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1.  MiR-324-5p Suppresses Hepatocellular Carcinoma Cell Invasion by Counteracting ECM Degradation through Post-Transcriptionally Downregulating ETS1 and SP1 
PLoS ONE  2015;10(7):e0133074.
Hepatocellular carcinoma (HCC) is one of the common malignancies, which is highly metastatic and the third common cause of cancer deaths in the world. The invasion and metastasis of cancer cells is a multistep and complex process which is mainly initiated by extracellular matrix (ECM) degradation. Aberrant expression of microRNA has been investigated in HCC and shown to play essential roles during HCC progression. In the present study, we found that microRNA-324-5p (miR-324-5p) was downregulated in both HCC cell lines and tissues. Ectopic miR-324-5p led to the reduction of HCC cells invasive and metastatic capacity, whereas inhibition of miR-324-5p promoted the invasion of HCC cells. Matrix metalloproteinase 2 (MMP2) and MMP9, the major regulators of ECM degradation, were found to be downregulated by ectopic miR-324-5p, while upregulated by miR-324-5p inhibitor. E26 transformation-specific 1 (ETS1) and Specificity protein 1 (SP1), both of which could modulate MMP2 and MMP9 expression and activity, were presented as the direct targets of and downregulated by miR-324-5p. Downregulation of ETS1 and SP1 mediated the inhibitory function of miR-324-5p on HCC migration and invasion. Our study demonstrates that miR-324-5p suppresses hepatocellular carcinoma cell invasion and might provide new clues to invasive HCC therapy.
PMCID: PMC4503725  PMID: 26177288
2.  The Blockage of KCa3.1 Channel Inhibited Proliferation, Migration and Promoted Apoptosis of Human Hepatocellular Carcinoma Cells 
Journal of Cancer  2015;6(7):643-651.
The intermediate conductance calcium-activated potassium channel KCa3.1 plays an important role in regulating cell proliferation and migration. However, the role of KCa3.1 channel in human hepatocellular carcinoma remained unknown. This study was therefore performed to investigate the effects of KCa3.1 potassium channel blocker on the proliferation, apoptosis and migration of human hepatocellular cancer cells HepG2. KCa3.1 mRNA and protein were detected in HepG2. Furthermore, KCa3.1 potassium channel blocker TRAM-34 was capable to inhibit the proliferation and induce the apoptosis of HepG2 cells, which can be partially attenuated by 1-EBIO, an activator of KCa3.1 channel. Moreover, the migration of HepG2 was obviously inhibited by TRAM-34. Consistently, knockdown of KCa3.1 channel using its siRNA was also able to induce apoptosis and suppress proliferation and migration of HepG2. Meanwhile, intracellular ROS level was found augmented in HepG2 treated with TRAM-34. More importantly, p53 protein was found translocation from the cytoplasm into the nuclei of HepG2. Collectively, inhibition of KCa3.1 channel suppressed the growth and migration, and promoted the apoptosis of human hepatocellular carcinoma cells by regulating intracellular ROS level and promoting p53 activation. This data suggests TRAM-34 as a promising anti-tumor drug for liver cancer.
PMCID: PMC4466414  PMID: 26078795
TRAM-34; Migration; Apoptosis; Proliferation; p53; ROS
3.  Whole genome sequence and comparative genomic analysis of multidrug-resistant Staphylococcus capitis subsp. urealyticus strain LNZR-1 
Gut Pathogens  2014;6:45.
Staphylococcus capitis is an emerging opportunistic pathogen of humans, and found as a colonizer of the human gut. Here, we report a case of S. capitis subsp. urealyticus infection. The strain LNZR-1 was isolated from the blood culture of a patient with sigmoid colon cancer. It was found to be resistant to some important antibiotics, such as linezolid, a highly effective antimicrobial against clinically important Staphylococci pathogens. However, data on the genetic resistance mechanisms in S. capitis subsp. urealyticus are only sparsely available.
