NF-κB target microRNAs and their target genes in TNFα-stimulated HeLa cells.

As a transcription factor, NF-κB was demonstrated to regulate the expressions of miRNAs. However, only a few miRNAs have been identified as its targets so far. In this study, by using ChIP-Seq, Genechip and miRNA-Seq techniques, we identified 14 NF-κB target miRNAs in TNFα-stimulated HeLa Cells, including miR-1276, miR-1286, miR-125b-1-3p, miR-219-1-3p, miR-2467-5p, miR-3200-3p, miR-449c-5p, miR-502-5p, miR-548d-5p, miR-30b-3p, miR-3620-5p, miR-340-3p, miR-4454 and miR-4485. Of these miRNAs, 8 detected miRNAs were also NF-κB target misRNAs in TNFα-stimulated HepG2 cells. We also identified 16 target genes of 6 miRNAs including miR-125b-1-3p, miR-1286, miR-502-5p, miR-1276, miR-219-1-3p and miR-30b-3p, in TNFα-stimulated HeLa cells. Target genes of miR-125b-1-3p and miR-1276 were validated in HeLa and HepG2 cells by transfecting their expression plasmids and mimics. Bioinformatic analysis revealed that two potential target genes of miR-1276, BMP2 and CASP9, were enriched in disease phenotypes. The former is enriched in osteoarthritis, and the latter is enriched in Type 2 diabetes and lung cancer, respectively. These findings suggested that this little known miRNA might play roles in these diseases via its two target genes of BMP2 and CASP9. The expression of miR-125b-1 regulated by NF-κB has been reported in diverse cell types under various stimuli, this study found that its expression was also significantly regulated by NF-κB in TNFα-stimulated HeLa and HepG2 cells. Therefore, this miRNA was proposed as a central mediator of NF-κB pathway. These findings provide new insights into the functions of NF-κB in its target miRNA-related biological processes and the mechanisms underlying the regulation of these miRNAs.

Subset of genes targeted by transcription factor NF-κB in TNFα-stimulated human HeLa cells.

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.

Rapid Visual LAMP Method for Detection of Genetically Modified Organisms.

We developed a novel loop-mediated isothermal amplification (LAMP) method using DNA captured on polyacrylamide microparticles (PAMMPs) as templates (PAMMPs@DNA-LAMP) for rapid qualitative detection of genetically modified organisms (GMOs). Here, DNA was extracted by a fast and cost-effective method using PAMMPs. Four LAMP primers were designed for the PAMMPs@DNA-LAMP method to detect the cauliflower mosaic virus 35S (CaMV35S) promotor in GMOs. We thus developed this method for rapid extraction of DNA (5-10 min) and fast amplification of DNA within ∼30 min at a constant temperature of 63 °C. Moreover, the DNA captured by PAMMPs (PAMMPs@DNA) could be effectively detected by both conventional and quantitative PCR (qPCR) and LAMP. The PAMMPs@DNA-LAMP method was validated with high specificity, sensitivity, and performance for practical sample analysis. This assay detected 0.01% target sequences, which had a high specificity like qPCR and better than the conventional PCR (cPCR). Furthermore, PAMMPs@DNA-LAMP was successfully used to extract and detect DNA from food samples of the major crops (soybean, maize, rice, etc.). In summary, a novel PAMMPs@DNA-LAMP assay has been developed, which has higher sensitivity and spends less time than the cPCR detection using the conventional DNA extracted process. This method offers a novel approach for rapid detection of GMOs in the field.

Function of the Mitochondrial Transport Protein BcMtp1 in Regulating Vegetative Development, Asexual Reproduction, Stress Response, Fungicide Sensitivity, and Virulence of Botrytis cinerea.

In model fungi, mitochondrial transport proteins (MTPs), also known as "mitochondrial carriers" (MC), are known to facilitate the exchange of biochemical substances across the mitochondrial inner membrane. In this study, we characterized an MTP in Botrytis cinerea homologous to the known MTPs in Saccharomyces cerevisiae designated BcMtp1. The BcMtp1 deletion mutant phenotype was strikingly defective in vegetative development, conidiation, and sclerotia production. In addition, ΔBcMtp1 showed increased sensitivity to osmotic stress, oxidative stress, and cell wall biogenesis inhibitors. In the pathogenicity assay, ΔBcMtp1 displayed compromised virulence on various host-plant tissues. The BcMtp1 deletion mutant phenotype was rescued by transforming the wild-type BcMtp1 variant into the mutant. Together, these data indicate that BcMtp1 is critical for vegetative development, asexual reproduction, stress tolerance, and virulence of B. cinerea.

Residual levels and dietary intake risk assessment of 11 pesticides in apricots from different ecological planting regions in China.

