Macrophage migration inhibitory factor (MIF) plays an important role in inflammation and immune response, as well as regulation of cell growth, apoptosis and tumorigenesis. MIF can inhibit Bim mediated cell apoptosis, but the signaling pathway and molecular mechanism remained unclear. BimEL was reported to directly bind to active ERK1/2, which promoted the phosphorylation of its Ser69 site and the following degradation via the ubiquitin-proteasome pathway. We proved that overexpression of MIF in HEK293 cells promoted ERK1/2 phosphorylation, which led to phosphorylation of Ser69 of BimEL. Downregulation of MIF reduced the activation of ERK1/2 pathway as well as serum stimulation effect of ERK1/2, which decreased the phosphorylation of Ser69 of BimEL. The results show that MIF promotes phosphorylation of BimEL Ser69 and degradation of BimEL by activation of ERK1/2 pathway. Our findings provide new insights into the molecular mechanism of MIF regulating Bim-mediated apoptosis.

AP-2 transcription factors are important sequence-specific DNA-binding regulators that are expressed in the neural crest and other tissues during mammalian development. The human AP-2 family of transcription factors consists of five members, AP-2α, -β, -γ, -δ and -ε, which have an important role in the regulation of gene expression during development and in the differentiation of multiple organs and tissues. The present study aimed to investigate the mechanism by which AP-2δ mediates heme oxygenase-1 (HMOX1) gene expression. It was identified that the human AP-2δ protein exhibited weak binding to a suboptimal AP-2 sequence, 5'-GCCN3GGC-3', to which all other AP-2 proteins bind in vitro, providing the first example of DNA target specificity amongst the AP-2 family. AP-2δ protein bound to an optimized AP-2 consensus DNA sequence, 5'-GCCTGAGGC-3', in vitro and transactivated gene expression in eukaryotic cells. The transactivation domain of Ap-2δ differs notably from those in the other AP-2 proteins as it lacks the PY motif (XPPXY) and several other conserved residues that are important for the transcriptional activity of AP-2 proteins, yet it functions as an equally strong activator.

Association studies between Alpha-1-antichymotrypsin (ACT)-17(A > T) polymorphisms and Alzheimer's disease (AD) susceptibility have shown conflicting results. In this investigation, we performed a meta-analysis to assess the purported associations. Subgroup analyses based on ethnicity (Caucasians, East-Asian and American mixed) were also performed including a total of 5,676 AD patients and 5,460 controls for ACT-17. Overall, allele contrast (A vs. T) of ACT -17 polymorphism produced significant results in the worldwide population [P(heterogeneity)=0.01, random-effects (RE) odds ratio (OR) 1.12; 95% CI 1.04-1.21, P=0.003] and in the Caucasian population [P(heterogeneity)=0.03, RE OR1.11 95% CI 1.01-1.24, P=0.04]. Meta-analyses of other genetic contrasts suggested that the A allele carriers are associated with increased susceptibility to AD in variant populations. No significant association was observed in the East-Asian subgroup analysis. In conclusion, ACT-17 variation presents a risk factor for AD in the worldwide population, especially in the Caucasian population.

Human NFB repressing factor (NKRF) is a negative regulation transcription factor, which is able to repress transcription by binding to the negative regulatory element (NRE) near the NFκB binding site in certain genes’ promoters. Current researches reveals that NKRF represses the activation of IFN-, IL-8, hiNOS and HIV-1 by NFκB. We used optical fiber beadchip to analysis the different gene expression patterns of RNAi mediated NKRF knockdown HEK293 cells and found that several genes showed significant change of expression levels. Real-time PCR was performed to verify the changes of expression of candidate genes. We analyzed the function of candidate genes by searching the gene ontology databases and publications and revealed that these genes functioned in cell cycle, cell proliferation, apoptosis, cell migration, DNA repair, transcription, metabolism, response to stimulus and signal transduction. This study provides new perspectives on NKRF’s potential multiple functions.

Human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9) is a specific substrate-recognition subunit of an elongin C-cullin-SOCS box E3 ubiquitin ligase complex. It recognizes its substrate, brain type creatine kinase (CKB), using the ankyrin repeat domain; and facilitates the polyubiquitination of CKB to mediate proteasomal degradation through the SOCS box domain. HASB9-2 is an isoform of hASB9 that contains one ankyrin repeat domain. In this study, the crystal structure of hASB9-2 is shown at 2.2-Å resolution using molecular replacement. Overall, hASB9-2 forms a slightly curved arch with a characteristic L-shaped cross-section. Amino acid substitution analysis based on docking experiments revealed that His103 and Phe107 in hASB9-2 are essential for binding to CKB. Analysis of truncation mutants demonstrated that the first six ankyrin repeats along with the N-terminal region of hASB9-2 contribute to the interaction with CKB.

