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author:("Zhang, lipins")
1.  SIRT1 counteracted the activation of STAT3 and NF-κB to repress the gastric cancer growth 
Sirtuin-1 (SIRT1) possesses apparently dual roles in regulation of tumor. Previous reports have documented the crosstalk between SIRT1 with signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-B (NF-κB) signaling in leukemia, lymphoma and myeloma. In this study, the purpose was to survey the regulatory effects of SIRT1 on gastric cancer (GC) cells (AGS and MKN-45) and the relationships between SIRT1 and activation of STAT3 and NF-κB in GC cells. We found the SIRT1 activator (resveratrol RSV) contributed to the repression of viability and increase of senescence, which were rescued by SIRT1 inhibitor (nicotinamide NA) and SIRT1 depletion by CCK-8 assay and SA-β-gal assay respectively. Further study found SIRT1 activation (RSV supplement) not only inhibited the activation of STAT3 including STAT3 mRNA level, c-myc mRNA level phosphorylated STAT3 (pSTAT3) proteins and acetylizad STAT3 (acSTAT3) proteins, but also repression of pNF-κB p65 and acNF-κB p65. NA reversed the effects of RSV. In addition, either RSV or NA application could not change the cellular viability and senescence in MKN-45 cells with STAT3 knockdown or NF-κB knockdown. Overall, our findings suggested SIRT1 activation could induced the loss of viability and increases of senescence in GC in vitro. Moreover, our observations revealed SIRT1 displayed growth inhibitory activity in GC cells highly associated with causing repression of activation of STAT3 and NF-κB proteins via deacetylation.
PMCID: PMC4307451  PMID: 25664004
Sirtuin-1; STAT3; NF-κB; gastric cancer; acetylation; activation
2.  Cannabinoid receptor 2 agonist attenuates pain related behavior in rats with chronic alcohol/high fat diet induced pancreatitis 
Molecular Pain  2014;10(1):66.
Chronic Pancreatitis (CP) is a complex and multifactorial syndrome. Many contributing factors result in development of dysfunctional pain in a significant number of patients. Drugs developed to treat a variety of pain states fall short of providing effective analgesia for patients with chronic pancreatitis, often providing minimal to partial pain relief over time with significant side effects. Recently, availability of selective pharmacological tools has enabled great advances in our knowledge of the role of the cannabinoid receptors in pathophysiology. In particular, cannabinoid receptor 2 (CB2) has emerged as an attractive target for management of chronic pain, as demonstrated in several studies with inflammatory and neuropathic preclinical pain models. In this study, the analgesic efficacy of a novel, highly selective CB2 receptor agonist, LY3038404 HCl, is investigated in a chronic pancreatitis pain model, induced with an alcohol/high fat (AHF) diet.
Rats fed the AHF diet developed visceral pain-like behaviors detectable by week 3 and reached a maximum at week 5 that persists as long as the diet is maintained. Rats with AHF induced chronic pancreatitis were treated with LY3038404 HCl (10 mg/kg, orally, twice a day for 9 days). The treated animals demonstrated significantly alleviated pain related behaviors after 3 days of dosing, including increased paw withdrawal thresholds (PWT), prolonged abdominal withdrawal latencies (ABWL), and decreased nocifensive responses to noxious 44°C hotplate stimuli. Terminal histological analysis of pancreatic tissue sections from the AHF chronic pancreatitis animals demonstrated extensive injury, including a global pancreatic gland degeneration (cellular atrophy), vacuolization (fat deposition), and fibrosis. After the LY3038404 HCl treatment, pancreatic tissue was significantly protected from severe damage and fibrosis. LY3038404 HCl affected neither open field exploratory behaviors nor dark/light box preferences as measures of higher brain and motor functions.
LY3038404 HCl, a potent CB2 receptor agonist, possesses tissue protective and analgesic properties without effects on higher brain function. Thus, activation of CB2 receptors is suggested as a potential therapeutic target for visceral inflammation and pain management.
PMCID: PMC4242547  PMID: 25403433
Alcohol; CB2; Pain; Ki67; Hyperalgesia; Pancreas; Behavioral testing; 44°C hotplate test; Tissue repair; High fat
3.  A recurrent deletion mutation in OPA1 causes autosomal dominant optic atrophy in a Chinese family 
Scientific Reports  2014;4:6936.
