It has been well-known that over activation of NF-κB has close relationship with hepatitis and hepatocellular carcinoma (HCC). However, the complete and exact underlying molecular pathways and mechanisms still remain not fully understood. By manipulating NF-κB activity with its recognized activator TNFα and using ChIP-seq and RNA-seq techniques, this study identified 699 NF-κB direct target genes (DTGs) in a widely used HCC cell line, HepG2, including 399 activated and 300 repressed genes. In these NF-κB DTGs, 216 genes (126 activated and 90 repressed genes) are among the current HCC gene signature. In comparison with NF-κB target genes identified in LPS-induced THP-1 and TNFα-induced HeLa cells, only limited numbers (24–46) of genes were shared by the two cell lines, indicating the HCC specificity of identified genes. Functional annotation revealed that NF-κB DTGs in HepG2 cell are mainly related with many typical NF-κB-related biological processes including immune system process, response to stress, response to stimulus, defense response, and cell death, and signaling pathways of MAPK, TNF, TGF-beta, Chemokine, NF-kappa B, and Toll-like receptor. Some NF-κB DTGs are also involved in Hepatitis C and B pathways. It was found that 82 NF-κB DTGs code secretory proteins, which include CCL2 and DKK1 that have already been used as HCC markers. Finally, the NF-κB DTGs were further confirmed by detecting the NF-κB binding and expression of 14 genes with ChIP-PCR and RT-PCR. This study thus provides a useful NF-κB DTG list for future studies of NF-κB-related molecular mechanisms and theranostic biomarkers of HCC.

Positively-charged nylon membrane (NM) is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM, RCA and NIRF, named NIRF-based solid phase RCA on nylon membrane (NM-NIRF-sRCA). The detection system of this method only need two kinds of nucleic acid molecules: target-specific probes with a RCA primer (P) at their 3′ end and a rolling circle (RC). The detection procedure consists of four steps: (1) immobilizing detected nucleic acids on NM by UV crosslinking; (2) hybridizing NM with specific probes and RC; (3) amplifying by a RCA reaction containing biotin-dUTP; (4) incubating NM with NIRF-labeled streptavidin and imaging with a NIRF imager. The method was fully testified by detecting oligonucleotides, L1 fragments of various HPV subtypes cloned in plasmid, and E.coli genomic DNA. This study thus provides a new facile method for detecting nucleic acid molecules.

In recent DNA microarray studies, we found that the transcription of the Id3 gene was significantly down-regulated in five cell lines (RAW264.7, Hepa1–6, THP-1, HepG2, and HL7702) treated with two doses (50 and 100 μg/mL) of a DMSA-coated magnetite nanoparticle. Given the regulatory roles of Id genes in the cell cycle, growth, and differentiation, we wanted to do more investigations on the effect of the nanoparticle upon the Id genes. This study detected the expression of Id genes in six cell lines (the above cell lines plus HeLa) treated with the nanoparticle at the same doses using quantitative PCR. The results revealed that the expression of Id genes was significantly affected by the nanoparticle in these cell lines. Under each treatment, the Id3 gene was significantly (p < 0.01) down-regulated in all cell lines, the Id1 gene was significantly down-regulated in all cell lines except the RAW264.7 cells, and the Id2 gene was significantly down-regulated in the HepG2, HL7702, and HeLa cells. Because the Id1, Id2, and Id3 genes were significantly down-regulated in three liver-derived cell lines (Hepa1–6, HepG2, and HL7702) in both microarray and PCR detections, this study then detected the expression of Id genes in the liver tissues of mice that were intravenously injected with the nanoparticle at two doses (2 and 5 mg/kg body weight). The results revealed that the expression of Id1, Id2, and Id3 genes was also significantly down-regulated in the liver tissues under each treatment. Another Id gene, Id4, was also significantly regulated in some cells or liver tissues treated with the nanoparticle. These results reveal that the nanoparticle exerts a significant effect on the in vitro and in vivo expression of Id genes. This study thus provides new insights into the Id-related nanotoxicity of the nanoparticle and the close relationship between the regulation of Id genes and iron.

The dimercaptosuccinic acid (DMSA) was widely used to coat iron oxide nanoparticles (FeNPs); however, its intracellular cytotoxicity remains to be adequately elucidated. This study analyzed the differentially expressed genes (DEGs) in four mammalian cells treated by a DMSA-coated magnetite FeNP at various doses at different times. The results revealed that about one-fourth of DEGs coded cysteine-rich proteins (CRPs) in all cells under each treatment, indicating that the nanoparticles greatly affected the expressions of CRP-coding genes. Additionally, about 26% of CRP-coding DEGs were enzyme genes in all cells, indicating that the nanoparticles greatly affected the expression of enzyme genes. Further experiments with the nanoparticles and a polyethylenimine (PEI)-coated magnetite FeNP revealed that the effect mainly resulted from DMSA carried into cells by the nanoparticles. This study thus first reported the cytotoxicity of DMSA at the gene transcription level as coating molecules of FeNPs. This study provides new insight into the molecular mechanism by which the DMSA-coated nanoparticles resulted in the transcriptional changes of many CRP-coding genes in cells. This study draws attention toward the intracellular cytotoxicity of DMSA as a coating molecule of nanoparticles, which has very low toxicity as an orally administered antidote due to its extracellular distribution.

