Publications by authors named "Liyi Wang"

21 Publications

  • Page 1 of 1

Microfluidic Model to Mimic Initial Event of Neovascularization.

J Vis Exp 2021 Apr 10(170). Epub 2021 Apr 10.

Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Chinese Education Ministry, School of Biological Science and Medical Engineering, Beihang University; School of Engineering Medicine, Beihang University;

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.
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http://dx.doi.org/10.3791/62003DOI Listing
April 2021

A model to predict the risk of mortality in severely ill COVID-19 patients.

Comput Struct Biotechnol J 2021 22;19:1694-1700. Epub 2021 Mar 22.

Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China.

Background: To investigate and select the useful prognostic parameters to develop and validate a model to predict the mortality risk for severely and critically ill patients with the coronavirus disease 2019 (COVID-19).

Methods: We established a retrospective cohort of patients with laboratory-confirmed COVID-19 (≥18 years old) from two tertiary hospitals: the People's Hospital of Wuhan University and Leishenshan Hospital between February 16, 2020, and April 14, 2020. The diagnosis of the cases was confirmed according to the WHO interim guidance. The data of consecutive severely and critically ill patients with COVID-19 admitted to these hospitals were analyzed. A total of 566 patients from the People's Hospital of Wuhan University were included in the training cohort and 436 patients from Leishenshan Hospital were included in the validation cohort. The least absolute shrinkage and selection operator (LASSO) and multivariable logistic regression were used to select the variables and build the mortality risk prediction model.

Results: The prediction model was presented as a nomograph and developed based on identified predictors, including age, chronic lung disease, C-reactive protein (CRP), D-dimer levels, neutrophil-to-lymphocyte ratio (NLR), creatinine, and total bilirubin. In the training cohort, the model displayed good discrimination with an AUC of 0.912 [95% confidence interval (CI): 0.884-0.940] and good calibration (intercept = 0; slope = 1). In the validation cohort, the model had an AUC of 0.922 [95% confidence interval (CI): 0.891-0.953] and a good calibration (intercept = 0.056; slope = 1.161). The decision curve analysis (DCA) demonstrated that the nomogram was clinically useful.

Conclusion: A risk score for severely and critically ill COVID-19 patients' mortality was developed and externally validated. This model can help clinicians to identify individual patients at a high mortality risk.
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http://dx.doi.org/10.1016/j.csbj.2021.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7983362PMC
March 2021

A small-molecule pan-class I glucose transporter inhibitor reduces cancer cell proliferation in vitro and tumor growth in vivo by targeting glucose-based metabolism.

Cancer Metab 2021 Mar 26;9(1):14. Epub 2021 Mar 26.

Department of Biological Sciences, Ohio University, Athens, OH, 45701, USA.

Background: Cancer cells drastically increase the uptake of glucose and glucose metabolism by overexpressing class I glucose transporters (GLUT1-4) to meet their energy and biomass synthesis needs and are very sensitive and vulnerable to glucose deprivation. Although targeting glucose uptake via GLUTs has been an attractive anticancer strategy, the relative anticancer efficacy of multi-GLUT targeting or single GLUT targeting is unclear. Here, we report DRB18, a synthetic small molecule, is a potent anticancer compound whose pan-class I GLUT inhibition is superior to single GLUT targeting.

Methods: Glucose uptake and MTT/resazurin assays were used to measure DRB18's inhibitory activities of glucose transport and cell viability/proliferation in human lung cancer and other cancer cell lines. Four HEK293 cell lines expressing GLUT1-4 individually were used to determine the IC values of DRB18's inhibitory activity of glucose transport. Docking studies were performed to investigate the potential direct interaction of DRB18 with GLUT1-4. Metabolomics analysis was performed to identify metabolite changes in A549 lung cancer cells treated with DRB18. DRB18 was used to treat A549 tumor-bearing nude mice. The GLUT1 gene was knocked out to determine how the KO of the gene affected tumor growth.

Results: DRB18 reduced glucose uptake mediated via each of GLUT1-4 with different ICs, which match with the docking glidescores with a correlation coefficient of 0.858. Metabolomics analysis revealed that DRB18 altered energy-related metabolism in A549 cells by changing the abundance of metabolites in glucose-related pathways in vitro and in vivo. DRB18 eventually led to G1/S phase arrest and increased oxidative stress and necrotic cell death. IP injection of DRB18 in A549 tumor-bearing nude mice at 10 mg/kg body weight thrice a week led to a significant reduction in the tumor volume compared with mock-treated tumors. In contrast, the knockout of the GLUT1 gene did not reduce tumor volume.