A thermostable GDSL family esterase-encoding gene, EstL5, was directly obtained from the genomic DNA of Geobacillus thermodenitrificans T2. Recombinant hexahistidine-tagged EstL5 was overexpressed, purified, and its biochemical properties were partially characterized. EstL5 was observed to be active within the temperature range of 0-80 degrees C, having maximal activity at 60 degrees C. Unlike most other thermostable enzymes, EstL5 displayed 24% of its highest activity at 0 degrees C. EstL5 exhibited a high level of activity within a pH range of 6.0-11.0, showing the highest activity at pH 8.0. EstL5 also retained 100% of its activity after a 12-h incubation at 55 degrees C. Furthermore, this enzyme was observed to be strongly inhibited by 10% (w/v) SDS and 0.1 mM PMSF. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

Trimethyl chitosan–cysteine conjugate (TMC–Cys) was evaluated as non-viral gene carriers to combine the advantages of TMC and thiolated chitosan. TMC–Cys with various molecular weights (30, 100, and 200kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanocomplexes with plasmid encoding enhanced green fluorescence protein (pEGFP), which demonstrated preferable diameters of below 200nm and zeta potentials of +15 to +20mV. Cell binding and uptake of TMC–Cys/pEGFP nanocomplexes (TMC–Cys NC) were enhanced 2.4–3.0 and 1.4–3.0 folds, respectively, compared to TMC/pEGFP nanocomplexes (TMC NC). pEGFP could be easily released from TMC–Cys NC at the intracellular glutathione concentration, which promoted its nuclear transport and accumulation. Consequently, TMC–Cys NC showed a 1.4 to 3.2-fold increase in the transfection efficiency in HEK293 cells as compared to TMC NC and the optimal TMC–Cys(100,30) NC showed a 1.5-fold enhancement than Lipofectamine2000. Such results were further confirmed by in vivo transfection with a 2.3-fold and 4.1-fold higher transfection efficiency of TMC–Cys(100,30) NC than TMC(100,30) NC and Lipofectamine2000, respectively. Therefore, TMC–Cys/DNA nanocomplexes could be a promising gene delivery system with in vitro and in vivo superiority to Lipofectamine2000.

LNX protein is the first described PDZ domain-containing member of the RING finger-type E3 ubiquitin ligase family. Studies have approved that LNX could participate in signal transduction, such as Notch pathway, and play an important role in tumorigenesis. In this study, we found that down-regulation of LNX resulted in G0/G1 cell cycle arrest in G0/G1 phase in HEK293 cells. To explore the molecular mechanism of this phenomenon, we employed expression microarray to comparatively analyze the genome-wide expression between the LNX-knockdown cells and the normal cells. We also used quantitative real-time PCR to further confirm the differential expression patterns of 25 transcripts involved in cell cycle. Combined with known information about genic functions, signal pathways and cell cycle machinery, we analyzed the role of endogenous LNX in cell cycle. The results suggest that down-regulation of LNX could result in cell cycle arrest in G0/G1 phase through inhibition of β-catenin, MAPK, NFκB, c-Myc-dependent pathway and activation of p53, TGF-β-dependent pathway. This study provides new perspectives on LNX’s pleiotropic activities, especially its essential role in cell proliferation and cell cycle.

To investigate genetic mechanisms of hepatocarcinogenesis and identify potential anticancer targets in hepatocellular carcinoma (HCC), we analyzed microarray gene expression profiles between 33 HCCs and their corresponding noncancerous liver tissues. Functional analysis of differentially-expressed genes in HCC indicated that cell cycle dysregulation plays an important role in hepatocarcinogenesis. Based on 14 differentially-expressed genes involved in cell cycle in HCC, we applied Structural Equation Modeling (SEM) to establish a potential genetic network which could assist understanding of HCC molecular mechanisms. siRNA-mediated knock-down of two significantly up-regulated genes, minichromosome maintenance protein 2 (MCM2) and cyclin B1 (CCNB1), in HCC cells (SMMC-7721 and QGY-7703) induced G2/M-phase arrest, apoptosis and antiproliferation in HCC. Some up-regulated cell cycle-related genes in HCC were down-regulated following specific depletion of MCM2 or/and CCNB1 in HCC cells, which might well validate and complement the reconstructed cell cycle network. This study may contribute to further disclose hepatocarcinogenesis mechanism through systematically analyzed the HCC-related-cell-cycle pathway. This study also shows that MCM2 and CCNB1 could be promising prognostic and therapeutic targets for HCC.