The draft genome of S. capitis subsp. urealyticus strain LNZR-1 was sequenced by using next-generation sequencing technologies. Sequence data assembly revealed a genome size of 2,595,865 bp with a G + C content of 32.67%. Genome annotation revealed the presence of antibiotic resistance genes conferring resistance against some of the tested antibiotics as well as non-tested antibiotics. The genome also possesses a lot of genes that may be related to multidrug resistance. Whole genome comparison of the LNZR-1 with five other S. capitis strains showed that some functional regions are highly homologous between the six assemblies made herein. The LNZR-1 genome has high similarity with the genomes of the strains VCU116 and CR01, although some short stretches present in the genomes of strains VCU116 and CR01 were absent in the strain LNZR-1.
The presence of a plethora of genes responsible for antibiotic resistance suggests that strain LNZR-1 could present a potential threat to human health. The comparative genomic analysis of S. capitis strains presented in this study is important for better understanding of multidrug resistance in S. capitis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13099-014-0045-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4310196  PMID: 25649186
Staphylococcus capitis subsp. urealyticus; Multidrug-resistant; Genome sequencing; Comparative genomic analysis
4.  A Statistical Parametric Mapping Toolbox Used for Voxel-Wise Analysis of FDG-PET Images of Rat Brain 
PLoS ONE  2014;9(9):e108295.
PET (positron emission tomography) imaging researches of functional metabolism using fluorodeoxyglucose (18F-FDG) of animal brain are important in neuroscience studies. FDG-PET imaging studies are often performed on groups of rats, so it is desirable to establish an objective voxel-based statistical methodology for group data analysis.
Material and Methods
This study establishes a statistical parametric mapping (SPM) toolbox (plug-ins) named spmratIHEP for voxel-wise analysis of FDG-PET images of rat brain, in which an FDG-PET template and an intracranial mask image of rat brain in Paxinos & Watson space were constructed, and the default settings were modified according to features of rat brain. Compared to previous studies, our constructed rat brain template comprises not only the cerebrum and cerebellum, but also the whole olfactory bulb which made the later cognitive studies much more exhaustive. And with an intracranial mask image in the template space, the brain tissues of individuals could be extracted automatically. Moreover, an atlas space is used for anatomically labeling the functional findings in the Paxinos & Watson space. In order to standardize the template image with the atlas accurately, a synthetic FDG-PET image with six main anatomy structures is constructed from the atlas, which performs as a target image in the co-registration.
The spatial normalization procedure is evaluated, by which the individual rat brain images could be standardized into the Paxinos & Watson space successfully and the intracranial tissues could also be extracted accurately. The practical usability of this toolbox is evaluated using FDG-PET functional images from rats with left side middle cerebral artery occlusion (MCAO) in comparison to normal control rats. And the two-sample t-test statistical result is almost related to the left side MCA.
We established a toolbox of SPM8 named spmratIHEP for voxel-wise analysis of FDG-PET images of rat brain.
PMCID: PMC4178133  PMID: 25259529
5.  RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells 
PLoS ONE  2014;9(6):e99625.
Asthma is a chronic inflammatory respiratory disease that affects over 300 million people worldwide. Glucocorticoids are a mainstay therapy for asthma because they exert anti-inflammatory effects in multiple lung tissues, including the airway smooth muscle (ASM). However, the mechanism by which glucocorticoids suppress inflammation in ASM remains poorly understood. Using RNA-Seq, a high-throughput sequencing method, we characterized transcriptomic changes in four primary human ASM cell lines that were treated with dexamethasone—a potent synthetic glucocorticoid (1 µM for 18 hours). Based on a Benjamini-Hochberg corrected p-value <0.05, we identified 316 differentially expressed genes, including both well known (DUSP1, KLF15, PER1, TSC22D3) and less investigated (C7, CCDC69, CRISPLD2) glucocorticoid-responsive genes. CRISPLD2, which encodes a secreted protein previously implicated in lung development and endotoxin regulation, was found to have SNPs that were moderately associated with inhaled corticosteroid resistance and bronchodilator response among asthma patients in two previously conducted genome-wide association studies. Quantitative RT-PCR and Western blotting showed that dexamethasone treatment significantly increased CRISPLD2 mRNA and protein expression in ASM cells. CRISPLD2 expression was also induced by the inflammatory cytokine IL1β, and small interfering RNA-mediated knockdown of CRISPLD2 further increased IL1β-induced expression of IL6 and IL8. Our findings offer a comprehensive view of the effect of a glucocorticoid on the ASM transcriptome and identify CRISPLD2 as an asthma pharmacogenetics candidate gene that regulates anti-inflammatory effects of glucocorticoids in the ASM.