Conclusions: DRB18 is a potent pan-class I GLUT inhibitor in vitro and in vivo in cancer cells. Mechanistically, it is likely to bind the outward open conformation of GLUT1-4, reducing tumor growth through inhibiting GLUT1-4-mediated glucose transport and metabolisms. Pan-class I GLUT inhibition is a better strategy than single GLUT targeting for inhibiting tumor growth.
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http://dx.doi.org/10.1186/s40170-021-00248-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004435PMC
March 2021

Flow shear stress controls the initiation of neovascularization via heparan sulfate proteoglycans within a biomimetic microfluidic model.

Lab Chip 2021 01 22;21(2):421-434. Epub 2020 Dec 22.

Key Laboratory for Biomechanics and Mechanobiology of the Ministry of Education, Beijing Advanced Innovation Centre for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.

Endothelial cells (ECs) in vivo are subjected to three forms of shear stress induced by luminal blood flow, transendothelial flow and interstitial flow simultaneously. It is controversial that shear stress, especially the component induced by luminal flow, was thought to inhibit the initialization of angiogenesis and trigger arteriogenesis. Here, we combined microfabrication techniques and delicate numerical simulations to reconstruct the initial physiological microenvironment of neovascularization in vitro, where ECs experience high luminal shear stress, physiological transendothelial flow and various vascular endothelial growth factor (VEGF) distributions simultaneously. With the biomimetic microfluidic model, cell alignment and endothelial sprouting assays were carried out. We found that luminal shear stress inhibits endothelial sprouting and tubule formation in a dose-dependent manner. Although a high concentration of VEGF increases EC sprouting, neither a positive nor a negative VEGF gradient additionally affects the degree of sprouting, and luminal shear stress significantly attenuates neovascularization even in the presence of VEGF. Heparinase was used to selectively degrade the heparan sulfate proteoglycan (HSPG) coating on ECs and messenger RNA profiles in ECs were analyzed. It turned out that HSPGs could act as a mechanosensor to sense the change of fluid shear stress, modulate multiple EC gene expressions, and hence affect neovascularization. In summary, distraction from the stabilized state, such as decreased luminal shear stress, increased VEGF and the destructed mechanotransduction of HSPGs would induce the initiation of neovascularization. Our study highlights the key role of the magnitude and forms of shear stress in neovascularization.
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http://dx.doi.org/10.1039/d0lc00493fDOI Listing
January 2021

LKB1 Differently Regulates Adipogenesis in Intramuscular and Subcutaneous Adipocytes through Metabolic and Cytokine-Related Signaling Pathways.

Cells 2020 12 4;9(12). Epub 2020 Dec 4.

College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.

Liver kinase B1 (LKB1) plays important and various roles in the differentiation and lipid metabolism of adipocytes. However, the current knowledge of the respective roles of LKB1 in subcutaneous fat (SCF) and intramuscular fat (IMF) adipocytes remains unclear. This study aimed to discover the different regulatory mechanisms of LKB1 in SCF and IMF adipocytes. We found that LKB1 overexpression inhibited adipogenesis in both SCF and IMF adipocytes, and SCF adipocytes were more sensitive to regulation by LKB1. Transcriptomics results showed that IMF adipocytes had many more differentially expressed genes (DEGs) than SCF adipocytes. Pathway analysis of the shared and distinct DEGs revealed that the main adipogenesis mechanism was similar between SCF and IMF adipocytes upon LKB1 overexpression, while regulatory and metabolic signaling pathways, such as MAPK, PPAR signaling pathways, were differently regulated by LKB1. Several cytokine-related pathways were only enriched in LKB1-overexpressing IMF adipocytes. Our study reveals different regulators and signaling pathways between SCF and IMF adipocytes under LKB1 overexpression, which may be potential targets to differentially control SCF and IMF deposition and improve our understanding of the regulatory mechanisms of IMF deposition.
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http://dx.doi.org/10.3390/cells9122599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761942PMC
December 2020

The regulatory role of dietary factors in skeletal muscle development, regeneration and function.

Crit Rev Food Sci Nutr 2020 Oct 6:1-19. Epub 2020 Oct 6.

College of Animal Sciences, Zhejiang University, Hangzhou, China.