Hypericin-mediated photodynamic therapy (HY-PDT) has become a potential treatment for tumors and nonmalignant disorders. Some studies reported that HY-PDT could lead to apoptosis in some carcinoma cells. However, the molecular mechanism of HY-PDT remains unknown. In this study, we evaluated the molecular mechanisms of hypericin associated with light-emitting diode irradiation on the poorly differentiated human nasopharyngeal carcinoma cell line CNE-2 in vitro. To comprehensively understand the effects of HY-PDT on CNE-2 cells, we detected cell viability, cell cycle, apoptosis, intracellular glutathione content, and intracellular caspase (caspase-9, caspase-3, and caspase-8) activity. Furthermore, we performed genome-wide expression analysis via microarrays at different time points in response to HY-PDT, and we found that differentially expressed genes were highly enriched in the pathways related to reactive oxygen species generation, mitochondrial activity, DNA replication and repair, cell cycle/proliferation, and apoptosis. These results were consistent with our cytology test results and demonstrated that caspase-dependent apoptosis occurred after HY-PDT. Taken together, both cellular and molecular data revealed that HY-PDT could inhibit the growth of CNE-2 cells and induce their apoptosis.

AIM:To construct a system for selecting reference genes (RGs) and to select the most optimal RGs for gene expression studies in nasopharyngeal carcinoma (NPC).METHODS:The total RNAs from 20 NPC samples were each labeled with Cy5-dUTP. To create a common control, the total RNA from 15 nasopharyngeal phlogistic (NP) tissues was mixed and labeled via reverse transcription with Cy3-dUTP. cDNA microarrays containing 14 112 genes were then performed. A mathematical approach was constructed to screen stably expressed genes from the microarray data. Using this method, three genes (YARS, EIF3S7, and PFDN1) were selected as candidate RGs. Furthermore, 7 commonly used RGs (HPRT1, GAPDH, TBP, ACTB, B2M, G6PDH, and HBB) were selected as additional potential RGs. Real-time PCR was used to detect these 10 candidate genes' expression levels and the geNorm program was used to find the optimal RGs for NPC studies.RESULTS:On the basis of the 10 candidate genes' expression stability level, geNorm analysis identified the optimal single RG (YARS or HPRT1) and the most suitable set of RGs (HPRT1, YARS, and EIF3S7) for NPC gene expression studies. In addition, this analysis determined that B2M, G6PDH, and HBB were not appropriate for use as RGs. Interestingly, ACTB was the least stable RG in our study, even though previous studies had indicated that it was one of the most stable RGs. Three novel candidate genes (YARS, EIF3S7, and PFDN1), which were selected from microarray data, were all identified as suitable RGs for NPC research. A RG-selecting system was then constructed, which combines microarray data analysis, a literature screen, real-time PCR, and bioinformatic analysis.CONCLUSION:We construct a RG-selecting system that helps find the optimal RGs. This process, applied to NPC research, determined the single RG (YARS or HPRT1) and the set of RGs (HPRT1, YARS, and EIF3S7) that are the most suitable internal controls.

Bim is a proapoptotic member of the Bcl-2 family and is primarily involved in the regulation of the intrinsic apoptotic pathway. However, the detail of regulation of Bim's proapoptotic activity has not been clarified yet. Using Bim L as bait, we screened a human fetal cDNA library for interacting proteins and identified Grb10 as an interactor. This interaction was verified by co-immunoprecipitation and intracellular co-localization studies. The potential segment of Bim L that binds Grb10 was identified via a yeast mating test. Grb10 interacted with the DBD (dynein binding domain) of Bim and inhibited apoptosis triggered by overexpression of DBD containing Bim isoforms. The putative phosphorylation sites on DBD of Bim play a role for the anti-proapoptotic activity of Grb10. Our results suggest that Grb10 interacts with Bim L and inhibits its proapoptotic activity in a phosphorylation-dependant manner.

