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1.  Quantitative survey of multiple CpGs from 5 genes identifies CpG methylation panel discriminating between high- and low-grade cervical intraepithelial neoplasia 
Clinical Epigenetics  2015;7(1):4.
Studies of methylation biomarkers for cervical cancer often involved only few randomly selected CpGs per candidate gene analyzed by methylation-specific PCR-based methods, with often inconsistent results from different laboratories. We evaluated the role of different CpGs from multiple genes as methylation biomarkers for high-grade cervical intraepithelial neoplasia (CIN).
We applied a mass spectrometry-based platform to survey the quantitative methylation levels of 34 CpG units from SOX1, PAX1, NKX6-1, LMX1A, and ONECUT1 genes in 100 cervical formalin-fixed paraffin-embedded (FFPE) tissues. We then used nonparametric statistics and Random Forest algorithm to rank significant CpG methylations and support vector machine with 10-fold cross validation and 200 times bootstrap resampling to build a predictive model separating CIN II/III from CIN I/normal subjects. We found only select CpG units showed significant differences in methylation between CIN II/III and CIN I/normal groups, while mean methylation levels per gene were similar between the two groups for each gene except PAX1. An optimal classification model involving five CpG units from SOX1, PAX1, NKX6-1, and LMX1A achieved 81.2% specificity, 80.4% sensitivity, and 80.8% accuracy.
Our study suggested that during CIN development, the methylation of CpGs within CpG islands is not uniform, with varying degrees of significance as biomarkers. Our study emphasizes the importance of not only methylated marker genes but also specific CpGs for identifying high-grade CINs. The 5-CpG classification model provides a promising biomarker panel for the early detection of cervical cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-014-0037-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4334603
Cervical cancer; Cervical intraepithelial neoplasia (CIN); DNA methylation; High-grade CIN; Biomarkers
2.  Characterization of a novel posttranslational modification in polypyrimidine tract-binding proteins by SUMO1 
BMB Reports  2014;47(4):233-238.
Polypyrimidine tract-binding protein 1 (PTBP1) and its brainspecific homologue, PTBP2, are associated with pre-mRNAs and influence pre-mRNA processing, as well as mRNA metabolism and transport. They play important roles in neural differentiation and glioma development. In our study, we detected the expression of the two proteins in glioma cells and predicted that they may be sumoylated using SUMOplot analyses. We confirmed that PTBP1 and PTBP2 can be modified by SUMO1 with co-immunoprecipitation experiments using 293ET cells transiently co-expressing SUMO1 and either PTBP1 or PTBP2. We also found that SUMO1 modification of PTBP2 was enhanced by Ubc9 (E2). The mutation of the sumoylation site (Lys137) of PTBP2 markedly inhibited its modification by SUMO1. Interestingly, in T98G glioma cells, the level of sumoylated PTBP2 was reduced compared to that of normal brain cells. Overall, this study shows that PTBP2 is posttranslationally modified by SUMO1. [BMB Reports 2014; 47(4): 233-238]
PMCID: PMC4163892  PMID: 24286314
Glioma; PTBP1; PTBP2; SUMO1; UBC9
3.  Specific Reduction of Glucose-6-Phosphate Transporter May Contribute to Down-regulation of Hepatic 11β-hydroxysteroid Dehydrogenase Type 1 in Diabetic Mice 
Pre-receptor activation of glucocorticoids via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) has been identified as an important mediator of the metabolic syndrome. Hexose-6-phosphate dehydrogenase (H6PDH) mediates 11β-HSD1 amplifying tissue glucocorticoid production by driving intracellular NADPH exposure to 11β-HSD1 and requires glucose-6-phosphate transporter (G6PT) to maintain its activity. However, the potential effects of G6PT on tissue glucocorticoid production in type 2 diabetes and obesity have not been yet defined. Here, we evaluated the possible role of G6PT antisense oligonucleotides (G6PT ASO) in the pre-receptor metabolism of glucocorticoids as related to glucose homeostasis and insulin tolerance by examining the production of 11β-HSD1 and H6PDH in both male db/+ and db/db mouse liver tissue. We observed that G6PT ASO treatment of db/db mice markedly reduced hepatic G6PT mRNA and protein levels and substantially diminished the activation of hepatic 11β-HSD1 and H6PDH. Reduction of G6PT expression was correlated with the suppression of both hepatic gluconeogenic enzymes G6Pase and PEPCK and corresponded to the improvement of hyperglycemia and insulin resistance in db/db mice. Addition of G6PT ASO to mouse hepa1-6 cells led to a dose-dependent decrease in 11β-HSD1 production. Knockdown of G6PT with RNA interference also impaired 11β-HSD1 expression and showed comparable effects to H6PDH siRNA on silencing of H6PDH and 11β -HSD1 expression in these intact cells. These findings suggest that G6PT plays an important role in the modulation of pre-receptor activation of glucocorticoids and provides new insights into the role of G6PT in the development of type 2 diabetes.
