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1.  Transcriptional analysis of porcine circovirus-like virus P1 
BMC Veterinary Research  2014;10(1):287.
Recently identified porcine circovirus-like virus P1 has the smallest DNA viral genome. In this study, we identified the viral genes and their corresponding mRNA transcripts.
The RNAs of P1, synthesized in porcine kidney cells, were examined with northern blotting and PCR analyses.
Eight virus-specific RNAs were detected. Four mRNAs (open reading frames (ORFs) 1, 2, 4, and 5) are encoded by the viral (−) strand and four (ORFs 3, 6, 7, and 8) are encoded by the viral (+) strand. All proteins encoded by the ORFs of the P1 virus are less than 50 amino acids in length, except that encoded by ORF1 (113 amino acids).
We show a very complex viral transcription pattern in P1-infected cells.
PMCID: PMC4258304  PMID: 25440084
Porcine circovirus like virus P1; Transcriptional analysis; Northern blotting; RACE
2.  miR-101 Suppresses Vascular Endothelial Growth Factor C That Inhibits Migration and Invasion and Enhances Cisplatin Chemosensitivity of Bladder Cancer Cells 
PLoS ONE  2015;10(2):e0117809.
The microRNA miR-101 is downregulated in several cancers, including bladder cancer. However, miR-101’s role in the invasion, metastasis, and chemosensitivity of bladder cancer cells remains unclear. This study was conducted to determine miR-101’s role on the lymphangiogenic molecule vascular endothelial growth factor C (VEGF-C) and their effects upon bladder cancer cell migration, invasion, and chemosensitivity to cisplatin.
Two bladder cancer cell lines (T24 and 5637) and the tool cell line 293T were employed here. Bladder cancer cells were transfected with either a miR-101 overexpression vector or a scrambled-sequence lentivirus, both of which exhibited a high transfection efficiency. Non-transfection was used as a mock negative control. Wound healing and Transwell assays were performed to measure cell migration and invasiveness. A luciferase reporter assay was performed to validate miR-101’s interaction with VEGF-C’s 3′ untranslated region followed by RT-PCR and Western blot confirmation. An MTS assay was used to evaluate the cisplatin sensitivity of the cell lines.
miR-101 overexpression significantly inhibited the migration and invasiveness while significantly enhancing cisplatin sensitivity. miR-101 negatively regulated VEGF-C protein expression, and VEGF-C overexpression rescued the effects of miR-101 overexpression, indicating that miR-101 negatively regulates VEGF-C protein expression post-transcriptionally. miR-101 and VEGF-C interference independently enhanced cisplatin cytotoxicity in bladder cancer cells.
miR-101 suppresses VEGF-C expression, inhibits cell migration and invasion, and increases cisplatin sensitivity in bladder cancer cells. This study provides new insight into miR-101’s role in bladder cancer and shows miR-101’s promise as a potential molecular target for bladder cancer.
PMCID: PMC4320037  PMID: 25658842
3.  Tuning LeSPL-CNR expression by SlymiR157 affects tomato fruit ripening 
Scientific Reports  2015;5:7852.
In plants, microRNAs (miRNAs) play essential roles in growth, development, yield, stress response and interactions with pathogens. However no miRNA has been experimentally documented to be functionally involved in fruit ripening although many miRNAs have been profiled in fruits. Here we show that SlymiR157 and SlymiR156 differentially modulate ripening and softening in tomato (Solanum lycopersicum). SlymiR157 is expressed and developmentally regulated in normal tomato fruits and in those of the Colourless non-ripening (Cnr) epimutant. It regulates expression of the key ripening gene LeSPL-CNR in a likely dose-dependent manner through miRNA-induced mRNA degradation and translation repression. Viral delivery of either pre-SlymiR157 or mature SlymiR157 results in delayed ripening. Furthermore, qRT-PCR profiling of key ripening regulatory genes indicates that the SlymiR157-target LeSPL-CNR may affect expression of LeMADS-RIN, LeHB1, SlAP2a and SlTAGL1. However SlymiR156 does not affect the onset of ripening, but it impacts fruit softening after the red ripe stage. Our findings reveal that working together with a ripening network of transcription factors, SlymiR157 and SlymiR156 form a critical additional layer of regulatory control over the fruit ripening process in tomato.