Skeletal muscle plays a crucial role in motor function, respiration, and whole-body energy homeostasis. How to regulate the development and function of skeletal muscle has become a hot research topic for improving lifestyle and extending life span. Numerous transcription factors and nutritional factors have been clarified are closely associated with the regulation of skeletal muscle development, regeneration and function. In this article, the roles of different dietary factors including green tea, quercetin, curcumin (CUR), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and resveratrol (RES) in regulating skeletal muscle development, muscle mass, muscle function, and muscle recovery have been summarized and discussed. We also reviewed the potential regulatory molecular mechanism of these factors. Based on the current findings, dietary factors may be used as a potential therapeutic agent to treat skeletal muscle dysfunction as well as its related diseases.
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http://dx.doi.org/10.1080/10408398.2020.1828812DOI Listing
October 2020

Factors inducing transdifferentiation of myoblasts into adipocytes.

J Cell Physiol 2021 Apr 28;236(4):2276-2289. Epub 2020 Sep 28.

College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China.

Fat infiltration in skeletal muscle is observed in several myopathies, is associated with muscular dysfunction, and is strongly correlated with insulin resistance, diabetes, obesity, and aging. In animal production, skeletal muscle fat (also known as intermuscular and intramuscular fat) is positively related to meat quality including tenderness, flavor, and juiciness. Thus, understanding the cell origin and regulation mechanism of skeletal muscle fat infiltration is important for developing therapies against human myopathies as well as for improving meat quality. Notably, age, sarcopenia, oxidative stress, injury, and regeneration can activate adipogenic differentiation potential in myoblasts and affect fat accumulation in skeletal muscle. In addition, several transcriptional and nutritional factors can directly induce transdifferentiation of myoblasts into adipocytes. In this review, we focused on the recent progress in understanding the muscle-to-adipocyte differentiation and summarized and discussed the genetic, nutritional, and physiological factors that can induce transdifferentiation of myoblasts into adipocytes. Moreover, the regulatory roles and mechanisms of these factors during the transdifferentiation process were also discussed.
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http://dx.doi.org/10.1002/jcp.30074DOI Listing
April 2021

The Monocyte to High-Density Lipoprotein Cholesterol Ratio in the Prediction for Atherosclerosis: A Retrospective Study in Adult Chinese Participants.

Lipids 2021 01 7;56(1):69-80. Epub 2020 Sep 7.

Department of cardiology, The Second Hospital of Hebei Medical University and Institute of Cardiocerebrovascular Disease of Hebei Province, Shijiazhuang, 050000, China.

The ratio of monocyte to high-density lipoprotein cholesterol level (MHR) was a newly proposed inflammatory and oxidative stress marker. This study aimed to explore the association between MHR and Brachial-ankle pulse wave velocity (Ba-PWV) in adult Chinese participants. A total of 2029 participants were divided into two groups according to the Ba-PWV: a high Ba-PWV group (Ba-PWV ≥1400 cm/s) and a low Ba-PWV group (Ba-PWV < 1400 cm/s). According to the cut-off points of quartile of MHR, the participants were divided into four groups. The relationship between MHR and Ba-PWV was analyzed. After adjusting for potential confounders, a non-linear relationship between MHR and Ba-PWV was found in the participants, and the inflection point was 7.78 in the non-linear curve. On the left of the inflection point, MHR had a positive correlation with Ba-PWV (OR = 1.17, 95% confidence interval (CI): 1.08 to 1.28, p < 0.01). However, there was no obvious relationship between MHR and Ba-PWV on the right of the inflection point (OR = 0.96, 95% CI: 0.90 to 1.01, p = 0.117). Further demographic analysis demonstrated that the positive relationship between MHR and Ba-PWV was found in the female participants with hypertension family history, but without a current history of hypertension, smoking, or drinking (p < 0.05). An increased MHR is a risk factor of atherosclerosis, which may predict the potential development of atherosclerosis. When the MHR is close to 7.78, it has the highest predictive value for the risk of atherosclerosis occurrence.
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http://dx.doi.org/10.1002/lipd.12276DOI Listing
January 2021

Myokines mediate the cross talk between skeletal muscle and other organs.

J Cell Physiol 2021 Apr 3;236(4):2393-2412. Epub 2020 Sep 3.

College of Animal Sciences, Zhejiang University, Hangzhou, China.

