Deletions of singular U1 snRNA gene significantly interfere with transcription and 3'-end mRNA formation.

Small nuclear RNAs (snRNAs) are structural and functional cores of the spliceosome. In metazoan genomes, each snRNA has multiple copies/variants, up to hundreds in mammals. However, the expressions and functions of each copy/variant in one organism have not been systematically studied. Focus on U1 snRNA genes, we investigated all five copies in Drosophila melanogaster using two series of constructed strains. Analyses of transgenic flies that each have a U1 promoter-driven gfp revealed that U1:21D is the major and ubiquitously expressed copy, and the other four copies have specificities in developmental stages and tissues. Mutant strains that each have a precisely deleted copy of U1-gene exhibited various extents of defects in fly morphology or mobility, especially deletion of U1:82Eb. Interestingly, splicing was changed at limited levels in the deletion strains, while large amounts of differentially-expressed genes and alternative polyadenylation events were identified, showing preferences in the down-regulation of genes with 1-2 introns and selection of proximal sites for 3'-end polyadenylation. In vitro assays suggested that Drosophila U1 variants pulled down fewer SmD2 proteins compared to the canonical U1. This study demonstrates that all five U1-genes in Drosophila have physiological functions in development and play regulatory roles in transcription and 3'-end formation.

Sirtuin 6 promotes transforming growth factor-ß1/H2O2/HOCl-mediated enhancement of hepatocellular carcinoma cell tumorigenicity by suppressing cellular senescence.

Sirtuin 6 (SIRT6) can function as a tumor suppressor by suppressing aerobic glycolysis and apoptosis resistance. However, the negative effect of SIRT6 on cellular senescence implies that it may also have the potential to promote tumor development. Here we report that the upregulation of SIRT6 expression was required for transforming growth factor (TGF)-ß1 and H2O2/HOCl reactive oxygen species (ROS) to promote the tumorigenicity of hepatocellular carcinoma (HCC) cells. Transforming growth factor-ß1/H2O2/HOCl could upregulate SIRT6 expression in HCC cells by inducing the sustained activation of ERK and Smad pathways. Sirtuin 6 in turn abrogated the inducing effect of TGF-ß1/H2O2/HOCl on cellular senescence of HCC cells, and was required for the ERK pathway to efficiently suppress the expression of p16 and p21. Sirtuin 6 altered the effect of Smad and p38 MAPK pathways on cellular senescence, and contributed to the inhibitory effect of the ERK pathway on cellular senescence. However, SIRT6 was inefficient in antagonizing the promoting effect of TGF-ß1/H2O2 HOCl on aerobic glycolysis and anoikis resistance. Intriguingly, if SIRT6 expression was inhibited, the promoting effect of TGF-ß1/H2O2/HOCl on aerobic glycolysis and anoikis resistance was not sufficient to enhance the tumorigenicity of HCC cells. Suppressing the upregulation of SIRT6 enabled TGF-ß1/H2O2/HOCl to induce cellular senescence, thereby abrogating the enhancement of HCC cell tumorigenicity by TGF-ß1/H2O2/HOCl. These results suggest that SIRT6 is required for TGF-ß1/H2O2/HOCl to enhance the tumorigenicity of HCC cells, and that targeting the ERK pathway to suppress the upregulation of SIRT6 might be a potential approach in comprehensive strategies for the therapy of HCC.

Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3-Regulated Histone Deacetylation and Transcription.

RNA splicing is crucial in the multilayer regulatory networks for gene expression, making functional interactions with DNA- and other RNA-processing machineries in the nucleus. However, these established couplings are all major spliceosome-related; whether the minor spliceosome is involved remains unclear. Here, through affinity purification using Drosophila lysates, an interaction is identified between the minor spliceosomal 65K/RNPC3 and ANKRD11, a cofactor of histone deacetylase 3 (HDAC3). Using a CRISPR/Cas9 system, Deletion strains are constructed and found that both Dm65KΔ/Δ and Dmankrd11Δ/Δ mutants have reduced histone deacetylation at Lys9 of histone H3 (H3K9) and Lys5 of histone H4 (H4K5) in their heads, exhibiting various neural-related defects. The 65K-ANKRD11 interaction is also conserved in human cells, and the HsANKRD11 middle-uncharacterized domain mediates Hs65K association with HDAC3. Cleavage under targets and tagmentation (CUT&Tag) assays revealed that HsANKRD11 is a bridging factor, which facilitates the synergistic common chromatin-binding of HDAC3 and Hs65K. Knockdown (KD) of HsANKRD11 simultaneously decreased their common binding, resulting in reduced deacetylation of nearby H3K9. Ultimately, this study demonstrates that expression changes of many genes caused by HsANKRD11-KD are due to the decreased common chromatin-binding of HDAC3 and Hs65K and subsequently reduced deacetylation of H3K9, illustrating a novel and conserved coupling mechanism that links the histone deacetylation with minor spliceosome for the regulation of gene expression.