Autosomal dominant optic atrophy (ADOA) is the most frequent form of hereditary optic neuropathy and occurs due to the degeneration of the retinal ganglion cells. To identify the genetic defect in a family with putative ADOA, we performed capture next generation sequencing (CNGS) to screen known retinal disease genes. However, six exons failed to be sequenced by CNGS in optic atrophy 1 gene (OPA1). Sequencing of those exons identified a 4 bp deletion mutation (c.2983-1_2985del) in OPA1. Furthermore, we sequenced the transcripts of OPA1 from the patient skin fibroblasts and found there is six-nucleotide deletion (c.2984-c.2989, AGAAAG). Quantitative-PCR and Western blotting showed that OPA1 mRNA and its protein expression have no obvious difference between patient skin fibroblast and control. The analysis of protein structure by molecular modeling suggests that the mutation may change the structure of OPA1 by formation of an alpha helix protruding into an existing pocket. Taken together, we identified an OPA1 mutation in a family with ADOA by filling the missing CNGS data. We also showed that this mutation affects the structural intactness of OPA1. It provides molecular insights for clinical genetic diagnosis and treatment of optic atrophy.
PMCID: PMC4221781  PMID: 25374051
4.  Clinical features of De Novo acute myeloid leukemia with concurrent DNMT3A, FLT3 and NPM1 mutations 
De novo acute myeloid leukemia (AML) with concurrent DNMT3A, FLT3 and NPM1 mutations (AMLDNMT3A/FLT3/NPM1) has been suggested to represent a unique AML subset on the basis of integrative genomic analysis, but the clinical features of such patients have not been characterized systematically.
We assessed the features of patients (n = 178) harboring mutations in DNMT3A, FLT3 and/or NPM1, including an index group of AMLDNMT3A/FLT3/NPM1 patients.
Patients with AMLDNMT3A/FLT3/NPM1 (n = 35) were significantly younger (median, 56.0 vs. 62.0 years; p = 0.025), mostly women (65.7% vs. 46.9%; p = 0.045), and presented with a higher percentage of bone marrow blasts (p < 0.001) and normal cytogenetics (p = 0.024) in comparison to patients within other mutation groups in this study. Among patients <60 years old, those with AMLDNMT3A/FLT3/NPM1 had a shorter event-free survival (EFS) (p = 0.047). DNMT3A mutations and not FLT3 or NPM1 mutations were independently associated with overall survival (OS) (p = 0.026). Within mutation subgroups, patients with AMLDNMT3A/NPM1 had a significantly shorter OS compared to those with AMLFLT3-ITD/NPM1 (p = 0.047) suggesting that the adverse impact of DNMT3A mutations is more pronounced than that of FLT3-ITD among patients with NPM1 mutation.
DNMT3A has a significant dominant effect on the clinical features and outcomes of de novo AML patients with concurrent DNMT3A, FLT3 and NPM1 mutations.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-014-0074-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4197326  PMID: 25281355
Acute myeloid leukemia; Next-generation sequencing; DNMT3A; FLT3; NPM1
5.  T cell immune abnormalities in immune thrombocytopenia 
Immune thrombocytopenia is an autoimmune disease with abnormal T cell immunity. Cytotoxic T cells, abnormal T regulatory cells, helper T cell imbalance, megakaryocyte maturation abnormalities and abnormal T cell anergy are involved in the pathogenesis of this condition. The loss of T cell-mediated immune tolerance to platelet auto-antigens plays a crucial role in immune thrombocytopenia. The induction of T cell tolerance is an important mechanism by which the pathogenesis and treatment of immune thrombocytopenia can be studied. Studies regarding the roles of the new inducible costimulator signal transduction pathway, the ubiquitin proteasome pathway, and the nuclear factor kappa B signal transduction pathway in the induction of T cell tolerance can help improve our understanding of immune theory and may provide a new theoretical basis for studying the pathogenesis and treatment of immune thrombocytopenia.
PMCID: PMC4189678  PMID: 25274611
Immune thrombocytopenia; T cell; Immune tolerance; Pathogenesis
6.  Stat3 Activation links a C/EBPδ to Myostatin Pathway to Stimulate Loss of Muscle Mass 
Cell metabolism  2013;18(3):368-379.