Myokines are muscle-derived cytokines and chemokines that act extensively on organs and exert beneficial metabolic functions in the whole-body through specific signal networks. Myokines as mediators provide the conceptual basis for a whole new paradigm useful for understanding how skeletal muscle communicates with other organs. In this review, we summarize and discuss classes of myokines and their physiological functions in mediating the regulatory roles of skeletal muscle on other organs and the regulation of the whole-body energy metabolism. We review the mechanisms involved in the interaction between skeletal muscle and nonmuscle organs through myokines. Moreover, we clarify the connection between exercise, myokines and disease development, which may contribute to the understanding of a potential mechanism by which physical inactivity affects the process of metabolic diseases via myokines. Based on the current findings, myokines are important factors that mediate the effect of skeletal muscle on other organ functions and whole-body metabolism.
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http://dx.doi.org/10.1002/jcp.30033DOI Listing
April 2021

Low Dietary n-6/n-3 PUFA Ratio Regulates Meat Quality, Reduces Triglyceride Content, and Improves Fatty Acid Composition of Meat in Heigai Pigs.

Animals (Basel) 2020 Sep 1;10(9). Epub 2020 Sep 1.

College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.

The objective of this study was to investigate the effects of dietary supplementation with different n-6/n-3 polyunsaturated fatty acid (PUFA) ratios on growth performance, meat quality, and fatty acid profile in Heigai pigs. A total of 54 Heigai finishing pigs (body weight: 71.59 ± 2.16 kg) were randomly divided into three treatments with six replications (three pigs per replication) and fed diets containing different n-6/n-3 PUFA ratios: 8:1, 5:1, and 3:1. Pigs fed the dietary n-6/n-3 PUFA ratio of 8:1 had the highest feed to gain ratio ( < 0.01), carcass weight ( < 0.05), redness a* ( < 0.01), and yellowness b* ( < 0.01). Fatty acid compositions in longissimus dorsi muscle (LDM) and subcutaneous adipose tissue (SAT) were significantly changed ( < 0.01). Notably, the meat from the pigs fed with the low dietary n-6/n-3 PUFA ratio had higher n-3 PUFA contents ( < 0.01) and lower n-6/n-3 PUFA ratio ( < 0.01). The triglyceride and total cholesterol contents were significantly decreased in SAT from the pigs fed with dietary n-6/n-3 PUFA ratios of 5:1 ( < 0.05) and 3:1 ( < 0.01). Reducing n-6/n-3 PUFA ratio upregulated the expression of HSL ( < 0.05), CPT1 ( < 0.01), and FABP4 ( < 0.01) but downregulated ATGL ( < 0.01) expression. These results demonstrate that the lower n-6/n-3 PUFA ratio regulates meat quality and enhances the deposition of n-3 PUFA in Heigai pigs.
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http://dx.doi.org/10.3390/ani10091543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552283PMC
September 2020

Isosteres of ester derived glucose uptake inhibitors.

Bioorg Med Chem Lett 2020 09 15;30(18):127406. Epub 2020 Jul 15.

Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA; Program of Molecular and Cellular Biology, Ohio University, Athens, OH 45701, USA. Electronic address:

Glucose transporters (GLUTs) facilitate glucose uptake and are overexpressed in most cancer cells. Inhibition of glucose transport has been shown to be an effective method to slow the growth of cancer cells both in vitro and in vivo. We have previously reported on the anticancer activity of an ester derived glucose uptake inhibitor. Due to the hydrolytic instability of the ester linkage we have prepared a series of isosteres of the ester moiety. Of all of the isosteres prepared, the amine linkage showed the most promise. Several additional analogues of the amine-linked compounds were also prepared to improve the overall activity.
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http://dx.doi.org/10.1016/j.bmcl.2020.127406DOI Listing
September 2020

Increased inheritance of structure and function of bacterial communities and pathogen propagation in plastisphere along a river with increasing antibiotics pollution gradient.

Environ Pollut 2020 Oct 21;265(Pt A):114641. Epub 2020 Apr 21.

Xinjiang Key Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, 830011, China; Key Laboratory of Microbial Technology for Industrial Pollution Control of Zhejiang Province, College of Environment, Zhejiang University of Technology, Hangzhou, 310014, China.