PMCID: PMC4297963  PMID: 25597857
4.  Complete Genome Sequence of Porcine Parvovirus N Strain Isolated from Guangxi, China 
Genome Announcements  2015;3(1):e01359-14.
We report here the complete genomic sequence of the porcine parvovirus (PPV) N strain, isolated in 1989 from the viscera of a stillborn fetus farrowed by a gilt in Guangxi, southern China. Phylogenetic analyses suggest that the PPV-N strain is closely related to attenuated PPV NADL-2 strains. The PPV-N strain has good immunogenicity, genetic stability, and safety.
PMCID: PMC4290985  PMID: 25573932
5.  Elucidating Pharmacological Mechanisms of Natural Medicines by Biclustering Analysis of the Gene Expression Profile: A Case Study on Curcumin and Si-Wu-Tang 
Natural medicines have attracted wide attention in recent years. It is of great significance to clarify the pharmacological mechanisms of natural medicines. In prior studies, we established a method for elucidating pharmacological mechanisms of natural products contained in connectivity map (cMap), in terms of module profiles of gene expression in chemical treatments. In this study, we explore whether this methodology is applicable to dissecting the pharmacological mechanisms of natural medicines beyond the agents contained in cMap. First, the gene expression profiles of curcumin (a typical isolated natural medicine) and Si-Wu-Tang (a classic traditional Chinese medicine formula) treatments were merged with those of cMap-derived 1309 agents, respectively. Then, a biclustering analysis was performed using FABIA method to identify gene modules. The biological functions of gene modules provide preliminary insights into pharmacological mechanisms of both natural medicines. The module profile can be characterized by a binary vector, which allowed us to compare the expression profiles of natural medicines with those of cMap-derived agents. Accordingly, we predicted a series of pharmacological effects for curcumin and Si-Wu-Tang by the indications of cMap-covered drugs. Most predictions were supported by experimental observations, suggesting the potential use of this method in natural medicine dissection.
PMCID: PMC4307259  PMID: 25551600
natural medicines; pharmacology; biclustering analysis; curcumin; Si-Wu-Tang
6.  The influence of fuel mass load, oxygen supply and burning rate on emission factor and size distribution of carbonaceous particulate matter from indoor corn straw burning 
The uncertainty in emission estimation is strongly associated with the variation in emission factor which could be influenced by a variety of factors, like fuel property, stove type, fire management and even methods used in measurements. The impacts of these factors were usually complicated and often interacted with each other. In the present study, controlled burning experiments were conducted to investigate the influence of fuel mass load, air supply and burning rate on the emission of carbonaceous particulate matter (PM) from indoor corn straw burning. Their impacts on PM size distribution were also studied. The results showed that EFs of PM (EFPM), organic carbon (EFOC) and element carbon (EFEC) was independent of the fuel mass load. The differences among them under different burning rates or air supply amounts were also found to be insignificant (p > 0.05) in the tested circumstances. PM from the indoor corn straw burning was dominated by fine PM, and PM with diameter less than 2.1 μm contributed about 86.4±3.9% of the total. The size distribution of PM was also influenced by the burning rate and changed air supply conditions. On average, EFPM, EFOC and EFEC for corn straw burned in a residential cooking stove were 3.84±1.02, 0.846±0.895 and 0.391±0.350 g/kg, respectively. EFPM, EFOC and EFEC were found to be positively correlated with each other, but they were not significantly correlated with EF of co-emitted CO, suggesting a special attention should be paid to the use of CO acting as a surrogate for other incomplete pollutants.