Catabolic conditions like chronic kidney disease (CKD) cause loss of muscle mass by unclear mechanisms. In muscle biopsies from CKD patients, we found activated Stat3 (p-Stat3) and hypothesized that p-Stat3 initiates muscle wasting. We created mice with muscle-specific knockout (KO) that prevents activation of Stat3. In these mice, losses of body and muscle weights were suppressed in models of CKD or acute diabetes. A small molecule that inhibits Stat3 activation produced similar responses suggesting a potential for translation strategies. Using C/EBPδ KO mice and C2C12 myotubes with knockdown of C/EBPδ or myostatin, we determined that p-Stat3 initiates muscle wasting via C/EBPδ, stimulating myostatin, a negative muscle growth regulator. C/EBPδ KO also improved survival of CKD mice. We verified that p-Stat3, C/EBPδ and myostatin were increased in muscles of CKD patients. The pathway from p-Stat3 to C/EBPδ to myostatin and muscle wasting could identify therapeutic targets that prevent muscle wasting.
PMCID: PMC3794464  PMID: 24011072
7.  Pathogenic Fungus Microsporum canis Activates the NLRP3 Inflammasome 
Infection and Immunity  2014;82(2):882-892.
Microsporum canis is a pathogenic fungus with worldwide distribution that causes tinea capitis in animals and humans. M. canis also causes invasive infection in immunocompromised patients. To defy pathogenic fungal infection, the host innate immune system is the first line of defense. As an important arm of innate immunity, the inflammasomes are intracellular multiprotein complexes that control the activation of caspase-1, which cleaves proinflammatory cytokine pro-interleukin-1β (IL-1β) into its mature form. To determine whether the inflammasome is involved in the host defense against M. canis infection, we challenged human monocytic THP-1 cells and mouse dendritic cells with a clinical strain of M. canis isolated from patients with tinea capitis. We found that M. canis infection triggered rapid secretion of IL-1β from both THP-1 cells and mouse dendritic cells. Moreover, by using gene-specific shRNA and competitive inhibitors, we determined that M. canis-induced IL-1β secretion was dependent on NLRP3. The pathways proposed for NLRP3 inflammasome activation, namely, cathepsin B activity, K+ efflux, and reactive oxygen species production, were all required for the inflammasome activation triggered by M. canis. Meanwhile, Syk, Dectin-1, and Card9 were found to be involved in M. canis-induced IL-1β secretion via regulation of pro-IL-1β transcription. More importantly, our data revealed that M. canis-induced production of IL-1β was dependent on the NLRP3 inflammasome in vivo. Together, this study unveils that the NLRP3 inflammasome exerts a critical role in host innate immune responses against M. canis infection, and our data suggest that diseases that result from M. canis infection might be controlled by regulating the activation of inflammasomes.
PMCID: PMC3911390  PMID: 24478101
8.  Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells 
Scientific Reports  2014;4:5405.
CRISPR/Cas9 -mediated DNA cleavage (CCMDC) is becoming increasingly used for efficient genome engineering. Proto-spacer adjacent motif (PAM) adjacent to target sequence is one of the key components in the design of CCMDC strategies. It has been reported that NAG sequences are the predominant non-canonical PAM for CCMDC at the human EMX locus, but it is not clear whether it is universal at other loci. In the present study, we attempted to use a GFP-reporter system to comprehensively and quantitatively test the efficiency of CCMDC with non-canonical PAMs in human cells. The initial results indicated that the effectiveness of NGA PAM for CCMDC is much higher than that of other 14 PAMs including NAG. Then we further designed another three pairs of NGG, NGA and NAG PAMs at different locations in the GFP gene and investigated the corresponding DNA cleavage efficiency. We observed that one group of NGA PAMs have a relatively higher DNA cleavage efficiency, while the other groups have lower efficiency, compared with the corresponding NAG PAMs. Our study clearly demonstrates that NAG may not be the universally predominant non-canonical PAM for CCMDC in human cells. These findings raise more concerns over off-target effects in CRISPR/Cas9-mediated genome engineering.