The purpose of this study was to identify and validate novel prognostic biomarkers in human hepatocellular carcinoma (HCC). We analyzed gene expression profiles not only between 33 HCCs and their corresponding noncancerous liver tissues, but also between 25 HCCs and pooled normal liver tissues using cDNA microarrays containing 12800 genes. Functional analysis of differentially expressed genes involved in HCC carcinogenesis and tumor progression revealed that up-regulated and down-regulated genes are mainly associated with cell cycle and immune response, respectively. We detected two regions of cytogenetic changes only in poorly-differentiated HCCs using the expression data. We identified a 9-gene expression signature, which was able to predict differentiation degree and survival of HCC samples. Among the 9 most discriminatory genes, minichromosome maintenance protein 2 (MCM2), a significantly up-regulated gene involved in cell cycle pathway, was selected for further analysis. Overexpression of MCM2 protein related to poor-differentiation in HCC was validated using tissue microarray-based immunohistochemistry containing 96 HCCs. Our studies show that the 9-gene expression signature may serve as promising prognostic biomarkers involved in hepatocarcinogenesis and tumor progression.

UBE1 plays an important role in the first step of ubiquitin–proteasome pathway to activate ubiquitin. Both the structure and biochemical property research of human UBE1 protein, and the activity analysis of those enzymes which are related with ubiquitination pathway, are based on high purity of UBE1 protein. To obtain human UBE1 protein, the full length of human UBE1 was expressed in E. coli and purified by Ni-NTA superflow sepharose and strep-tactin sepharose which based on UB–UBE1 high-energy thioester bonded intermediate complex. It was demonstrated that purified UBE1 could activate and conjugate UB to ubiquitin-conjugating enzyme E2s. The purified large amount of UBE1 could be used for in vitro studies of ubiquitin pathway and structural studies.

BACKGROUND:Recently, microRNAs (miRNAs) have taken centre stage in the field of human molecular oncology. Several studies have shown that miRNA profiling analyses offer new possibilities in cancer classification, diagnosis and prognosis. However, the function of miRNAs that are dysregulated in tumours remains largely a mystery. Global analysis of miRNA-target gene expression has helped illuminate the role of miRNAs in developmental gene expression programs, but such an approach has not been reported in cancer transcriptomics.RESULTS:In this study, we globally analysed the expression patterns of miRNA target genes in prostate cancer by using several public microarray datasets. Intriguingly, we found that, in contrast to global mRNA transcript levels, putative miRNA targets showed a reduced abundance in prostate tumours relative to benign prostate tissue. Additionally, the down-regulation of these miRNA targets positively correlated with the number of types of miRNA target-sites in the 3' untranslated regions of these targets. Further investigation revealed that the globally low expression was mainly driven by the targets of 36 specific miRNAs that were reported to be up-regulated in prostate cancer by a miRNA expression profiling study. We also found that the transcript levels of miRNA targets were lower in androgen-independent prostate cancer than in androgen-dependent prostate cancer. Moreover, when the global analysis was extended to four other cancers, significant differences in transcript levels between miRNA targets and total mRNA backgrounds were found.CONCLUSION:Global gene expression analysis, along with further investigation, suggests that miRNA targets have a significantly reduced transcript abundance in prostate cancer, when compared with the combined pool of all mRNAs. The abnormal expression pattern of miRNA targets in human cancer could be a common feature of the human cancer transcriptome. Our study may help to shed new light on the functional roles of miRNAs in cancer transcriptomics.

BACKGROUND:The identification of gene differential co-expression patterns between cancer stages is a newly developing method to reveal the underlying molecular mechanisms of carcinogenesis. Most researches of this subject lack an algorithm useful for performing a statistical significance assessment involving cancer progression. Lacking this specific algorithm is apparently absent in identifying precise gene pairs correlating to cancer progression.RESULTS:In this investigation we studied gene pair co-expression change by using a stochastic process model for approximating the underlying dynamic procedure of the co-expression change during cancer progression. Also, we presented a novel analytical method named 'Stochastic process model for Identifying differentially co-expressed Gene pair' (SIG method). This method has been applied to two well known prostate cancer data sets: hormone sensitive versus hormone resistant, and healthy versus cancerous. From these data sets, 428,582 gene pairs and 303,992 gene pairs were identified respectively. Afterwards, we used two different current statistical methods to the same data sets, which were developed to identify gene pair differential co-expression and did not consider cancer progression in algorithm. We then compared these results from three different perspectives: progression analysis, gene pair identification effectiveness analysis, and pathway enrichment analysis. Statistical methods were used to quantify the quality and performance of these different perspectives. They included: Re-identification Scale (RS) and Progression Score (PS) in progression analysis, True Positive Rate (TPR) in gene pair analysis, and Pathway Enrichment Score (PES) in pathway analysis. Our results show small values of RS and large values of PS, TPR, and PES; thus, suggesting that gene pairs identified by the SIG method are highly correlated with cancer progression, and highly enriched in disease-specific pathways. From this research, several gene interaction networks inferred could provide clues for the mechanism of prostate cancer progression.CONCLUSION:The SIG method reliably identifies cancer progression correlated gene pairs, and performs well both in gene pair ontology analysis and in pathway enrichment analysis. This method provides an effective means of understanding the molecular mechanism of carcinogenesis by appropriately tracking down the process of cancer progression.