Plastic debris provides a stable substrate and novel ecological niche for microorganisms in the aquatic environment, which was referred to as "Plastisphere". Little is known about distribution patterns and responses of ecological function and structure of microbial communities in the plastisphere along rivers which usually have antibiotics pollution gradient. In this study, the differences in the community structure between the plastisphere and the planktonic bacteria, and their spatial variation of the community structure and function along a river with increased antibiotics pollution gradient was investigated at the watershed scale. The diversity of bacteria colonized on most plastic debris was higher than in surrounding water. Plastic debris could accumulate a higher abundance of some potential pathogens than surrounding water even at high antibiotics concentrations. The source tracking results showed that downstream plastisphere inherited much higher proportions of bacterial taxa from upstream than planktonic bacteria. About 92.3-99.7% of bacteria communities in downstream water were not from upstream but from the input of downstream human activities. On the contrary, high proportions of bacterial taxa in downstream plastisphere were closely connected to upstream. The plastisphere possesses higher ecological functional diversity than the planktonic bacteria. Seventy nine functional groups across plastisphere were predicted using functional annotation of prokaryotic taxa and only 65 functional groups were found in the planktonic bacteria. Plastisphere also acts as hotspot for biogeochemical cycling of nutrients such as N and S. Intensive human activities of urban and downstream agriculture and aquaculture had great effects on microbial community structure and functional groups of the Urumqi River. Pastisphere communities are much more resistant to human disturbance than planktonic bacteria. Compared to surrounding water, plastisphere increased inheritance from upstream microbial structure and function and also increased survival and propagation of pathogens in the downstream water with high concentrations of antibiotics.
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http://dx.doi.org/10.1016/j.envpol.2020.114641DOI Listing
October 2020

Bacterial community rather than metals shaping metal resistance genes in water, sediment and biofilm in lakes from arid northwestern China.

Environ Pollut 2019 Nov 10;254(Pt A):113041. Epub 2019 Aug 10.

University of Chinese Academy of Science, Beijing, 100049, China; Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, China; School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK. Electronic address:

Lakes in arid northwestern China are valuable freshwater resources that drive socioeconomic development. Environmental pollution can significantly influence the composition of microbial communities and the distribution of functional genes in lakes. This study investigated heavy metal pollution to identify possible correlations with metal resistance genes (MRGs) and bacterial community composition in water, sediment and biofilm samples from Bosten Lake and Ebi Lake in northwestern China. High levels of zinc were detected in all samples. However, the metals detected in the sediment samples of both lakes were determined to be at low risk levels according to an ecological index. The mercury resistance gene subtype merP had the greatest average abundance (4.61 × 10 copies per 16S rRNA) among all the samples, followed by merA and merC. The high abundance of merA in the pelagic zone rather than in benthic sediment suggests that the pelagic microbial community was important in mercury reduction. Proteobacteria were the main phylum found in the microbial communities in all samples. However, microbial communities in most of the water, sediment and biofilm samples had different compositions, indicating that the habitat niche plays an important role in shaping the bacterial communities in lakes. The microbial community, rather than the heavy metals, was the main driver of MRG distribution. The abundances of some bacterial genera involved in the decomposition of organic matter and the terrestrial nitrogen cycle were negatively correlated with heavy metals. This result suggests that metal pollution can adversely affect the biogeochemical processes that occur in lakes.
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http://dx.doi.org/10.1016/j.envpol.2019.113041DOI Listing
November 2019

Distinct miRNA profiles in normal and gastric cancer myofibroblasts and significance in Wnt signaling.

Am J Physiol Gastrointest Liver Physiol 2016 05 3;310(9):G696-704. Epub 2016 Mar 3.

Departments of Cellular and Molecular Physiology and

Stromal cells influence epithelial function in both health and disease. Myofibroblasts are abundant stromal cells that influence the cellular microenvironment by release of extracellular matrix (ECM) proteins, growth factors, proteases, cytokines, and chemokines. Cancer-associated myofibroblasts (CAMs) differ from adjacent tissue (ATMs) and normal tissue myofibroblasts (NTMs), but the basis of this is incompletely understood. We report now the differential expression of miRNAs in gastric cancer CAMs. MicroRNA arrays identified differences in the miRNA profile in gastric and esophageal NTMs and in CAMs from stomach compared with NTMs. miR-181d was upregulated in gastric CAMs. Analysis of differentially regulated miRNAs indicated an involvement in Wnt signaling. Examination of a microarray data set then identified Wnt5a as the only consistently upregulated Wnt ligand in gastric CAMs. Wnt5a stimulated miR-181d expression, and knockdown of miR-181d inhibited Wnt5a stimulation of CAM proliferation and migration. Analysis of miR-181d targets suggested a role in chemotaxis. Conditioned medium from CAMs stimulated gastric cancer cell (AGS) migration more than that from ATMs, and miR-181d knockdown reduced the effect of CAM-CM on AGS cell migration but had no effect on AGS cell responses to ATM conditioned media. The data suggest that dysregulation of miRNA expression in gastric CAMs, secondary to Wnt5a signaling, accounts at least in part for the effect of CAMs in promoting cancer cell migration.
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http://dx.doi.org/10.1152/ajpgi.00443.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867324PMC
May 2016

Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

Mol Cancer Res 2014 Sep 21;12(9):1233-43. Epub 2014 May 21.

Molecular Biology Graduate Program and Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado

Unlabelled: Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing.

Implications: This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes.
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http://dx.doi.org/10.1158/1541-7786.MCR-14-0149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4163527PMC
September 2014

HIFs enhance the transcriptional activation and splicing of adrenomedullin.

Mol Cancer Res 2014 May 12;12(5):728-41. Epub 2014 Feb 12.

Authors' Affiliations: Molecular Biology Graduate Program, 2Department of Craniofacial Biology School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Unlabelled: Adrenomedullin (ADM) is important for tumor angiogenesis, tumor cell growth, and survival. Under normoxic conditions, the ADM gene was found to produce two alternative transcripts, a fully spliced transcript that produces AM and PAMP peptides and intron-3-retaining transcript that produces a less functionally significant PAMP peptide only. ADM is a well-established hypoxia inducible gene; however, it is not clear which ADM isoform is induced by hypoxia. In this study, it was determined that various cancer and normal cells express two predominant types of ADM transcripts, a AM/PAMP peptide producing full-length transcript in which all introns are removed, and a nonprotein producing I1-3 transcript in which all introns are retained. Interestingly, hypoxia preferentially induced the full-length isoform. Moreover, hypoxia-inducible factors (HIF), but not hypoxia per se, are necessary and sufficient to increase splicing of ADM pre-mRNA. ADM splicing reporters confirmed that transcriptional activation by HIF or other transcription factors is sufficient to enhance splicing. However, HIFs are more potent in enhancing ADM pre-mRNA splicing than other transcriptional activators. Thus, ADM intron retention is not a consequence of abnormal splicing, but is an important mechanism to regulate ADM expression. These results demonstrate a novel function of HIFs in regulating ADM expression by enhancing its pre-mRNA splicing. Importantly, using endogenous and cloned ADM gene, further evidence is provided for the coupling of transcription and RNA splicing.

Implications: Here, a novel function of HIFs in regulating ADM gene expression is identified by enhancing ADM pre-mRNA splicing.
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http://dx.doi.org/10.1158/1541-7786.MCR-13-0607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020993PMC
May 2014

STAT3 or USF2 contributes to HIF target gene specificity.

PLoS One 2013 21;8(8):e72358. Epub 2013 Aug 21.

Molecular Biology Graduate Program, School of Dental Medicine University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072358PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749168PMC
June 2014

BRG1 and BRM chromatin-remodeling complexes regulate the hypoxia response by acting as coactivators for a subset of hypoxia-inducible transcription factor target genes.

Mol Cell Biol 2013 Oct 29;33(19):3849-63. Epub 2013 Jul 29.

Molecular Biology Graduate Program.

Chromatin remodeling is an active process, which represses or enables the access of transcription machinery to genes in response to external stimuli, including hypoxia. However, in hypoxia, the specific requirement, as well as the molecular mechanism by which the chromatin-remodeling complexes regulate gene expression, remains unclear. In this study, we report that the Brahma (BRM) and Brahma-related gene 1 (BRG1) ATPase-containing SWI/SNF chromatin-remodeling complexes promote the expression of the hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α genes and also promote hypoxic induction of a subset of HIF1 and HIF2 target genes. We show that BRG1 or BRM knockdown in Hep3B and RCC4T cells reduces hypoxic induction of HIF target genes, while reexpression of BRG1 or BRM in BRG1/BRM-deficient SW13 cells increases HIF target gene activation. Mechanistically, HIF1 and HIF2 increase the hypoxic induction of HIF target genes by recruiting BRG1 complexes to HIF target gene promoters, which promotes nucleosome remodeling of HIF target gene promoters in a BRG1 ATPase-dependent manner. Importantly, we found that the function of BRG1 complexes in hypoxic SW13 and RCC4T cells is dictated by the HIF-mediated hypoxia response and could be opposite from their function in normoxic SW13 and RCC4T cells.
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http://dx.doi.org/10.1128/MCB.00731-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811865PMC
October 2013

Distinct reactivity of Morita-Baylis-Hillman acetates as a novel C(2) component in amine-catalyzed [2 + 2 + 2] and [2 + 4] annulations.