PMCID: PMC4280911  PMID: 23923424
indoor corn straw burning; emission factor; size distribution; influencing factor
7.  Emissions of parent, nitrated, and oxygenated polycyclic aromatic hydrocarbons from indoor corn straw burning in normal and controlled combustion conditions 
Emission factors (EFs) of parent polycyclic aromatic (pPAHs), nitrated PAHs (nPAHs), and oxygenated PAHs (oPAHs) were measured for indoor corn straw burned in a cooking brick stove in both normal and controlled burning conditions. EFs of total 28 pPAHs, 6 nPAHs and 4 oPAHs were 7.9±3.4, 6.5±1.6×10-3, and 6.1±1.4×10-1 mg/kg, respectively. By controlling the burning conditions, it was found that the influence of fuel charge size on EFs of the pPAHs and derivatives was insignificant. Measured EFs increased significantly in a fast burning mainly because of the oxygen deficient atmosphere formed in the stove chamber with a small volume. In both restricted and enhance air supply conditions, EFs of pPAHs, nPAHs and oPAHs were significantly higher than those measured in normal burning conditions. Though EFs varied in different burning conditions, the composition profiles and calculated isomer ratios were similar without significant differences. The results from the stepwise regression model showed that fuel burning rate, air supply amount, and modified combustion efficiency were three most significant influencing factors, explaining 72-85% of the total variations.
PMCID: PMC4280912  PMID: 24494494
polycyclic aromatic hydrocarbons; PAH derivatives; emission factor; influencing factor; indoor crop straw burning
8.  The Influence of Fuel Moisture, Charge Size, Burning Rate and Air Ventilation Conditions on Emissions of PM, OC, EC, Parent PAHs, and Their Derivatives from Residential Wood Combustion 
Controlled combustion experiments were conducted to investigate the influence of fuel charge size, moisture, air ventilation and burning rate on the emission factors (EFs) of carbonaceous particulate matter, parent polycyclic aromatic hydrocarbons (pPAHs) and their derivatives from residential wood combustion in a typical brick cooking stove. Measured EFs were found to be independent of fuel charge size, but increased with increasing fuel moisture. Pollution emissions from a normal burning under an adequate air supply condition were the lowest for most pollutants, while more pollutants were emitted when the oxygen deficient atmosphere was formed in stove chamber during fast burning. The impact of these 4 factors on particulate matter size distribution was also studied. Modified combustion efficiency and the four investigated factors explained 68, 72, and 64% of total variations in EFs of PM, organic carbon, and oxygenated PAHs, respectively, but only 36, 38 and 42% of the total variations in EFs of elemental carbon, pPAHs and nitro-PAHs, respectively.
PMCID: PMC4278645  PMID: 24520723
residential wood combustion; influencing factor; carbonaceous particulate matter; polycyclic aromatic hydrocarbons; emission factor
9.  Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus 
To investigate whether the enhanced green fluorescent protein (EGFP) reporter gene could be transferred into human trabecular meshwork (HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.
The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection (MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1×108 transducing unit (TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.
The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo. Immunohistochemistry staining revealed that 88.19% EGFP-positive trabecular meshwork (TM) cells were observed in the human anterior segment. Nevertheless, the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group (P>0.05).
EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.
PMCID: PMC4270982  PMID: 25540740
gene transfer; trabecular meshwork; HIV-based lentivirus; glaucoma
10.  Construction of a high-density genetic map based on large-scale markers developed by specific length amplified fragment sequencing (SLAF-seq) and its application to QTL analysis for isoflavone content in Glycine max 
BMC Genomics  2014;15(1):1086.
Quantitative trait locus (QTL) mapping is an efficient approach to discover the genetic architecture underlying complex quantitative traits. However, the low density of molecular markers in genetic maps has limited the efficiency and accuracy of QTL mapping. In this study, specific length amplified fragment sequencing (SLAF-seq), a new high-throughput strategy for large-scale SNP discovery and genotyping based on next generation sequencing (NGS), was employed to construct a high-density soybean genetic map using recombinant inbred lines (RILs, Luheidou2 × Nanhuizao, F5:8). With this map, the consistent QTLs for isoflavone content across various environments were identified.
In total, 23 Gb of data containing 87,604,858 pair-end reads were obtained. The average coverage for each SLAF marker was 11.20-fold for the female parent, 12.51-fold for the male parent, and an average of 3.98-fold for individual RILs. Among the 116,216 high-quality SLAFs obtained, 9,948 were polymorphic. The final map consisted of 5,785 SLAFs on 20 linkage groups (LGs) and spanned 2,255.18 cM in genome size with an average distance of 0.43 cM between adjacent markers. Comparative genomic analysis revealed a relatively high collinearity of 20 LGs with the soybean reference genome. Based on this map, 41 QTLs were identified that contributed to the isoflavone content. The high efficiency and accuracy of this map were evidenced by the discovery of genes encoding isoflavone biosynthetic enzymes within these loci. Moreover, 11 of these 41 QTLs (including six novel loci) were associated with isoflavone content across multiple environments. One of them, qIF20-2, contributed to a majority of isoflavone components across various environments and explained a high amount of phenotypic variance (8.7% - 35.3%). This represents a novel major QTL underlying isoflavone content across various environments in soybean.