To evaluate the relationship between methionine synthase reductase gene(MTRR) A66G polymorphism and cancer risk by Meta-analysis,relevant articles were identified by literature retrieval.A total of 13901 cancer patients and 20385 controls from 25 articles(38 studies) were investigated.Overall,a significantly increased cancer risk was observed in allele G vs.A contrast(OR,1.05;P=0.006;95% CI,1.01—1.08;Pheterogeneity=0.193).The pooled ORs were consistently significant by omitting one study or one kind of tumors,suggesting robustness of our results.Subgroup analysis and Meta-regression indicated that ethnicity and study design didn't contribute to the heterogeneity,and only tumor site was the significant source of heterogeneity.In conclusion,MTRR gene A66G polymorphism may be a cancer risk factor,especially for lymphoid leukemia and Meningioma.

Polymorphisms in methionine synthase (MTR) gene may be involved in carcinogenesis by affecting DNA methylation. However, association studies on MTR A2756G polymorphism in cancers have reported conflicting results. Therefore we performed a meta-analysis to better assess the associations. A total of 24 896 cancer patients and 33 862 controls from 52 articles for MTR A2756G were investigated. Overall, individuals carrying MTR 2756GG genotype had a subtly reduced cancer risk under a recessive genetic model (odds ratio (OR), 0.92; P=0.053; 95% confidence interval (95% CI), 0.84–1.00; I2=0.0%; Pheterogeneity=0.61). In the subgroup analyses by ethnicity, 2756GG was associated with a significantly reduced cancer risk in European populations (OR, 0.83; P=0.001; 95% CI, 0.74–0.93; I2=0.0%; Pheterogeneity=0.99). However, in Asian populations, a significantly elevated association between 2756GG genotype and cancer risk was observed (OR, 1.33; P=0.012; 95% CI, 1.06–1.65; I2=0.0%; Pheterogeneity=0.50). In studies stratified by tumor site, there was a significantly reduced risk of acute lymphoblastic leukemia (ALL) (OR, 0.54; P=0.049; 95% CI, 0.29–1.00; I2=10.7%; Pheterogeneity=0.33) and colorectal cancer (OR, 0.63; P=0.004; 95% CI, 0.47–0.87; I2=0.0%; Pheterogeneity=0.73) in European populations. Our study indicates that MTR A2756G polymorphism is a candidate gene polymorphism for cancer susceptibility regardless of environmental factors. Large-scale, well-designed, and population-based studies are required to further investigate gene–gene and gene–environment interactions on MTR A2756G polymorphism and tissue-specific cancer risk in an ethnicity-specific population.

Many time series microarray experiments have relatively short (less than ten) time points and lack in repeats, weakening the confidence of results. Combining the microarray data from different groups may improve the statistical power of detecting differentially expressed genes. However, few efforts have been taken to combine or compare the time-course array datasets generated by independent groups. Here we demonstrated a suitable strategy for meta-analysis of short time series microarray datasets and implemented this strategy on four published heat shock microarray datasets of Saccharomyces Cerevisiae. We first assessed the significance of each gene in each datasets based on area calculation and the null distribution of the areas. Then the similarity of significance values across datasets was assessed with meta-analysis methods, yielding a set of transient heat shock stress sensitive genes. Following correlation calculation helped us to combine the transformed data at the same time points of each gene. Further bioinformatic investigation showed the significance of our strategy, and also indicated some interesting features of regulatory systems in S. cerevisiae during transient heat stress.

[目的]探索和研究谷胱甘肽过氧化酶(GPX1)的198号位点的多态性与肺癌和乳腺癌易感性之间的关系.[方法]收集2007年12月31日前有关GPX1基因198位点多态性与肺癌和乳腺癌易感性关系的病例对照文献,进行Meta分析.[结果]通过文献检索和筛选,共得到10篇文献.在Meta分析中肺癌和乳腺癌的合并OR值(杂合型加上突变纯合型对比于野生纯合型)分别为1.35和1.06,95%可信区间分别为(0.92-1.17)和(0.91-2.01).[结论]GPX1198化点多态性和乳腺癌易感性无关;但是GPX1 198位点多态性和肺癌易感性有关,Lue198基因型携带者比其他人多35%的肺癌发生概率.