PMCID: PMC4057123  PMID: 24926665
6.  The Role of Circulating MicroRNA-126 (miR-126): A Novel Biomarker for Screening Prediabetes and Newly Diagnosed Type 2 Diabetes Mellitus 
Recent studies suggested an association of endothelial microRNA-126 (miR-126) with type 2 diabetes mellitus (T2DM). In the current study, we examined whether circulating miR-126 is associated with T2DM and pre-diabetic syndrome. The study included 82 subjects with impaired glucose tolerance (IGT), 75 subjects with impaired fasting glucose (IFG), 160 patients with newly diagnosed T2DM, and 138 healthy individuals. Quantitative polymerase chain reaction (qPCR) was used to examine serum miR-126. Serum miR-126 was significantly lower in IGT/IFG subjects and T2DM patients than in healthy controls (p < 0.05). After six months of treatment (diet control and exercise in IGT/IFG subjects, insulin plus diet control and exercise in T2DM patients), serum miR-126 increased significantly (p < 0.05). An analysis based on serum miR-126 in the sample revealed a significantly higher odds ratio (OR) for the subjects with the lowest 1/3 of serum miR-126 for T2DM (OR: 3.500, 95% confidence interval: 1.901–6.445, p < 0.05) than subjects within the highest 1/3 of serum miR-126. Such an association was still apparent after adjusting for other major risk factors. The area under the curve (AUC) for the receiver-operating characteristic (ROC) analysis was 0.792 (95% confidence interval: 0.707–0.877, p < 0.001). These results encourage the use of serum miR-126 as a biomarker for pre-diabetes and diabetes mellitus, as well as therapeutic response.
PMCID: PMC4100169  PMID: 24927146
microRNA-126; biomarker; IGT/IFG; pre-diabetes; type 2 diabetes mellitus
7.  Stability of miR-126 in Urine and Its Potential as a Biomarker for Renal Endothelial Injury with Diabetic Nephropathy 
Background. The purpose of the present study was to assess the feasibility of using miR-126 in the urine as a biomarker for diabetic nephropathy. Methods. miRNAs were extracted from the urine samples of T2DM patients with diabetic nephropathy (DN; n = 92), T2DM without DN (n = 86), and 85 healthy volunteers using quantitative reverse transcriptase polymerase chain reaction (real-time polymerase chain reaction) analysis. Stability of urinary miR-126 and factors that affected the stability were assessed. A subgroup analysis was also carried out to compare the urinary miR-126 level in T2DM patients well controlled by the treatment versus those who were not well controlled. Results. Urinary miR-126 was stable when the urine samples were kept at room temperature for extended period of time, 4°C, −20°C, and −80°C for up to 12 hours or subjected to 10 freeze-and-thaw cycle. Urinary miR-126 was significantly higher in T2DM patients with DN (5.76 ± 0.33 versus 3.25 ± 0.45 in T2DM patients without DN). Successful treatment significantly reduced urinary miR-126 in T2DM patients with DN to 3.89 ± 0.52 (P < 0.05). Conclusion. miR-126 in the urine is stable and it could be used as a biomarker of DN and to monitor the treatment response.
PMCID: PMC4016912  PMID: 24864140
8.  MicroRNA-590-5p Regulates Proliferation and Invasion in Human Hepatocellular Carcinoma Cells by Targeting TGF-β RII 
Molecules and Cells  2012;33(6):545-551.