Herein, we reported a high-density genetic map for soybean. This map exhibited high resolution and accuracy. It will facilitate the identification of genes and QTLs underlying essential agronomic traits in soybean. The novel major QTL for isoflavone content is useful not only for further study on the genetic basis of isoflavone accumulation, but also for marker-assisted selection (MAS) in soybean breeding in the future.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1086) contains supplementary material, which is available to authorized users.
PMCID: PMC4320444  PMID: 25494922
High-density genetic map; Isoflavone content; QTL; SLAF-seq; Soybean [Glycine max (L.) Merr.]
11.  Upregulation of microRNA-106b is associated with poor prognosis in hepatocellular carcinoma 
Diagnostic Pathology  2014;9(1):226.
MicroRNA-106b (miR-106b) is a member of the miR-106b ~ 25 cluster. It has been reported that miR-106b acts as an oncogene and is upregulated in many human cancers. However, the prognostic value of miR-106b in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the clinical significance of miR-106b expression in HCC.
We determined the expression level of miR-106b in 104 cases of paired HCC and adjacent non-tumor tissues by quantitative real-time PCR (qRT-PCR). The correlation between miR-106b expression and prognosis of HCC was studied by univariate and multivariate analysis. Multivariate analysis of the prognostic factors was performed with Cox proportional hazards model.
MiR-106b expression was significantly upregulated in as high as 76.0% of HCC tissues, compared with their non-tumor counterparts (P < 0.001). High miR-106b expression was significantly associated with large tumor size (P = 0.019) and vascular invasion (P = 0.016). Kaplan-Meier analysis showed that patients with high miR-106b expression had a worse overall survival than patients with low miR-106b expression (log-rank P = 0.004). The multivariate Cox regression analysis indicated that miR-106b expression was an independent prognostic factor for overall survival (HR, 2.002; 95% CI, 1.130-6.977; P = 0.027).
Our data indicated that miR-106b expression was significantly upregulated in HCC and could serve as a potential unfavorable prognostic biomarker.
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PMCID: PMC4261545  PMID: 25466449
Hepatocellular carcinoma; miR-106b; Prognosis
12.  A Hybrid Positioning Strategy for Vehicles in a Tunnel Based on RFID and In-Vehicle Sensors 
Sensors (Basel, Switzerland)  2014;14(12):23095-23118.
Many intelligent transportation system applications require accurate, reliable, and continuous vehicle positioning. How to achieve such positioning performance in extended GPS-denied environments such as tunnels is the main challenge for land vehicles. This paper proposes a hybrid multi-sensor fusion strategy for vehicle positioning in tunnels. First, the preliminary positioning algorithm is developed. The Radio Frequency Identification (RFID) technology is introduced to achieve preliminary positioning in the tunnel. The received signal strength (RSS) is used as an indicator to calculate the distances between the RFID tags and reader, and then a Least Mean Square (LMS) federated filter is designed to provide the preliminary position information for subsequent global fusion. Further, to improve the positioning performance in the tunnel, an interactive multiple model (IMM)-based global fusion algorithm is developed to fuse the data from preliminary positioning results and low-cost in-vehicle sensors, such as electronic compasses and wheel speed sensors. In the actual implementation of IMM, the strong tracking extended Kalman filter (STEKF) algorithm is designed to replace the conventional extended Kalman filter (EKF) to achieve model individual filtering. Finally, the proposed strategy is evaluated through experiments. The results validate the feasibility and effectiveness of the proposed strategy.