The frequent and massive use of pesticides has led to pesticide residues in apricot, threatening food safety and human health. A reliable and simple modified QuEChERS method with ultra-performance liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of 11 pesticides in apricot. Method validation indicated that satisfied linearity (R2 ≥ 0.9959), accuracy (recoveries of 72-119%), sensitivity (limits of detection, 0.03-0.30 µg/kg; limits of quantification, 0.13-1.00 µg/kg), and precision (relative standard deviations ≤ 11.9%), and matrix effects were 0.89-1.13. Apricot samples from different ecological regions in China were collected and tested using the proposed methods. Monitoring results were used to assess the dietary intake risk of Chinese populations of different ages and genders. Dietary risk assessment revealed that the risk quotients were 0.003-1.184% for different gender and age groups in China, indicating none unacceptable public health risk for general population. This work was thus significant in developing a simpler, more efficient and economical analysis method and food safety risks of the 11 pesticides on apricot and facilitated the establishment of maximum residue limits.

[Antiproliferative effects mechanism of beta-sitosterul in hepatoma HepG2 cells].

OBJECTIVE: To study the antiproliferative effects of beta-sitosterul and its mechanism in hepatoma HepG2 cells. METHOD: Cell proliferation was assessed by MTT assay. Cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by high content screening (HCS). The protein expression of caspase-3, caspase-8, caspase-9, Bcl-2, Bax, tBid and cytochrome c in the HepG2 cells were evaluated by Western Blots. RESULT: beta-Sitosterul exerted significant antiproliferative effects in HepG2 cells. Furthermore, beta-sitosterul also induced HepG2 cells apoptosis, lost mitochondrial membrane potential, activated caspase-3, caspase-8 and caspase-9, up-regulate Bax, tBid protein, down-regulation Bcl-2 protein. However, beta-sitosterul had hardly any effects on QSG7701 cells. CONCLUSION: beta-Sitosterul exerted antiproliferative effects and induced HepG2 cells apoptosis via mitochondrial pathway and membrane death receptor pathway.

Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection.

The fast and cost-effective DNA extraction is critical for all DNA-based detections. Here, we fabricated a new kind of polyacrylamide microsphere (PAMMP) in various sizes with two methods, spot polymerization (large size but low yield) and modified inverse microemulsion polymerization (small size but high yield). The fabricated PAMMPs have strong autofluorescence (fPAMMPs), including both visible fluorescence (VF) and near-infrared fluorescence (NIRF), which can remain very stable in various stringent conditions including strong acid and alkali and high temperature. The fabricated fPAMMPs were also highly positively charged, which could be used to effectively capture various biomolecules such as IRDye 800-labeled streptavidin and DNA. We thus developed a new method for rapid extraction (3-5 min) of DNA from various samples including bacteria, mammalian cells, plant and animal solid tissues, and human blood plasma using fPAMMPs. Moreover, the DNA captured on fPAMMPs (fPAMMP@DNA) could be effectively detected by both normal and quantitative PCR amplifications. Finally, we showed that NaBH4 treatment removed autofluorescence in fPAMMPs (PAMMPs), which could also be applied to DNA extraction and PCR detection. In conclusion, we here fabricated new kinds of fPAMMPs and PAMMPs, developed a new rapid DNA extraction method, and demonstrated their useful applications in PCR detection.

Characterization of genome-wide binding of NF-κB in TNFα-stimulated HeLa cells.

This study characterized the genome-wide binding of NF-κB RelA with ChIP-Seq and explored its effects on the gene transcription with DNA microarray. It was found that NF-κB showed several significant binding characteristics, including the inter- and intra-chromosomal differential high-fold enrichment binding, the dominant intronic binding to vast majority of target genes through multiple ChIP-seq peaks and κB sites, extensively binding to large number of genes in the human genome, and binding its target genes more broadly through noncanonical κB sites than canonical κB sites. These in vivo genome-wide binding characteristics exerted effects on the transcription of its direct target genes in genome, reflecting some important traits of this protein which acts as a stimulatory transcription factor involving in many biological processes and responding to various internal and external stimuli.

NBPF is a potential DNA-binding transcription factor that is directly regulated by NF-κB.

The neuroblastoma breakpoint family (NBPF) has been reported to play potential roles in the development of neuroblastoma and human evolution. However, the exact regulation and function of this family is still unknown. In this study, the genes of NBPF family were found to be densely covered by many high-confidence ChIP-Seq peaks of NF-κB. The expressions of NBPF genes were thus deduced to be regulated by this transcription factor. The activities of NF-κB in HeLa, HepG2 and ECa109 cells were then manipulated with NF-κB activator (TNFα) and inhibitors (BAY11-7082 or p65 siRNA), and the expressions of NBPF genes in these cells were checked. As result, it was found that the expressions of NBPF genes were regulated by NF-κB in HeLa and HepG2 cells. Therefore, the genes of NBPF family were identified as new bona fide direct target genes of NF-κB. In addition, NBPF was also identified as a nuclear protein by in silico prediction and immunolocalization. Finally, the bioinformatics analysis revealed that most of NBPF proteins contained classical nuclear localization signals (NLSs) and a conserved DNA-binding domain similar to the transcription factor stat3b/dna complex or stat-1/dna complex in their N-terminals. Therefore, this study concluded that NBPF was nuclear protein that contained classical NLSs and conserved known DNA-binding domain, and its expression was regulated by another important transcription factor, NF-κB. These findings suggest that NBPF may function as DNA-binding transcription factor in nucleus, which provides important new insight into the functions of NBPF genes in the human cells.