PMCID: PMC4066725  PMID: 24956376
9.  A focused review of hematopoietic neoplasms occurring in the therapy-related setting 
Hematological neoplasms developed in patients with a history of cytotoxic therapies comprise a group of diseases with a poor clinical outcome, and collectively categorized as “therapy-related myeloid neoplasms” (t-MN) in the 2008 World Health Organization (WHO) Classification. In recent years, numerous publications have emerged, and these studies have greatly expanded the scope of our understanding in this field. We here focused our review on several selected areas including secondary malignancies occurring in patients with autoimmune diseases; radiation therapy alone as a causative agent; the similarity and differences between therapy-related myelodysplastic syndromes (t-MDS) and acute myeloid leukemia (t-AML); clinical behavior and treatment outcome of t-AML patients with favorable cytogenetics; the incidence and clinical features of myelodysplastic/myeloproliferative neoplasms, as well as acute lymphoblastic leukemia and myeloproliferative neoplasms in patients with prior cytotoxic exposure. These recent studies have shown that therapy-related hematopoietic neoplasms are heterogeneous, and may manifest in various forms, more complex than we have recognized previously. Cytogenetic abnormalities and underlying mutations are likely to be the major factors dictating prognosis.
PMCID: PMC4128965  PMID: 25120730
Therapy-related myeloid neoplasm; autoimmune disease; myelodysplastic syndromes; acute myeloid leukemia; acute lymphoblastic leukemia; radiation; myelodysplastic/myeloproliferative neoplasm; myeloproliferative neoplasm
10.  Altered Modular Organization of Functional Connectivity Networks in Cirrhotic Patients without Overt Hepatic Encephalopathy 
BioMed Research International  2014;2014:727452.
Minimal hepatic encephalopathy (MHE) is associated with changes in functional connectivity. To investigate the patterns of modular changes of the functional connectivity in the progression of MHE, resting-state functional magnetic resonance imaging was acquired in 24 MHE patients, 31 cirrhotic patients without minimal hepatic encephalopathy (non-HE), and 38 healthy controls. Newman's metric, the modularity Q value, was maximized and compared in three groups. Topological roles with the progression of MHE were illustrated by intra- and intermodular connectivity changes. Results showed that the Q value of MHE patients was significantly lower than that of controls (P < 0.01) rather than that of non-HE patients (P > 0.05), which was correlated with neuropsychological test scores rather than the ammonia level and Child-Pugh score. Less intrasubcortical connections and more isolated subcortical modules were found with the progression of MHE. The non-HE patients had the same numbers of connect nodes as controls and had more hubs compared with MHE patients and healthy controls. Our findings supported that both intra- and intermodular connectivity, especially those related to subcortical regions, were continuously impaired in cirrhotic patients. The adjustments of hubs and connector nodes in non-HE patients could be a compensation for the decreased modularity in their functional connectivity networks.
PMCID: PMC4066720  PMID: 25165713
11.  Homozygous inv(11)(q21q23) and MLL gene rearrangement in two patients with myeloid neoplasms 
Rearrangements of the MLL gene located at chromosome 11q23 are common chromosomal abnormalities associated with acute leukemias. In vast majority of cases with MLL gene rearrangements, only one chromosome 11 or a single MLL allele got involved. We report two very unusual cases of myeloid neoplasms with homozygous inv(11)(q21q23) and biallelic MLL rearrangement. Both patients, a 12-year old boy and a 29-year old woman, presented initially with T lymphoblastic leukemia/lymphoma (T-ALL), achieved complete remission with intensive chemotherapy, then recurred as acute myeloid leukemia in one patient and therapy-related myelodysplastic syndromes in the other patient, 24 and 15 months after initial T-ALL diagnosis, respectively. In both cases, biallelic MLL gene rearrangements were confirmed by fluorescence in situ hybridization. Mastermind like 2 gene was identified as MLL partner gene in one case. To our knowledge, homozygous inv(11)(q21q23) with two MLL genes rearrangement are extremely rare; it is likely a result of acquired uniparental disomy.
PMCID: PMC4097224  PMID: 25031740
Inv(11)(q21q23); MLL-MAML2 fusion; T lymphoblastic leukemia; myeloid neoplasms; acquired uniparental disomy; homozygosity
Src family tyrosine kinases (SFKs) are often coincidently expressed but few studies have dissected their individual functions in the same cell during development. Using the classical embryonic lens as our model we investigated SFK signaling in the regulation of both differentiation initiation and morphogenesis, and the distinct functions of c-Src and Fyn in these processes.