PMCID: PMC4299054  PMID: 25490581
vehicle positioning; sensor fusion; tunnel; RFID; interactive multiple model
13.  Biochemical changes of oxidative stress in premature ovarian insufficiency induced by tripterygium glycosides 
Premature ovarian insufficiency (POI) is one of clinical manifestations of ovarian damage. This study is to evaluate biochemical changes of oxidative stress in POI induced by tripterygium glycosides (TG) via subcutaneous injection. 24 female KM mice were assigned to two groups: control group and TG group. The mice in TG group were subjected to 50 TG for 35 days, while these in control group were fed with parallel volume of sterile water. Blood samples were separately obtained in day 15, 22, 29, 36 and 43. Ovarian histopathological changes were determined when finished the administration and observed under optical microscope. Serum levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and anti-mullerian hormone (AMH) and ovarian homogenates levels of MDA, SOD, GSH-Px and AMH were assessed by ELISA. AMH expression in the ovaries was analyzed by immunohistochemistry. Compared with control group, the results in TG group showed a significant reduction of serum levels of SOD and GSH-Px in day 15, 22, 29, 43 and increase of MDA in day 22, 36. They also presented decreased SOD and GSH-Px levels and increased MDA level in ovarian homogenates. Our data suggested that oxidative stress was involved in POI and might be the potential pathogenesis of POI induced by TG.
PMCID: PMC4313959
Premature ovarian insufficiency; tripterygium glycosides; oxidative stress; malondialdehyde; superoxide dismutase; glutathione peroxidase
14.  Downregulation of microRNA-100 protects apoptosis and promotes neuronal growth in retinal ganglion cells 
BMC Molecular Biology  2014;15(1):25.
Retinal ganglion cells (RGCs) are preferentially lost in glaucoma or optic neuritis. In the present study, we investigated the protective effect of mircoRNA 100 (miR-100) against oxidative stress induced apoptosis in RGC-5 cells.
Rat RGC-5 cells were cultured in plates and H2O2 was added to induce oxidative stress. TUNEL assay and qRT-PCR showed H2O2 induced apoptosis and up-regulated miR-100 in a dose-dependent manner in RGC-5 cells. Conversely, lentiviral-mediated miR-100 down-regulation protected H2O2 induced apoptosis, promoted neurite growth and activated AKT/ERK and TrkB pathways through phosphorylation. Luciferase assay confirmed that IGF1R was directly regulated by miR-100 in RGC-5 cells, and siRNA-mediated IGF1R knockdown activated AKT protein through phosphorylation, down-regulated miR-100, therefore exerted a protective effect on RGC-5 apoptosis.
Down-regulating miR-100 is an effective method to protect H2O2 induced apoptosis in RGC-5 cells, possible associated with IGF1R regulation.
PMCID: PMC4271342  PMID: 25406880
Retinal ganglion; miR-100; Oxidative stress; Apoptosis; IGF-1
15.  Selective antegrade cerebral perfusion reduces brain injury following deep hypothermic circulatory arrest in the piglets’ model by decreasing the levels of protein SUMO2/3-ylation 
Selective antegrade cerebral perfusion (SACP), which was adopted by many surgical groups for complex neonatal cardiac surgery, especially aortic arch repair, is a proven adjunct for neuroprotection during deep hypothermic circulatory arrest (DHCA). Several recent studies suggest that SUMO2/3 modification of proteins is markedly activated during deep hypothermia and believed to be an endogenous neuroprotective stress response. Here, we report that SACP reduces the increasing degree of SUMO2/3 conjugation following DHCA. Piglets were subjected to 1 h SACP and/or 1 h DHCA. DHCA was sufficient to markedly increase in protein SUMOylation by SUMO2/3 both in the hippocampus and cerebral cortex. SACP, especially at flow rate of 50 ml/kg/min, reduces the increasing degree of SUMO2/3 conjugation and also reduces levels of pro-apoptotic factors, Bax and Caspase 3, and increases levels of antiapoptotic factors, Bcl-2, following DHCA both in the hippocampus and cerebral cortex. This suggests that SACP at flow rate of 50 ml/kg/min is more appropriate for neuroprotection during DHCA in the pig model and level of protein SUMO2/3-ylation maybe an indicator of the degree of brain injury.
PMCID: PMC4276245  PMID: 25550987
Selective antegrade cerebral perfusion; SUMO2/3; deep hypothermic circulatory arrest; piglet; apoptosis; brain injury
16.  A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design 
BMC Biotechnology  2014;14(1):93.
Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.