MicroRNAs (miRNAs) are regulatory small non-coding RNAs that can regulate gene expression by binding to gene elements, such as the gene promotor 5′UTR, mainly in the 3′UTR of mRNA. One miRNA targets many mRNAs, which can be regulated by many miRNAs, leading to a complex metabolic network. In our study, we found that the expression level of miR-590-5p is higher in the human hepatocellular carcinoma cell line HepG2 than in the normal hepatocellular cell line L02. Downregulation of miR-590-5p inhibited proliferation and invasion of hepatocellular carcinoma cells (HCCs). We also showed that expression of TGF-beta RII, which has been regarded as a regulator of tumor proliferation, invasion, and migration in hepatocellular carcinoma, is regulated by miRNA-590-5p. In addition, miR-590-5p downregulated the expression of TGF-beta RII by targeting the 3′UTR of mRNA. We also found that downregulation of miR-590-5p was associated with an elevation of TGF-beta RII and inhibition of proliferation and invasion in HepG2 cells. Furthermore, overexpression of miR-590-5p was associated with upregulation of TGF-beta RII and could promote proliferation and invasion in L02 cells. In conclusion, we determined that TGF-beta RII is a novel target of miRNA-590-5p. Thus, the role of TGF-beta RII in regulating proliferation and invasion of human HCCs is controlled by miR-590-5p. In other words, miR-590-5p promotes proliferation and invasion in human HCCs by directly targeting TGF-beta RII.
PMCID: PMC3887758  PMID: 22684895
HepG2; invasion; L02; miR-590-5p; proliferation; TGF-beta RII
9.  Genome-Wide Association Analysis in Asthma Subjects Identifies SPATS2L as a Novel Bronchodilator Response Gene 
PLoS Genetics  2012;8(7):e1002824.
Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function (i.e. FEV1) before and after the administration of a short-acting β2-agonist, the most common rescue medications used for the treatment of asthma. BDR also serves as a test of β2-agonist efficacy. BDR is a complex trait that is partly under genetic control. A genome-wide association study (GWAS) of BDR, quantified as percent change in baseline FEV1 after administration of a β2-agonist, was performed with 1,644 non-Hispanic white asthmatic subjects from six drug clinical trials: CAMP, LOCCS, LODO, a medication trial conducted by Sepracor, CARE, and ACRN. Data for 469,884 single-nucleotide polymorphisms (SNPs) were used to measure the association of SNPs with BDR using a linear regression model, while adjusting for age, sex, and height. Replication of primary P-values was attempted in 501 white subjects from SARP and 550 white subjects from DAG. Experimental evidence supporting the top gene was obtained via siRNA knockdown and Western blotting analyses. The lowest overall combined P-value was 9.7E-07 for SNP rs295137, near the SPATS2L gene. Among subjects in the primary analysis, those with rs295137 TT genotype had a median BDR of 16.0 (IQR = [6.2, 32.4]), while those with CC or TC genotypes had a median BDR of 10.9 (IQR = [5.0, 22.2]). SPATS2L mRNA knockdown resulted in increased β2-adrenergic receptor levels. Our results suggest that SPATS2L may be an important regulator of β2-adrenergic receptor down-regulation and that there is promise in gaining a better understanding of the biological mechanisms of differential response to β2-agonists through GWAS.
Author Summary
Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function before and after the administration of short-acting β2-agonists, common medications used for asthma treatment. We performed a genome-wide association study of BDR with 1,644 white asthmatic subjects from six drug clinical trials and attempted to replicate these findings in 1,051 white subjects from two independent cohorts. The most significant associated variant was near the SPATS2L gene. We knocked down SPATS2L mRNA in human airway smooth muscle cells and found that β2-adrenergic receptor levels increased, suggesting that SPATS2L may be a regulator of BDR. Our results highlight the promise of pursuing GWAS results that do not necessarily reach genome-wide significance and are an example of how results from pharmacogenetic GWAS can be studied functionally.