Ethylidene-Tethered Chromene-Pyrone Hybrids as Potential Plant-Growth Regulators from an Endolichenic Phaeosphaeria Species.

Phaeosphaeria sp., a lichen-associated fungus, produced six skeletally new dimeric spiciferones (1-6) and four known metabolites (7-10). The new structures were elucidated by spectroscopic analysis, and their absolute configurations were determined by electronic circular dichroism calculations. Compounds 1 and 3-6 represent the first examples of ethylidene-bridged dimers from the building blocks 4H-chromene-4,7(8H)-dione and α-pyrone, and 2 is a unique homodimer of spiciferone. Compounds 1, 2, and 5-9 significantly inhibited the growth of weed-like dicot Arabidopsis thaliana at 100.0 µM. Notably, 8 showed the strongest inhibitory activity against the fresh weight and root elongation of A. thaliana with the IC50 values of 32.04 and 26.78 µM, respectively, whereas 1, 8, and 9 stimulated the growth of A. thaliana at lower concentrations. Meanwhile, compounds 2 and 6 exhibited weak inhibitory effects on the root elongation of monocot rice, while 1 and 8 exhibited growth-promoting effects on the shoot and root elongation of rice in a roughly dose-dependent manner.

Transcriptional up-regulation of FoxM1 in response to hypoxia is mediated by HIF-1.

The proliferation-specific Forkhead box M1 (FoxM1) transcription factor is overexpressed in cancer cells and acts as an important regulator of cancer cell growth and survival. Here, we show the molecular mechanisms by which hypoxia regulate FoxM1 expression in cancer cells. When cells were subjected to hypoxia (1% O2), the mRNA and protein levels of FoxM1 had a significant increase in cancer cells (HepG2, MCF-7, and HeLa). Such increase was due to the direct binding of hypoxia-inducible factor 1 (HIF-1) to the HIF-1 binding sites in the FoxM1 promoter. By deletion and mutation assays, we demonstrated that the HIF1-1 and HIF1-3/4 binding sites on the FoxM1 promoter were essential for transcriptional activation of FoxM1 by hypoxia. We also demonstrated that HIF-1alpha directly bound to the promoter of FoxM1 and the binding was specific, as revealed by HIF-1 binding/competition assay and chromatin immunoprecipitation assay. Consequently, the up-regulation of FoxM1 accelerated the growth of hypoxic cancer cells by decreasing nuclear levels of p21 and increasing expression of cyclin B1 and cyclin D1. These findings provide a new insight into how tumor cells overcome hypoxic stress and survive, and also disclose a new regulatory mechanism of FoxM1 expression in tumor cells.

[Hypoxia induces heat shock protein HSP70-2 expression in a HIF-1 dependent manner].

OBJECTIVES: To investigate role of hypoxia inducible factor 1 (HIF-1) in the transcriptional activation of heat shock protein 70-2 (HSP70-2) in hepatocellular carcinoma (HCC) cells under hypoxic conditions. METHODS: HCC cells were exposed to reduced oxygen atmosphere (1% O2), or treated with YC-1 or HIF-1 alpha siRNA, the expression of HIF-1 alpha and HSP70-2 were detected by Western blot analysis. Serial deletions of the HSPA2 promoter were cloned in the reporter pGL3-Basic plasmid. These reporter plasmids were co-transfected with HIF-1 alpha siRNA, and the promoter activities were detected with the dual luciferase assay. RESULTS: Western blot analysis showed that both HIF-1 alpha and HSP70-2 proteins were strongly increased after HCC cells were exposed to hypoxic conditions (1% O2) for 6 h, and the expression level of HSP70-2 was increased in a time-dependent manner. Treatment of HepG2 cells with YC-1 or HIF-1 alpha siRNA significantly inhibited the expression of HIF-1 alpha and HSP70-2. In silico analysis of the HSP70-2 promoter using the Gene2 Promoter software revealed the presence of two putative hypoxic response element (HRE) consensus at -446bp (HRE1) and -238bp (HRE2). Depletion of promoter sequence between -653 and -385 led to a dramatic reduction of promoter activity, whereas further deletion to position -201 did not reduce the activity further. These data suggested that HRE1 plays an important role in hypoxia-induced activation of the HSPA2 promoter. Site-directed mutagenesis further confirmed these results. Mutation of HRE1 but not of HRE2 abrogated the sensitivity of the HSP70-2 promoter to hypoxia. CONCLUSIONS: HSP70-2 expression is up-regulated in response to hypoxia and a HIF-1 binding site (HRE1) in the HSP70-2 promoter is involved in this response.