This study describes a method for analyzing transcription factor (TF) activity, near-infrared fluorescent solid-phase rolling circle amplification (NIRF-sRCA). This method analyzes TF activity in four steps: (i) incubate DNA with protein sample and isolate TF-bound DNA, (ii) hybridize the TF-bound DNA and rolling circle to DNA microarray, (iii) amplify the TF-bound DNA with sRCA that contains biotin-labeled dUTP, and (iv) detect sRCA products by binding of NIRF-labeled streptavidin and NIRF imaging. This method was validated by proof-of-concept detection of purified TF protein and cell nuclear extract. Detection of purified TF protein demonstrated that NIRF-sRCA could quantitatively detect NF-κB p50 protein, and as little as 6.94 ng (∼140 fmol) of this protein was detected. Detection of nuclear extract revealed that NIRF-sRCA could specifically and quantitatively detect NF-κB p50 activity in HeLa cell nuclear extracts, and the activity of this TF in as little as 0.625 μg of nuclear extracts could be detected. Detection of nuclear extract also revealed that NIRF-sRCA could detect the relative activities of multiple TFs in HeLa cell nuclear extracts and the fold induction of multiple TFs in the TNFα-induced HeLa cell nuclear extracts. Therefore, this study provides a new tool for studying TFs.

Nuclear factor κB (NF-κB) is a ubiquitous transcription factor that plays a pivotal role in controlling important cellular processes, ranging from normal cell growth and differentiation to apoptosis and cancer. In recent years, many new target genes of NF-κB have been identified in several cell lines that were treated with various stimuli using chromatin immunoprecipitation (ChIP)-based high-throughput techniques. However, the target genes from various cell lines and stimuli are not identical, and many of them are cell or stimulus specific. This suggests that it is necessary to investigate different cell lines and stimuli for identifying all target genes of this transcription factor. In this study, the direct target genes (DTGs) of NF-κB in the TNFα-stimulated HeLa cells were identify by using ChIP-Seq, RNAi, and gene expression profiling techniques. As a result, 584 DTGs were identified, in which 266 were activated and 318 were repressed. The κB motif searching revealed that 50 % of these genes contained canonical κB sites in their ChIP peaks and 90 % contained non-canonical κB sites in their ChIP peaks. In comparison with target genes identified in LPS-treated U937 and THP-1, only limited numbers (10∼23) of target genes were shared by each of two cell lines, and only two gene (NFKB2 and STAT5A) were commonly shared by three cell lines.

In recent years, near-infrared fluorescence (NIRF)-labeled iron nanoparticles have been synthesized and applied in a number of applications, including the labeling of human cells for monitoring the engraftment process, imaging tumors, sensoring the in vivo molecular environment surrounding nanoparticles and tracing their in vivo biodistribution. These studies demonstrate that NIRF-labeled iron nanoparticles provide an efficient probe for cell labeling. Furthermore, the in vivo imaging studies show excellent performance of the NIR fluorophores. However, there is a limited selection of NIRF-labeled iron nanoparticles with an optimal wavelength for imaging around 800 nm, where tissue autofluorescence is minimal. Therefore, it is necessary to develop additional alternative NIRF-labeled iron nanoparticles for application in this area.This study manufactured 12-nm DMSA-coated Fe3O4 nanoparticles labeled with a near-infrared fluorophore, IRDye800CW (excitation/emission, 774/789 nm), to investigate their applicability in cell labeling and in vivo imaging. The mouse macrophage RAW264.7 was labeled with IRDye800CW-labeled Fe3O4 nanoparticles at concentrations of 20, 30, 40, 50, 60, 80 and 100 μg/ml for 24 h. The results revealed that the cells were efficiently labeled by the nanoparticles, without any significant effect on cell viability. The nanoparticles were injected into the mouse via the tail vein, at dosages of 2 or 5 mg/kg body weight, and the mouse was discontinuously imaged for 24 h. The results demonstrated that the nanoparticles gradually accumulated in liver and kidney regions following injection, reaching maximum concentrations at 6 h post-injection, following which they were gradually removed from these regions. After tracing the nanoparticles throughout the body it was revealed that they mainly distributed in three organs, the liver, spleen and kidney. Real-time live-body imaging effectively reported the dynamic process of the biodistribution and clearance of the nanoparticles in vivo.IRDye800CW-labeled Fe3O4 nanoparticles provide an effective probe for cell-labeling and in vivo imaging.