Blocking SFK activity with the highly specific inhibitor PP1 induced initiation of the lens differentiation program but blocked lens fiber cell elongation and organization into mini lens-like structures called lentoids. These dichotomous roles for SFK signaling were discovered to reflect distinct functions of c-Src and Fyn and their differentiation-state-specific recruitment to and action at N-cadherin junctions. c-Src was highly associated with the nascent N-cadherin junctions of undifferentiated lens epithelial cells. Its siRNA knockdown promoted N-cadherin junctional maturation, blocked proliferation, and induced lens cell differentiation. In contrast, Fyn was recruited to mature N-cadherin junctions of differentiating lens cells and siRNA knockdown suppressed differentiation-specific gene expression and blocked morphogenesis.
Through inhibition of N-cadherin junction maturation c-Src promotes lens epithelial cell proliferation and the maintenance of the lens epithelial cell undifferentiated state, while Fyn, signaling downstream of mature N-cadherin junctions, promotes lens fiber cell morphogenesis.
PMCID: PMC3882121  PMID: 23361870
Src family kinases; c-Src; Fyn; lens differentiation; morphogenesis
13.  First confirmation of imported dengue virus serotype 2 complete genome in urine from a Chinese traveler returning from India 
Virology Journal  2014;11:56.
Dengue virus (DENV) is a mosquito-borne virus that has four serotypes. Collection of serum from patients is time- and labor- consuming, and presents a high injury risk for infants and children. The genomic and serological diagnosis of imported dengue fever from a urine sample was used as a non-invasive diagnostic method in this study. A serum sample was collected on disease day 5, and a serum and urine sample were collected on disease day 8 and 18. The results of serological tests for DENV IgM revealed that the serum samples were positive for DENV. The results of RT-qPCR assay revealed that the serum sample collected on day 5 was DENV-positive; however, the serum sample collected on day 8 and 18 were negative for DENV. The urine sample collected on day 8 and 18 were DENV-positive. We also sequenced the complete DENV genome (10723 bp) from the urine sample (GenBank KF479233). The results of phylogenetic and epidemiological analysis indicated strong confirmation that the strain was located within the DENV-2 group with a 100% bootstrap value. In this report, we (1) provided the first evidence of a DENV infection that was imported from India to a non-endemic city of China, (2) investigated the DENV genome detection having a longer timeframe for positive detection in urine sample compared to previous studies, (3) provided the sequence results for the complete DENV-2 genome from a concentrated urine sample (4) discussed how virus-typing results could be used to manage the risk of sero-specific and re-infected travel-associated dengue fever.
PMCID: PMC3986945  PMID: 24666930
DENV-2; Dengue virus serotype 2; Imported disease; Complete genome; Dengue fever; Secondary infection; Travel-associated disease; Urine
14.  MicroRNA-29 induces cellular senescence in aging muscle through multiple signaling pathways 
Aging (Albany NY)  2014;6(3):160-175.
The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.
PMCID: PMC4012934  PMID: 24659628
p50; p16Ink4A; RB; B-myb; sarcopenia; p85; IGF-1
15.  Potential therapeutic targets for hypoxia-induced pulmonary artery hypertension 
Hypoxic pulmonary artery hypertension (PAH) as a severe pulmonary disease is characterized by changes of pulmonary vascular reconstruction. Mitochondrial ATP-sensitive potassium channel (mitoKATP) was considered as one of factors responsible for the proliferation of hypoxic pulmonary arterial smooth muscle cells (PASMCs), although the exact mechanisms remain unclear.
Pulmonary artery hypertension was induced in rats with or without 5-hydroxydecanoate (5-HD). The mean pulmonary artery pressure, morphologic changes, mRNA and protein expressions of voltage-gated potassium channels (Kv1.5 channel), were measured. The concentrations of monocyte chemo-attractant protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-β1) were detected. Furthermore, pulmonary arterial smooth muscle cells (PASMCs) were isolated and cultured with or without hypoxia pretreated with or without 5-HD or/and Kv1.5 inhibitor 4-aminopyridine (4-AP). Mitochondrial membrane potential (Δψm) and the proliferation of PASMCs were detected.
5-HD significantly prevented the development of PAH by blocking the mitochondrial membrane depolarization, increased the expression of voltage-gated potassium channels, and reduced pulmonary hypertension mediated by TGF-β1 or MCP-1 signaling pathway.
The MitoKATP plays an important role in the development of PAH and may be therapeutic target for the treatment of disease.