Here, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the known SHMT from Escherichia coli. The obtained protein AnSHMT showed the optimal activity at 40°C and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum’s SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position.
This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.
PMCID: PMC4260256  PMID: 25394480
Arthrobacter nicotianae; SHMT; Characterization; Site-directed mutagenesis; Catalytic efficiency
17.  Malignant gangliocytic paraganglioma of the duodenum with distant metastases and a lethal course 
World Journal of Gastroenterology : WJG  2014;20(41):15454-15461.
Gangliocytic paraganglioma (GP) is rare and has been regarded as benign in general with a good prognosis. We present a patient with duodenal GP showing a malignant and lethal clinical course. A 47-year-old male patient was found to have a duodenal tumor and enlarged regional lymph nodes. The patient initially underwent a pancreaticoduodenectomy to resect the tumor and involved lymph nodes completely. Histological and immunohistochemical analyses showed findings typical of GP. However, the distant metastatic lesions in the liver and pelvic cavity were rapidly observed after surgery. The patient underwent chemotherapy and radiotherapy, as well as a second surgery to partly remove the metastatic mass in the pelvic cavity. The histological examination revealed no significant difference in histological features between the primary duodenal tumor and the metastatic pelvic mass. However, the patient finally died of the tumor due to the recurrence of the residual pelvic lesion and increased liver mass. To our knowledge, this is the first report of lethal GP with multifocal metastases. Our case confirms that GP should be regarded as a malignant potential tumor with behavior code of “1”, rather than a benign tumor of “0”.
PMCID: PMC4223280  PMID: 25386095
Duodenal neoplasms; Gangliocytic neoplasms; Paraganglioma; Lymphatic metastasis; Treatment outcome
18.  Validation of Bmi1 as a Therapeutic Target of Hepatocellular Carcinoma in Mice 
Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. Our previous studies have revealed that Bmi1 acts as an oncogene in hepatic carcinogenesis in an INK4a/ARF locus independent manner. However, whether Bmi1 can be used as a potential target for hepatocellular carcinoma treatment has not been fully confirmed yet. Here, we show that perturbation of Bmi1 expression by using short hairpin RNA can inhibit the tumorigenicity and tumor growth of hepatocellular carcinoma cells both in vitro and in vivo. Importantly, Bmi1 knockdown can block the tumor growth, both in the initiating stages and the fast growing stages. Cellular biology analysis revealed that Bmi1 knockdown induces cell cycle arrest and apoptosis. Our findings verify Bmi1 as a qualified treatment target for hepatocellular carcinoma (HCC) and support Bmi1 targeting treatment with chemotherapeutic agents.
PMCID: PMC4264152  PMID: 25372945
Bmi1; HCC; knockdown; proliferation; treatment
19.  I(f) current channel inhibitor (ivabradine) deserves cardioprotective effect via down-regulating the expression of matrix metalloproteinase (MMP)-2 and attenuating apoptosis in diabetic mice 
Ivabradine (IVBD), a novel I(f)-channel inhibitor and specific heart rate-lowering agent, is known to have anti-oxidative activity that promotes endothelial function. However, the molecular mechanism through which IVBD acts on cardiac function has yet to be elucidated, especially in experimental diabetic animals.
For this reason, twenty diabetic mice were randomly assigned to IVBD-treated (10 mg/kg/day) and control (saline) groups. After a 3-month treatment, microarray assay was performed to identify differentia expressed genes, and cardiac function was measured by echocardiography, with subsequent immunohistochemistry analysis and western blotting.
Our results showed that ivabradine treatment attenuated the expression and staining score of matrix metalloproteinase (MMP)-2, induced the dephosphorylation of caspase 3, BAX and MMP-2, and enhanced the phosphorylation of NF-κB. Ivabradine treatment led to a significant improvement in cardiac function.
Ivabradine significantly improved cardiac function by attenuating apoptosis and inhibiting the expression and activity of MMP-2 in diabetic mice, which underscored the novel clinical implications of ivabradine for diabetic patients.