PMCID: PMC3763023  PMID: 23267038
11β-HSD1; H6PDH; G6PT; G6PT ASO; H6PDH siRNA; G6Pase; PEPCK; obesity; Insulin sensitivity; type 2 diabetes
4.  Necl-4/SynCAM-4 Is Expressed in Myelinating Oligodendrocytes but Not Required for Axonal Myelination 
PLoS ONE  2013;8(5):e64264.
The timing and progression of axonal myelination are precisely controlled by intercellular interactions between neurons and glia in development. Previous in vitro studies demonstrated that Nectin like 4 (Necl-4, also known as cell adhesion molecule Cadm-4 or SynCAM-4) plays an essential role in axonal myelination by Schwann cells in the peripheral nervous system (PNS). However, the role of Necl-4 protein in axonal myelination in the developing central nervous system (CNS) has remained unknown. In this study, we discovered upregulation of Necl-4 expression in mature oligodendrocytes at perinatal stages when axons undergo active myelination. We generated Necl4 gene knockout mice, but found that disruption of Necl-4 gene did not affect oligodendrocyte differentiation and myelin formation in the CNS. Surprisingly, disruption of Necl-4 had no significant effect on axonal myelination in the PNS either. Therefore, our results demonstrated that Necl-4 is dispensable for axonal myelination in the developing nervous system.
PMCID: PMC3659047  PMID: 23700466
5.  RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3  
The Journal of Clinical Investigation  2013;123(5):2103-2118.
PCBP2 is a member of the poly(C)-binding protein (PCBP) family, which plays an important role in posttranscriptional and translational regulation by interacting with single-stranded poly(C) motifs in target mRNAs. Several PCBP family members have been reported to be involved in human malignancies. Here, we show that PCBP2 is upregulated in human glioma tissues and cell lines. Knockdown of PCBP2 inhibited glioma growth in vitro and in vivo through inhibition of cell-cycle progression and induction of caspase-3–mediated apoptosis. Thirty-five mRNAs were identified as putative PCBP2 targets/interactors using RIP-ChIP protein-RNA interaction arrays in a human glioma cell line, T98G. Four-and-a-half LIM domain 3 (FHL3) mRNA was downregulated in human gliomas and was identified as a PCBP2 target. Knockdown of PCBP2 enhanced the expression of FHL3 by stabilizing its mRNA. Overexpression of FHL3 attenuated cell growth and induced apoptosis. This study establishes a link between PCBP2 and FHL3 proteins and identifies a new pathway for regulating glioma progression.
PMCID: PMC3635714  PMID: 23585479
6.  microRNA-214-mediated UBC9 expression in glioma 
BMB Reports  2012;45(11):641-646.
It has been reported that ubiquitin-conjugating enzyme 9 (Ubc9), the unique enzyme2 in the sumoylation pathway, is up-regulated in many cancers. However, the expression and regulation of UBC9 in glioma remains unknown. In this study, we found that Ubc9 was up-regulated in glioma tissues and cell lines compared to a normal control. UBC9 knockdown by small interfering RNA (siRNA) affected cell proliferation and apoptosis in T98G cells. Further experiments revealed that microRNA (miR)-214 directly targeted the 3' untranslated region (UTR) of UBC9 and that there was an inverse relationship between the expression levels of miR-214 and UBC9 protein in glioma tissues and cells. miR-214 overexpression suppressed the endogenous UBC9 protein and affected T98G cell proliferation. These findings suggest that miR-214 reduction facilitates UBC9 expression and is involved in the regulation of glioma cell proliferation. [BMB Reports 2012; 45(11): 641-646]
PMCID: PMC4133799  PMID: 23187003
Cell proliferation; Glioma; miR-214; Sumoylation; UBC9
7.  The CREB-miR-9 Negative Feedback Minicircuitry Coordinates the Migration and Proliferation of Glioma Cells 
PLoS ONE  2012;7(11):e49570.