PMCID: PMC3946029  PMID: 24507703
5-hydroxydecanoate; Mitochondrial ATP-sensitive potassium channel; Hypoxia; Kv1.5 channel; Pulmonary artery hypertension
16.  Signal regulatory protein-α interacts with the insulin receptor contributing to muscle wasting in chronic kidney disease 
Kidney international  2013;84(2):308-316.
Insulin resistance from chronic kidney disease (CKD) stimulates muscle protein wasting but mechanisms causing this resistance are controversial. To help resolve this, we used microarray analyses to identify initiators of insulin resistance in the muscles of mice with CKD, glucose intolerance and insulin resistance. CKD raised mRNAs of inflammatory cytokines in muscles and there was a 5.2-fold increase in signal regulatory protein-α (SIRP-α), a transmembrane glycoprotein principally present in muscle membranes. By immunoprecipitation we found it interacts with the insulin receptor and insulin receptor substrate-1 (IRS-1). Treatment of myotubes with a mixture of inflammatory cytokines showed that SIRP-α expression was increased by a NF-κB-dependent pathway. Blockade of NF-κB using a small molecule chemical inhibitor or a dominant-negative IKKβ reduced cytokine-induced SIRP-α expression. The overexpression of SIRP-α in myotubes impaired insulin signaling and raised proteolysis while SIRP-α knockdown with siRNAs in skeletal muscle cells increased tyrosine phosphorylation of the insulin receptor and IRS-1 despite inclusion of cytokines. This led to increased p-Akt and suppression of protein degradation. Thus, SIRP-α is part of a novel mechanism for inflammation-mediated insulin resistance in muscle. In catabolic conditions with impaired insulin signaling, targeting SIRP-α may improve insulin sensitivity and prevent muscle atrophy.
PMCID: PMC3777429  PMID: 23515050
17.  HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells 
PLoS ONE  2014;9(1):e81730.
Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs (miRNAs). The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC), but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152) by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells.
MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR). Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting.
HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT–PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001), miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels.
These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1, which might, at least partially lead to cell proliferation of liver cancer cells. MiR-152 may have a therapeutic potential to suppress liver cancer proliferation.
PMCID: PMC3886937  PMID: 24416131
18.  Prevalence and Predictors of Mild Cognitive Impairment in Xi’an: A Community-Based Study among the Elders 
PLoS ONE  2014;9(1):e83217.
Mild cognitive impairment (MCI) is an intermediate stage between normal cognitive function and dementia among aging individuals. This study was designed to estimate the prevalence of MCI and explore the possible risk factors including gender disparities among community-dwelling older individuals. The study was conducted in Xi’an, China. This is a cross-sectional study. A total of 815 individuals, 60 years and older were selected by stratified random cluster sampling. Cognitive function was measured using the mini-mental status examination (MMSE), the Chinese version of the Dementia Rating Scales (CDRS) was used to apply the diagnostic of non-dementia, and activities of daily living (ADL) and instrumental activities of daily living (IADL) systems were used to functional status. The association between sociodemographic characteristics, lifestyle, history of chronic diseases and MCI were evaluated separately for men and women using the Pearson χ2- test and binary logistic regression. Of the 815 community-dwelling individuals, 145 were found to have MCI. Overall, the prevalence of MCI was 18.5%, with a prevalence of 19.6% in women (105/535), and 15.3% (40/261) in men. The results of the binary logistical regression analysis indicated that age and history of stroke were associated with MCI in men. For women, the risk factors were lower level of educational and lack of religious attendance. Results suggested that the factors capable of influencing MCI differed profoundly between older men and older women. For this reason, different preventative measures should be adopted to delay or reverse cognitive impairment among community-dwelling older men and women.
PMCID: PMC3885430  PMID: 24421876
19.  Activation of IL-27 signalling promotes development of postinfluenza pneumococcal pneumonia 
EMBO Molecular Medicine  2013;6(1):120-140.
