammation

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