PMCID: PMC4230832  PMID: 25361902
Diabetes; Gene; Microarray; Signal pathway; Immunohistochemistry; Apoptosis
20.  Extracts and compounds with anti-diabetic complications and anti-cancer activity from Castanea mollissina Blume (Chinese chestnut) 
Castanea mollissima Blume (Chinese chestnut), as a food product is known for its various nutrients and functional values to the human health. The present study was carried out to analyze the anti-diabetic complications and anti-cancer activities of the bioactive compounds present in C. mollissima.
The kernels (CK), shells (CS) and involucres (CI) parts of C. Blume were extracted with 90% alcohol. The water suspension of these dried alcohol extracts were extracted using EtOAc and n-BuOH successively. The n-BuOH fraction of CI (CI-B) was isolated by silica gel column, Sephadex LH 20 column and preparative HPLC. The isolated compounds were identified by 1H-NMR, 13C-NMR, HMBC, HMQC and ESI-Q-TOF MS, All the fractions and compounds isolated were evaluated on human recombinant aldose reductase (HR-AR) assay, advanced glycation end products (AGEs) formation assay and human COLO 320 DM colon cancer cells inhibitory assay.
CI-B was found to show a significant inhibitory effect in above biological screenings. Six flavonoids and three polyphenolic acids were obtained from CI-B. They were identified as kaempferol (1), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (2), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-β-D-galactopyranoside (3), kaempferol-3-O-[2''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (4), kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-β-D-glucopyranoside (5) and kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-β-D-galactopyranoside (6), casuariin (7), casuarinin (8) and castalagin (9). Compounds 2–9 were found to show higher activity than quercetin (positive control) in the AR assay. Compounds 3–6, 8, and 9 showed stronger inhibitory effects than amino guanidine (positive control) on AGEs production. Compounds 4–6, 7, and 8 showed much higher cytotoxic activity than 5-fluorouracil (positive control) against the human COLO 320 DM colon cancer cells.
Our results suggest that flavonoids and polyphenolic acids possesses anti-diabetes complications and anti-cancer properties, and they were presumed to be the bioactive components of Castanea mollissima Blume.
Electronic supplementary material
The online version of this article (doi:10.1186/1472-6882-14-422) contains supplementary material, which is available to authorized users.
PMCID: PMC4226895  PMID: 25351676
Anti-diabetic complications; Anti-cancer; Castanea mollissina Blume; Phenolic acids; Flavonoids
21.  MOFzyme: Intrinsic protease-like activity of Cu-MOF 
Scientific Reports  2014;4:6759.
The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.
PMCID: PMC4208042  PMID: 25342169
22.  Inhibitory effects of C-type natriuretic peptide on the differentiation of cardiac fibroblasts, and secretion of monocyte chemoattractant protein-1 and plasminogen activator inhibitor-1 
Molecular Medicine Reports  2014;11(1):159-165.
The present study aimed to investigate the effect of C-type natriuretic peptide (CNP) on the function of cardiac fibroblasts (CFs). Western blotting was used to investigate the expression of myofibroblast marker proteins: α-smooth muscle actin (α-SMA), extra domain-A fibronectin, collagen I and collagen III, and the activity of extracellular signal-regulated kinase 1/2 (ERK1/2). Immunofluorescence was used to examine the morphological changes; a transwell assay was used to analyze migration, and reverse transcription-quantitative polymerase chain reaction and ELISA were employed to determine the mRNA expression and protein secretion of monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1). The results demonstrated that CNP significantly reduced the protein expression of α-SMA, fibronectin, collagen I and collagen III, and suppressed the migratory ability of CFs. Additionally, the mRNA and protein expression of MCP-1 and PAI-1 was inhibited under the CNP treatment; and this effect was mediated by the inhibition of the ERK1/2 activity. In conclusion, CNP inhibited cardiac fibroblast differentiation and migration, and reduced the secretion of MCP-1 and PAI-1, which demonstrates novel mechanisms to explain the antifibrotic effect of CNP.
PMCID: PMC4237089  PMID: 25352084
cardiac fibroblast cell; cardiac fibrosis; C-type natriuretic peptide; differentiation; monocyte chemoattractant protein-1; plasminogen activator inhibitor-1
23.  Comparative characterization of two intracellular Ca2+-release channels from the red flour beetle, Tribolium castaneum 
Scientific Reports  2014;4:6702.
Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) are members of a family of tetrameric intracellular Ca2+-release channels (CRCs). While it is well known in mammals that RyRs and IP3Rs modulate multiple physiological processes, the roles of these two CRCs in the development and physiology of insects remain poorly understood. In this study, we cloned and functionally characterized RyR and IP3R cDNAs (named TcRyR and TcIP3R) from the red flour beetle, Tribolium castaneum. The composite TcRyR gene contains an ORF of 15,285 bp encoding a protein of 5,094 amino acid residues. The TcIP3R contains an 8,175 bp ORF encoding a protein of 2,724 amino acids. Expression analysis of TcRyR and TcIP3R revealed significant differences in mRNA expression levels among T. castaneum during different developmental stages. When the transcript levels of TcRyR were suppressed by RNA interference (RNAi), an abnormal folding of the adult hind wings was observed, while the RNAi-mediated knockdown of TcIP3R resulted in defective larval–pupal and pupal–adult metamorphosis. These results suggested that TcRyR is required for muscle excitation-contraction (E-C) coupling in T. castaneum, and that calcium release via IP3R might play an important role in regulating ecdysone synthesis and release during molting and metamorphosis in insects.
PMCID: PMC4204029  PMID: 25330781
24.  Regulatory role of tetR gene in a novel gene cluster of Acidovorax avenae subsp. avenae RS-1 under oxidative stress 
Acidovorax avenae subsp. avenae is the causal agent of bacterial brown stripe disease in rice. In this study, we characterized a novel horizontal transfer of a gene cluster, including tetR, on the chromosome of A. avenae subsp. avenae RS-1 by genome-wide analysis. TetR acted as a repressor in this gene cluster and the oxidative stress resistance was enhanced in tetR-deletion mutant strain. Electrophoretic mobility shift assay demonstrated that TetR regulator bound directly to the promoter of this gene cluster. Consistently, the results of quantitative real-time PCR also showed alterations in expression of associated genes. Moreover, the proteins affected by TetR under oxidative stress were revealed by comparing proteomic profiles of wild-type and mutant strains via 1D SDS-PAGE and LC-MS/MS analyses. Taken together, our results demonstrated that tetR gene in this novel gene cluster contributed to cell survival under oxidative stress, and TetR protein played an important regulatory role in growth kinetics, biofilm-forming capability, superoxide dismutase and catalase activity, and oxide detoxicating ability.
PMCID: PMC4204640  PMID: 25374564
horizontal gene transfer; tetR; repressor; oxidative stress; EMSA; proteome profiling
25.  miR-150 Modulates Cisplatin Chemosensitivity and Invasiveness of Muscle-Invasive Bladder Cancer Cells via Targeting PDCD4 In Vitro 
Chemotherapeutic insensitivity and tumor cell invasiveness are major obstacles to effectively treating muscle-invasive bladder cancer (MIBC). Recent reports show that microRNAs (miRNAs) play an important role in the chemotherapeutic response and disease progression of MIBC. Therefore, here we investigated the role of miR-150 in MIBC cells in vitro.
miR-150 expression was quantified by qRT-PCR in two MIBC cell lines (5637 and T24). After successful miR-150 inhibition by transfection, MTS and transwell assays were used to assess the MIBC’s cisplatin sensitivity and cell invasiveness, respectively. The TargetScan database and a luciferase reporter system were used to identify whether the programmed cell death 4 protein (PDCD4) is a direct target of miR-150 in MIBC cells.
miR-150 expression was found to be significantly increased in both MIBC cell lines, and treatment with a miR-150 inhibitor significantly sensitized MIBC cells to cisplatin and inhibited MIBC cell invasiveness. PDCD4 was identified as a direct target of miR-150 in MIBC cells, and increased PDCD4 expression via transfection with the pLEX-PDCD4 plasmid efficiently sensitized MIBC cells to cisplatin chemotherapy and inhibited MIBC cell invasiveness.
This study provides novel evidence that miR-150 functions as a tumor promoter in reducing chemosensitivity and promoting invasiveness of MIBC cells via targeting PDCD4. Thus, modulation of the miR-150-PDCD4 axis shows promise as a therapeutic strategy for MIBC.
PMCID: PMC4199406  PMID: 25287716
Cisplatin; MicroRNAs; Urinary Bladder Neoplasms

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