Postinfluenza pneumococcal pneumonia is a common cause of death in humans. However, the role of IL-27 in the pathogenesis of secondary pneumococcal pneumonia after influenza is unknown. We now report that influenza infection induced pulmonary IL-27 production in a type I IFN-α/β receptor (IFNAR) signalling-dependent manner, which sensitized mice to secondary pneumococcal infection downstream of IFNAR pathway. Mice deficient in IL-27 receptor were resistant to secondary pneumococcal infection and generated more IL-17A-producing γδ T cells but not αβ T cells, thereby leading to enhanced neutrophil response during the early phase of host defence. IL-27 treatment could suppress the development of IL-17A-producing γδ T cells activated by Streptococcus pneumoniae and dendritic cells. This suppressive activity of IL-27 on γδ T cells was dependent on transcription factor STAT1. Finally, neutralization of IL-27 or administration of IL-17A restored the role of γδ T cells in combating secondary pneumococcal infection. Our study defines what we believe to be a novel role of IL-27 in impairing host innate immunity against pneumococcal infection.
PMCID: PMC3936494  PMID: 24408967
influenza virus; IL-17; IL-27; Streptococcus pneumoniae; γδ T cells
20.  Global RNA sequencing reveals that genotype-dependent allele-specific expression contributes to differential expression in rice F1 hybrids 
BMC Plant Biology  2013;13:221.
Extensive studies on heterosis in plants using transcriptome analysis have identified differentially expressed genes (DEGs) in F1 hybrids. However, it is not clear why yield in heterozygotes is superior to that of the homozygous parents or how DEGs are produced. Global allele-specific expression analysis in hybrid rice has the potential to answer these questions.
We report a genome-wide allele-specific expression analysis using RNA-sequencing technology of 3,637–3,824 genes from three rice F1 hybrids. Of the expressed genes, 3.7% exhibited an unexpected type of monoallelic expression and 23.8% showed preferential allelic expression that was genotype-dependent in reciprocal crosses. Those genes exhibiting allele-specific expression comprised 42.4% of the genes differentially expressed between F1 hybrids and their parents. Allele-specific expression accounted for 79.8% of the genes displaying more than a 10-fold expression level difference between an F1 and its parents, and almost all (97.3%) of the genes expressed in F1, but non-expressed in one parent. Significant allelic complementary effects were detected in the F1 hybrids of rice.
Analysis of the allelic expression profiles of genes at the critical stage for highest biomass production from the leaves of three different rice F1 hybrids identified genotype-dependent allele-specific expression genes. A cis-regulatory mechanism was identified that contributes to allele-specific expression, leading to differential gene expression and allelic complementary effects in F1 hybrids.
PMCID: PMC3878109  PMID: 24358981
Allele-specific expression; Complementary effects; Differentially expressed genes; Genotype-dependent monoallelic expression; Rice hybrids
21.  MiR-103 Controls Milk Fat Accumulation in Goat (Capra hircus) Mammary Gland during Lactation 
PLoS ONE  2013;8(11):e79258.
Milk is the primary source of nutrition for young mammals including humans. The nutritional value of milk is mainly attributable to fats and proteins fractions. In comparison to cow milk, goat milk contains greater amounts of total fat, including much higher levels of the beneficial unsaturated fatty acids. MicroRNAs (miRNAs), a well-defined group of small RNAs containing about 22 nucleotides (nt), participate in various metabolic processes across species. However, little is known regarding the role of miRNAs in regulating goat milk composition. In the present study, we performed high-throughput sequencing to identify mammary gland-enriched miRNAs in lactating goats. We identified 30 highly expressed miRNAs in the mammary gland, including miR-103. Further studies revealed that miR-103 expression correlates with the lactation. Further functional analysis showed that over-expression of miR-103 in mammary gland epithelial cells increases transcription of genes associated with milk fat synthesis, resulting in an up-regulation of fat droplet formation, triglyceride accumulation, and the proportion of unsaturated fatty acids. This study provides new insight into the functions of miR-103, as well as the molecular mechanisms that regulate milk fat synthesis.
PMCID: PMC3823599  PMID: 24244462
22.  Crystallization and preliminary X-ray analysis of the RPB5 subunit of human RNA polymerase II 
The recombinant RPB5 subunit of human RNA polymerase II has been crystallized by vapour diffusion in hanging drops.
RPB5 is an essential subunit of eukaryotic RNA polymerase II. It has been proposed to interact with DNA and several key transcription factors during transcription. These interactions are crucial for transcription and its regulation. Here, prior to obtaining complex structures of human RPB5 and its binding partners, recombinant human RPB5 was crystallized alone by vapour diffusion in hanging drops. A complete data set was collected from a single frozen crystal employing an in-house X-ray source. The crystal diffracted to 2.8 Å resolution and belonged to space group P43212. The likely Matthews coefficient and solvent content of 2.67 Å3 Da−1 and 53.92%, respectively, suggested the presence of two protein subunits in the asymmetric unit. The structure was solved using molecular replacement.