Migration-proliferation dichotomy is a common mechanism in gliomagenesis; however, an understanding of the exact molecular mechanism of this “go or grow” phenomenon remains largely incomplete. In the present study, we first found that microRNA-9 (miR-9) is highly expressed in glioma cells. MiR-9 inhibited the proliferation and promoted the migration of glioma cells by directly targeting cyclic AMP response element-binding protein (CREB) and neurofibromin 1 (NF1), respectively. Our data also suggested a migration-inhibitory role for CREB through directly regulating the transcription of NF1. Furthermore, we found that the transcription of miR-9-1 is under CREB's control, forming a negative feedback minicircuitry. Taken together, miR-9 inhibits proliferation but promotes migration, whereas CREB plays a pro-proliferative and anti-migratory role, suggesting that the CREB-miR-9 negative feedback minicircuitry plays a critical role in the determination of “go or grow” in glioma cells.
PMCID: PMC3502497  PMID: 23185366
8.  Clock-controlled mir-142-3p can target its activator, Bmal1 
BMC Molecular Biology  2012;13:27.
microRNAs (miRNAs) are shown to be involved in the regulation of circadian clock. However, it remains largely unknown whether miRNAs can regulate the core clock genes (Clock and Bmal1).
In this study, we found that mir-142-3p directly targeted the 3’UTR of human BMAL1 and mouse Bmal1. The over-expression (in 293ET and NIH3T3 cells) and knockdown (in U87MG cells) of mir-142-3p reduced and up-regulated the Bmal1/BMAL1 mRNA and protein levels, respectively. Moreover, the expression level of mir-142-3p oscillated in serum-shocked NIH3T3 cells and the results of ChIP and luciferase reporter assays suggested that the expression of mir-142-3p was directly controlled by CLOCK/BMAL1 heterodimers in NIH3T3 cells.
Our study demonstrates that mir-142-3p can directly target the 3’UTR of Bmal1. In addition, the expression of mir-142-3p is controlled by CLOCK/BMAL1 heterodimers, suggesting a potential negative feedback loop consisting of the miRNAs and the core clock genes. These findings open new perspective for studying the molecular mechanism of circadian clock.
PMCID: PMC3482555  PMID: 22958478
mir-142-3p; Bmal1; Circadian clock
9.  PCBP2 Enhances the Antiviral Activity of IFN-α against HCV by Stabilizing the mRNA of STAT1 and STAT2 
PLoS ONE  2011;6(10):e25419.
Interferon-α (IFN-α) is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV) during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b). However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3′Untranslated Region (UTR) of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3′UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.
PMCID: PMC3191149  PMID: 22022391
10.  Disruption of Nectin-Like 1 Cell Adhesion Molecule Leads to Delayed Axonal Myelination in the CNS 
Nectin-like 1 (Necl-1) is a neural-specific cell adhesion molecule that is expressed in both the CNS and PNS. Previous in vitro studies suggested that Necl-1 expression is essential for the axon-glial interaction and myelin sheath formation in the PNS. To investigate the in vivo role of Necl-1 in axonal myelination of the developing nervous system, we generated the Necl-1 mutant mice by replacing axons 2–5 with the LacZ reporter gene. Expression studies revealed that Necl-1 is exclusively expressed by neurons in the CNS. Disruption of Necl-1 resulted in developmental delay of axonal myelination in the optic nerve and spinal cord, suggesting that Necl-1 plays an important role in the initial axon-oligodendrocyte recognition and adhesion in CNS myelination.
PMCID: PMC2728619  PMID: 19036974
cell adhesion molecule; gene targeting; myelination; spinal cord; optic nerve; knock-out
11.  Characterization of the New World Monkey Homologues of Human Poliovirus Receptor CD155▿  
Journal of Virology  2008;82(14):7167-7179.