PMCID: PMC3390407  PMID: 22792082
10.  Potential clinical significance of plasma-based KRAS mutation analysis using the COLD-PCR/TaqMan® -MGB probe genotyping method 
Despite the improved ability to detect mutations in recent years, tissue specimens cannot always be procured in a clinical setting, particularly from patients with recurrence of tumors or metastasis. Therefore, the aim of this study was to investigate whether plasma is able to be used for mutation analysis instead of tissue specimens. We collected plasma from 62 patients with colorectal cancer (CRC) prior to treatment. DNA extracted from plasma and matched tumor tissues were obtained. Mutations in KRAS were amplified from the tissue specimens and sequenced by regular polymerase chain reaction (PCR) and co-amplification at lower denaturation temperature (COLD)-PCR. Plasma KRAS gene mutation on codon 12 (GGT>GAT) was detected using a nested COLD-PCR/TaqMan® -MGB probe. Mutations in plasma and matched tumors were compared. KRAS mutation on codon 12 (GGT>GAT) was found in 13 (21.0%) plasma specimens and 12 (19.4%) matched tumor tissues. The consistency of KRAS mutations between plasma and tumors was 75% (9/12), which indicated a high correlation between the mutations detected in plasma DNA and the mutations detected in the corresponding tumor DNA (P<0.001; correlation index, k=0.649). Notably, four (6.5%) patients with plasma DNA mutations had no detectable KRAS mutations in the corresponding primary tumors, and three (4.8%) patients with tumor DNA mutations had no detectable KRAS mutations in the corresponding plasma DNA samples. Thus, KRAS mutations in plasma DNA correlate with the mutation status in matched tumor tissues of patients with CRC. Our study provides evidence to suggest that plasma DNA may be used as a potential medium for KRAS mutation analysis in CRC using the COLD-PCR/TaqMan-MGB probe method.
PMCID: PMC3460285  PMID: 23060932
KRAS mutation; plasma; colorectal cancer; COLD-PCR
11.  CD44 variant isoforms are specifically expressed on peripheral blood lymphocytes from asthmatic patients 
Asthma is a disease characterized by chronic airway inflammation, and Th2 cells play a critical role in initiating and sustaining asthmatic inflammation. It has been shown that CD44 expressed on CD4+ T cells plays a critical role in the accumulation of antigen-specific Th2 cells in the development of airway hyperresponsiveness induced by antigen challenge in the airways. The aim of this study was to determine whether there are specific CD44 variant isoforms (CD44v) expressed on lymphocytes from asthmatic patients. We collected whole blood samples from 103 normal subjects, 165 subjects with asthma and 104 with pneumonia. Peripheral blood lymphocyte isolation was performed, and total RNA was extracted from the isolated lymphocytes, using nested PCR for specific CD44v amplification on lymphocytes. Demographic variables were analyzed using linear regression in order to determine whether the expression of CD44v was correlated with these demographic features. The nested PCR results revealed that CD44v5 was expressed by 55.2% of asthma patients, which was significantly higher than levels of expression in the other groups. Lower percentages of individuals in the normal subject group exhibited expression of CD44v5 and CD44v6. The data demonstrated that the percentage of individuals in the pneumonia group expressing CD44v5 was 29.0%, but a higher percentage of these patients expressed CD44v6. CD44v5 expression was positively correlated with IgE levels (p=0.032) in the asthmatic patient group, and CD44v6 was significantly positively correlated with the neutrophil count (p<0.05). CD44v5 was expressed by a higher proportion of asthmatic patients than other subjects and thus may play an important role in the pathogenesis of asthma. These findings may offer a new target for the diagnosis and treatment of asthma and may also provide insights into the mechanisms of asthma development.
PMCID: PMC3460314  PMID: 23060926
asthma; peripheral blood lymphocytes; CD44v5; CD44v6
12.  More expressions of BDNF and TrkB in multiple hepatocellular carcinoma and anti-BDNF or K252a induced apoptosis, supressed invasion of HepG2 and HCCLM3 cells 
Brain-derived neurotrophic factor (BDNF) and its receptor Tropomysin-related kinase B (TrkB) are commonly up-regulated in a variety of human tumors. However, the roles of BDNF/TrkB in hepatocellular carcinoma (HCC) have been poorly investigated.