To develop an EMSA-free assay approach for analyzing the sequence-specific DNA-binding proteins (DBPs), an easy cost-effective dsDNA-coupled plate (dcPlate) was developed in our lab for this purpose. In this paper, the assay conditions of such dcPlate were fully optimized for detecting an important transcription factor, NF-κB. The optimized parameters of dcPlate for assay of NF-κB were as follows: immobilized DNA probe at the concentration of 25 pmol/100 μL-well, incubation time of 90 min for NF-κB binding to dcPlate, primary and secondary antibody concentration of 0.1 μL/100 μL dilution, incubation time of 90 min for primary antibody binding to NF-κB, temperature of 25 °C for the above process, colorimetric developing time for 30 min. After optimization, the signal was improved three times higher than that from not optimized conditions. The linear colorimetric detection ranges of the purified recombinant NF-κB p50 and the cell nuclear extract were from 0.59 to 75 ng/well and 0.313 to 10 μg/well, respectively.

Fe3O4 magnetic nanoparticles (MNPs) coated with 2,3-dimercaptosuccinnic acid (DMSA) are considered to be a promising nanomaterial with biocompatibility. In the present study, the effects of DMSA-coated Fe3O4 MNPs on the expression of all identified mouse genes, which regulate various cellular biological processes, were determined to establish whether this nanoparticle is cytotoxic to mammalian cells. Mouse macrophage RAW264.7 cells were treated with 100 μg/ml of DMSA-coated Fe3O4 MNPs for 4, 24 and 48 h, and the global gene expression was detected via Affymetrix Mouse Genome 430 2.0 GeneChips® microarrays. It was found that gene expression of 711, 545 and 434 transcripts was significantly altered by 4-, 24- and 48-h treatments, respectively. Of these genes, 27 were consistently upregulated and 6 were consistently downregulated at the three treatment durations. Bioinformatic analysis of all differentially expressed genes revealed that this nanoparticle can strongly activate inflammatory and immune responses and can inhibit the biosynthesis and metabolism of RAW264.7 cells at a dose of 100 μg/ml. These results demonstrated that DMSA-coated Fe3O4 MNPs display cytotoxicity in this type of macrophage at high doses.Highlights► We prepared water-soluble DMSA-coated Fe3O4 magnetic nanoparticles (MNPs). ► We investigated their effects on the global gene expression of RAW264.7 cell. ► The expression of many genes was altered by the treatments of this nanoparticle. ► The inflammatory and immune responses of RAW264.7 cells were strongly activated at a dose of 100 μg/ml. ► This nanoparticles display cytotoxicity in this type of macrophage at high doses.

•NF-κB is proposed to regulate cancer cellular energetic metabolism.•NF-κB binds tricarboxylic acid cycle-related genes in cancer cells.•NF-κB regulates tricarboxylic acid cycle-related genes in cancer cells.•NF-κB regulates expression of IDH1, IDH3A, ACO2, and SUCLA2 genes.NF-κB may promote tumor progression by altering cell metabolism. Hence, finding its target genes that are involved in cell metabolism is helpful for understanding its role in tumor growth. Here we discovered four metabolism-related target genes of this transcription factor. By analyzing a chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) data that characterizing the global binding sites (BSs) of NF-κB RelA in the TNFα-stimulated HeLa cells, we found that four genes that encode core enzymes of the tricarboxylic acid (TCA) cycle, including IDH1, IDH3A, ACO2, and SUCLA2, were multiply bound by this transcription factor. The subsequent bioinformatic analysis revealed that the NF-κB BSs contained many canonical κB sequences and the NF-κB-like DNA-binding motifs. Detection of ChIPed DNA with polymerase chain reaction (ChIP-PCR) also indicated that the NF-κB BSs were bound by NF-κB in both TNFα-treated HeLa and HepG2 cells. The reporter construct showed that the NF-κB BSs could activate the luciferase expression in cells in a NF-κB-specific manner. The quantitative PCR and Western blot detections demonstrated that NF-κB could regulate the expressions of IDH1, IDH3A, and ACO2 genes at both mRNA and protein levels and that of SUCLA2 gene at mRNA level in the TNFα-treated HeLa and HepG2 cells. Based on these investigations we identified the four genes as new target genes of NF-κB. The finding provides new insights into the role of NF-κB in cellular energetic metabolism, which may be beneficial for understanding the metabolic physiology of tumor growth.Download high-res image (198KB)Download full-size image