Chem Commun (Camb) 2013 May;49(34):3543-5

The State Key Laboratory of Elemento-Organic Chemistry and Department of Chemistry, Nankai University, Tianjin 300071, PR China.

Amine-catalyzed [2 + 2 + 2] and [2 + 4] annulations of Morita-Baylis-Hillman (MBH) acetates with cyano activated alkenes and 1,3-azadienes have been developed to provide cyclohexanes and tetrahydropyridines. In the annulations, MBH acetates serve as a novel C(2) component with an inactive homoallylic methyl involved in the bond formation.
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http://dx.doi.org/10.1039/c3cc41419aDOI Listing
May 2013

Upstream stimulatory factor 2 and hypoxia-inducible factor 2α (HIF2α) cooperatively activate HIF2 target genes during hypoxia.

Mol Cell Biol 2012 Nov 10;32(22):4595-610. Epub 2012 Sep 10.

Molecular Biology Graduate Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

While the functions of hypoxia-inducible factor 1α (HIF1α)/aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF2α/ARNT (HIF2) proteins in activating hypoxia-inducible genes are well established, the role of other transcription factors in the hypoxic transcriptional response is less clear. We report here for the first time that the basic helix-loop-helix-leucine-zip transcription factor upstream stimulatory factor 2 (USF2) is required for the hypoxic transcriptional response, specifically, for hypoxic activation of HIF2 target genes. We show that inhibiting USF2 activity greatly reduces hypoxic induction of HIF2 target genes in cell lines that have USF2 activity, while inducing USF2 activity in cells lacking USF2 activity restores hypoxic induction of HIF2 target genes. Mechanistically, USF2 activates HIF2 target genes by binding to HIF2 target gene promoters, interacting with HIF2α protein, and recruiting coactivators CBP and p300 to form enhanceosome complexes that contain HIF2α, USF2, CBP, p300, and RNA polymerase II on HIF2 target gene promoters. Functionally, the effect of USF2 knockdown on proliferation, motility, and clonogenic survival of HIF2-dependent tumor cells in vitro is phenocopied by HIF2α knockdown, indicating that USF2 works with HIF2 to activate HIF2 target genes and to drive HIF2-depedent tumorigenesis.
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http://dx.doi.org/10.1128/MCB.00724-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486188PMC
November 2012

The N-terminal transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1alpha and HIF-2alpha.

Mol Biol Cell 2007 Nov 5;18(11):4528-42. Epub 2007 Sep 5.

Abramson Family Cancer Research Institute,University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

The basic helix-loop-helix-Per-ARNT-Sim-proteins hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha are the principal regulators of the hypoxic transcriptional response. Although highly related, they can activate distinct target genes. In this study, the protein domain and molecular mechanism important for HIF target gene specificity are determined. We demonstrate that although HIF-2alpha is unable to activate multiple endogenous HIF-1alpha-specific target genes (e.g., glycolytic enzymes), HIF-2alpha still binds to their promoters in vivo and activates reporter genes derived from such targets. In addition, comparative analysis of the N-terminal DNA binding and dimerization domains of HIF-1alpha and HIF-2alpha does not reveal any significant differences between the two proteins. Importantly, replacement of the N-terminal transactivation domain (N-TAD) (but not the DNA binding domain, dimerization domain, or C-terminal transactivation domain [C-TAD]) of HIF-2alpha with the analogous region of HIF-1alpha is sufficient to convert HIF-2alpha into a protein with HIF-1alpha functional specificity. Nevertheless, both the N-TAD and C-TAD are important for optimal HIF transcriptional activity. Additional experiments indicate that the ETS transcription factor ELK is required for HIF-2alpha to activate specific target genes such as Cited-2, EPO, and PAI-1. These results demonstrate that the HIF-alpha TADs, particularly the N-TADs, confer HIF target gene specificity, by interacting with additional transcriptional cofactors.
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http://dx.doi.org/10.1091/mbc.e06-05-0419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2043574PMC
November 2007