In contrast to Old World monkeys, most New World monkeys (NWMs) are not susceptible to poliovirus (PV), regardless of the route of infection. We have investigated the molecular basis of restricted PV pathogenesis of NWMs with two kidney cell lines of NWMs, TMX (tamarin) and NZP-60 (marmoset), and characterized their PV receptor homologues. TMX cells were susceptible to infection by PV1 (Mahoney) and PV3 (Leon) but not by PV2 (Lansing). Binding studies to TMX cells indicated that the formation of PV/receptor complexes increased when measured first at 4°C and then at 25°C, whereas PV2 did not significantly bind to TMX cells at either temperature. On the other hand, NZP-60 cells were not susceptible to infection by any of the PV serotypes. However, a low amount of PV1 bound to NZP-60 cells at 4°C, but there was no increase of binding at 25°C. In contrast, both NWM cell lines supported genome replication and virion formation when transfected with viral RNAs of either serotype, an observation indicating that infection was blocked in receptor-virus interaction. To overcome the receptor block, we substituted 3 amino acids in the marmoset receptor (nCD155), H80Q, N85S, and P87S, found in the human PV receptor, hCD155. Cells expressing the mutant receptor (L-nCD155mt) were now susceptible to infection with PV1, which correlated with an increase in PV1-bound receptor complexes from 4°C to 25°C. L-nCD155mt cells were, however, still resistant to PV2 and PV3. These data show that an increase in the formation of PV/receptor complexes, when measured at 4°C and at 25°C, correlates with and is an indicator of successful infection at 37°C, suggesting that the complex formed at 25°C may be an intermediate in PV uptake.
PMCID: PMC2446954  PMID: 18480448
12.  Cooperation between EZH2, NSPc1-mediated histone H2A ubiquitination and Dnmt1 in HOX gene silencing 
Nucleic Acids Research  2008;36(11):3590-3599.
An intricate interplay between DNA methylation and polycomb-mediated gene silencing has been highlighted recently. Here we provided evidence that Nervous System Polycomb 1 (NSPc1), a BMI1 homologous polycomb protein, plays important roles in promoting H2A ubiquitination and cooperates with DNA methylation in HOX gene silencing. We showed that NSPc1 stimulates H2A ubiquitination in vivo and in vitro through direct interaction with both RING2 and H2A. RT-PCR analysis revealed that loss of NSPc1, EZH2 or DNA methyltransferase 1 (Dnmt1), or inhibition of DNA methylation in HeLa cells de-represses the expression of HOXA7. Chromatin immunoprecipitation (ChIP) assays demonstrated that NSPc1, EZH2 and Dnmt1 bind to the promoter of HOXA7, which is frequently hypermethylated in tumors. Knockdown of NSPc1 results in significant reduction of H2A ubiquitination and DNA demethylation as well as Dnmt1 dissociation in the HOXA7 promoter. Meanwhile Dnmt1 deficiency affects NSPc1 recruitment and H2A ubiquitination, whereas on both cases EZH2-mediated H3K27 trimethylation remains unaffected. When EZH2 was depleted, however, NSPc1 and Dnmt1 enrichment was abolished concomitant with local reduction of H3K27 trimethylation, H2A ubiquitination and DNA methylation. Taken together, our findings indicated that NSPc1-mediated H2A ubiquitination and DNA methylation, both being directed by EZH2, are interdependent in long-term target gene silencing within cancer cells.
PMCID: PMC2441805  PMID: 18460542
13.  A novel method for high accuracy sumoylation site prediction from protein sequences 
BMC Bioinformatics  2008;9:8.
Protein sumoylation is an essential dynamic, reversible post translational modification that plays a role in dozens of cellular activities, especially the regulation of gene expression and the maintenance of genomic stability. Currently, the complexities of sumoylation mechanism can not be perfectly solved by experimental approaches. In this regard, computational approaches might represent a promising method to direct experimental identification of sumoylation sites and shed light on the understanding of the reaction mechanism.
Here we presented a statistical method for sumoylation site prediction. A 5-fold cross validation test over the experimentally identified sumoylation sites yielded excellent prediction performance with correlation coefficient, specificity, sensitivity and accuracy equal to 0.6364, 97.67%, 73.96% and 96.71% respectively. Additionally, the predictor performance is maintained when high level homologs are removed.
By using a statistical method, we have developed a new SUMO site prediction method – SUMOpre, which has shown its great accuracy with correlation coefficient, specificity, sensitivity and accuracy.