We evaluated the expressions of BDNF and TrkB in 65 cases of HCC by immunohistochemical staining. Moreover, in human HCC cell lines of HepG2 and high metastatic HCCLM3, the secretory BDNF in supernatant was measured by ELISA, the effects of BDNF neutralizing antibody or Trk tyrosine kinase inhibitor K252a on apoptosis and invasion were examined by flow cytometry and transwell assay respectively.
Higher expression of BDNF (63.1%) or positive expression of TrkB (55.4%) was found in HCC specimens, which was significantly correlated with multiple and advanced stage of HCC. BDNF secretory level in HCCLM3 was higher than that in HepG2 cells. Both anti-BDNF and K252a effectively induced apoptosis and suppressed invasion of HepG2 and HCCLM3 cells.
These findings suggested that BDNF/TrkB are essential for HCC cells survival and invasion. BDNF/TrkB signaling should probably be an effective target to prevent HCC advancement.
PMCID: PMC3212909  PMID: 21999199
13.  Acetylation of H2AX on lysine 36 plays a key role in the DNA double-strand break repair pathway 
FEBS letters  2010;584(13):2926-2930.
Phosphorylation of H2AX functions to recruit DNA repair complexes to sites of DNA damage. Here, we report that H2AX is constitutively acetylated on lysine 36 (H2AXK36Ac) by the CBP/p300 acetyltransferases. H2AXK36Ac is required for cells to survive exposure to ionizing radiation; however, H2AXK36Ac levels are not increased by DNA damage. Further, acetylation of H2AX did not affect phosphorylation of H2AX or the formation of DNA damage foci. Finally, cells with a double mutation in both the H2AX acetylation and phosphorylation sites were more radiosensitive than cells containing individual mutations. H2AXK36Ac is therefore a novel, constitutive histone modification located within the histone core region which regulates radiation sensitivity independently of H2AX phosphorylation.
PMCID: PMC2887596  PMID: 20488183
H2AX; acetylation; chromatin; DNA-double strand breaks; Histone acetyltransferase; IR
14.  The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair 
The Journal of Cell Biology  2010;191(1):31-43.
p400 unwinds chromatin from nucleosomes flanking double-strand breaks to facilitate recruitment of the DNA repair components brca1 and 53BP1.
The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage.
PMCID: PMC2953432  PMID: 20876283
15.  Tip60: Connecting chromatin to DNA damage signaling 
Cell cycle (Georgetown, Tex.)  2010;9(5):930-936.
Cells are constantly exposed to genotoxic events that can damage DNA. To counter this, cells have evolved a series of highly conserved DNA repair pathways to maintain genomic integrity. The ATM protein kinase is a master regulator of the DNA double-strand break (DSB) repair pathway. DSBs activate ATM's kinase activity, promoting the phosphorylation of proteins involved in both checkpoint activation and DNA repair. Recent work has revealed that 2 DNA damage response proteins, the Tip60 acetyltransferase and the mre11-rad50-nbs1 (MRN) complex, co-operate in the activation of ATM in response to DSBs. MRN functions to target ATM and the Tip60 acetyltransferase to DSBs. Tip60's chromodomain then interacts with histone H3 trimethylated on lysine 9, activating Tip60's acetyltransferase activity and stimulating the subsequent acetylation and activation of ATM's kinase activity. These results underscore the importance of chromatin structure in regulating DNA damage signaling and emphasize how histone modifications co-ordinate DNA repair. In addition, human tumors frequently exhibit altered patterns of histone methylation. This rewriting of the histone methylation code in tumor cells may impact the efficiency of DSB repair, increasing genomic instability and contributing to the initiation and progression of cancer.
PMCID: PMC2901859  PMID: 20160506
Tip60; ATM; histone methylation; H3K9me3; chromodomain; DNA repair; lysine demethylase

Results 1-16 (16)