PMCID: PMC3212458  PMID: 22102239
RNA polymerase II; RPB5 subunit
23.  The Role of MicroRNA-146a in the Pathogenesis of the Diabetic Wound-Healing Impairment 
Diabetes  2012;61(11):2906-2912.
The impairment in diabetic wound healing represents a significant clinical problem. Chronic inflammation is thought to play a central role in the pathogenesis of this impairment. We have previously shown that treatment of diabetic murine wounds with mesenchymal stem cells (MSCs) can improve healing, but the mechanisms are not completely defined. MicroRNA-146a (miR-146a) has been implicated in regulation of the immune and inflammatory responses. We hypothesized that abnormal miRNA-146a expression may contribute to the chronic inflammation. To test this hypothesis, we examined the expression of miRNA-146a and its target genes in diabetic and nondiabetic mice at baseline and after injury. MiR-146a expression was significantly downregulated in diabetic mouse wounds. Decreased miR-146a levels also closely correlated with increased gene expression of its proinflammatory target genes. Furthermore, the correction of the diabetic wound-healing impairment with MSC treatment was associated with a significant increase in the miR-146a expression level and decreased gene expression of its proinflammatory target genes. These results provide the first evidence that decreased expression of miR-146a in diabetic wounds in response to injury may, in part, be responsible for the abnormal inflammatory response seen in diabetic wounds and may contribute to wound-healing impairment.
PMCID: PMC3478555  PMID: 22851573
24.  Hydrogen Sulfide Improves Drought Tolerance in Arabidopsis thaliana by MicroRNA Expressions 
PLoS ONE  2013;8(10):e77047.
Hydrogen sulfide (H2S) is a gasotransmitter and plays an important role in many physiological processes in mammals. Studies of its functions in plants are attracting ever growing interest, for example, its ability to enhance drought resistance in Arabidopsis. A general role of microRNAs (miRNAs) in plant adaptive responses to drought stress has thereby increased our interest to delve into the possible interplay between H2S and miRNAs. Our results showed that treating wild type (WT) Arabidopsis seedlings with polyethylene glycol 8000 (PEG8000) to simulate drought stress caused an increase in production rate of endogenous H2S; and a significant transcriptional reformation of relevant miRNAs, which were also triggered by exogenous H2S in WT. When lcd mutants (with lower H2S production rate than WT) were treated with PEG8000, they showed lower levels of miRNA expression changes than WT. In addition, we detected significant changes in target gene expression of those miRNAs and the corresponding phenotypes in lcd, including less roots, retardation of leaf growth and development and greater superoxide dismutase (SOD) activity under drought stress. We thereby conclude that H2S can improve drought resistance through regulating drought associated miRNAs in Arabidopsis.
PMCID: PMC3806758  PMID: 24194857
25.  Impact of a regional acute care surgery model on patient access and outcomes 
Canadian Journal of Surgery  2013;56(5):318-324.
The consolidation of acute care surgery (ACS) services at 3 of 6 hospitals in a Canadian health region sought to alleviate a relative shortage of surgeons able to take emergency call. We examined how this affected patient access and outcomes.
Using the generalized linear model and statistical process control, we analyzed ACS-related episodes that occurred between 39 months prior to and 17 months after the model’s implementation (n = 14 713).
Time to surgery increased after the consolidation. Wait times increased primarily for patients presenting at nonreferral hospitals who were likely to require transfer to a referral hospital. Although ACS teams enabled referral hospitals to handle a much higher volume of patients without increasing within-hospital wait times, overall system wait times were lengthened by the growing frequency of patient transfers. Wait times for inpatient admission were difficult to interpret because there was a trend toward admitting patients directly to the ACS service, bypassing the emergency department (ED). For patients who did go through the ED, wait times for inpatient admission increased after the consolidation; however, this trend was cancelled out by the apparently zero waits of patients who bypassed the ED. Regionalization showed no impact on length of stay, readmissions, mortality or complications.
Consolidation enabled the region to ensure adequate surgical coverage without harming patients. The need to transfer patients who presented at nonreferral hospitals led to longer waits.
PMCID: PMC3788010  PMID: 24067516

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