PMCID: PMC2245905  PMID: 18179724
14.  NSPc1 is a cell growth regulator that acts as a transcriptional repressor of p21Waf1/Cip1 via the RARE element 
Nucleic Acids Research  2006;34(21):6158-6169.
The mammalian polycomb group proteins play an important role in cell cycle control and tumorigenesis. Nervous system polycomb 1 (NSPc1) is a newly identified transcription repressor, highly homologous with PcG protein Bmi-1. In this article, we showed that NSPc1 could promote tumor cell cycle progression and cell proliferation. Semi-quantitative RT–PCR showed that NSPc1 did not affect the expression levels of most Cyclin-depentent kinases (CDK) inhibitors except for p21Waf1/Cip1. Repression activity assays, chromatin immunoprecipitation (ChIP) and DNA pulldown assays all verified that NSPc1 represses the expression of p21Waf1/Cip1 by binding to the (−1357 to −1083) region of the p21Waf1/Cip1 promoter in vivo, and the repression effect is dependent on the retinoid acid response element (RARE element) within the above region of the p21Waf1/Cip1 promoter. Further analysis showed that NSPc1 could compete the RARE element site with RA receptors both in vitro and in vivo. Taken together, our results support the hypothesis that NSPc1 has a positive role in tumor cell growth by down-regulating p21Waf1/Cip1 via the RARE element, which directly connects transcriptional repression of PcGs to CDKIs and RA signaling pathways.
PMCID: PMC1693893  PMID: 17088287
15.  Human Bex2 interacts with LMO2 and regulates the transcriptional activity of a novel DNA-binding complex 
Nucleic Acids Research  2005;33(20):6555-6565.
Human Bex2 (brain expressed X-linked, hBex2) is highly expressed in the embryonic brain, but its function remains unknown. We have identified that LMO2, a LIM-domain containing transcriptional factor, specifically interacts with hBex2 but not with mouse Bex1 and Bex2. The interaction was confirmed both by pull-down with GST-hBex2 and by coimmunoprecipitation assays in vivo. Using electrophoretic mobility shift assay, we have demonstrated the physical interaction of hBex2 and LMO2 as part of a DNA-binding protein complex. We have also shown that hBex2 can enhance the transcriptional activity of LMO2 in vivo. Furthermore, using mammalian two-hybrid analysis, we have identified a neuronal bHLH protein, NSCL2, as a novel binding partner for LMO2. We then showed that LMO2 could up-regulate NSCL2-dependent transcriptional activity, and hBex2 augmented this effect. Thus, hBex2 may act as a specific regulator during embryonic development by modulating the transcriptional activity of a novel E-box sequence-binding complex that contains hBex2, LMO2, NSCL2 and LDB1.
PMCID: PMC1298925  PMID: 16314316
16.  Complexes of Poliovirus Serotypes with Their Common Cellular Receptor, CD155 
Journal of Virology  2003;77(8):4827-4835.
Structures of all three poliovirus (PV) serotypes (PV1, PV2, and PV3) complexed with their cellular receptor, PV receptor (PVR or CD155), were determined by cryoelectron microscopy. Both glycosylated and fully deglycosylated CD155 exhibited similar binding sites and orientations in the viral canyon for all three PV serotypes, showing that all three serotypes use a common mechanism for cell entry. Difference maps between the glycosylated and deglycosylated CD155 complexes determined the sites of the carbohydrate moieties that, in turn, helped to verify the position of the receptor relative to the viral surface. The proximity of the CD155 carbohydrate site at Asn105 to the viral surface in the receptor-virus complex suggests that it might interfere with receptor docking, an observation consistent with the properties of mutant CD155. The footprints of CD155 on PV surfaces indicate that the south rim of the canyon dominates the virus-receptor interactions and may correspond to the initial CD155 binding state of the receptor-mediated viral uncoating. In contrast, the interaction of CD155 with the north rim of the canyon, especially the region immediately outside the viral hydrophobic pocket that normally binds a cellular “pocket factor,” may be critical for the release of the pocket factor, decreasing the virus stability and hence initiating uncoating. The large area of the CD155 footprint on the PV surface, in comparison with other picornavirus-receptor interactions, could be a potential limitation on the viability of PV escape mutants from antibody neutralization. Many of these are likely to have lost their ability to bind CD155, resulting in there being only three PV serotypes.
PMCID: PMC152153  PMID: 12663789

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