An integrated cell printing system for the construction of heterogeneous tissue models.

A new three-dimensional (3D) cell printing system was developed and investigated to organize multiple cells/biomaterials with a control precision within 100 µm. This system can be used for the in vitro construction of heterogeneous tissue models. The proposed printing system was achieved by the integration of extrusion printing and alternating viscous and inertial force jetting (AVIFJ) techniques using dual-nozzle switching. In this technique, hydrogels containing high cell densities were extruded using extrusion printing, while droplets containing single cells were precisely manipulated using AVIFJ. The droplets that contained single cells were at the scale of pico-liters and could be accurately positioned at the micron scale. Stable hydrogel structures with adjustable diameters were also printed, with cell viabilities exceeding 90% after printing. A heterogeneous tumor model that contained spheroids and human umbilical vein endothelial cells (HUVECs) was then constructed using the established integrated cell printing system in a stepwise or simultaneous fashion. HUVEC-loaded droplets were observed to locate around the preformed tumor spheroids as designed. Cells and spheroids in the model maintained high cell viability and sustained growth throughout the culture period. The ELISA results of albumin production also proved that the spheroids maintained increased cellular function during the culture. These results demonstrated the feasibility of this integrated 3D printing system for the engineering of in vitro heterogeneous tissue models for future biological and pathological studies. STATEMENT OF SIGNIFICANCE: Addressing the challenge of multi-scale printing in the construction of heterogeneous tissue models, a new 3D cell printing system was developed to organize cells/biomaterials of a control precision within 100 µm. AVIFJ was integrated with extrusion printing, thereby achieving the construction of cell interactions between single cells and spheroids, the manipulation of single cells in a 3D microenvironment with high accuracy, and the real-time on-demand printing. The printed heterogeneous tumor model maintained cell viability, sustained cell growth, and increased cell function during 7 days of culture. We believed that this work would benefit the production of functional artificial tissues, enabling the construction of more biomimetic cell arrangements and microenvironment to support cell functions.

Dalmanol biosyntheses require coupling of two separate polyketide gene clusters.

Polyketide-polyketide hybrids are unique natural products with promising bioactivity, but the hybridization processes remain poorly understood. Herein, we present that the biosynthetic pathways of two immunosuppressants, dalmanol A and acetodalmanol A, result from an unspecific monooxygenase triggered hybridization of two distinct polyketide (naphthalene and chromane) biosynthetic gene clusters. The orchestration of the functional dimorphism of the polyketide synthase (ChrA) ketoreductase (KR) domain (shortened as ChrA KR) with that of the KR partner (ChrB) in the bioassembly line increases the polyketide diversity and allows the fungal generation of plant chromanes (e.g., noreugenin) and phloroglucinols (e.g., 2,4,6-trihydroxyacetophenone). The simultaneous fungal biosynthesis of 1,3,6,8- and 2-acetyl-1,3,6,8-tetrahydroxynaphthalenes was addressed as well. Collectively, the work may symbolize a movement in understanding the multiple-gene-cluster involved natural product biosynthesis, and highlights the possible fungal generations of some chromane- and phloroglucinol-based phytochemicals.

Dalestones A and B, two anti-inflammatory cyclopentenones from Daldinia eschscholzii with an edited strong promoter for the global regulator LaeA-like gene.

Replacement of the native promoter of theglobal regulator LaeA-like gene of Daldinia eschscholzii by a strong gpdA promoter led to the generation of two novel cyclopentenone metabolites, named dalestones A and B, whose structures were assigned by a combination of spectroscopic analysis, modified Mosher's reaction, and electronic circular dichroism (ECD). Dalestones A and B inhibit the gene expression of TNF-α and IL-6 in LPS-induced RAW264.7 macrophages.

Processing of ß-glucosidase-silk fibroin nanoparticle bioconjugates and their characteristics.

Silk fibroin derived from Bombyx mori is a biomacromolecular protein with excellent biocompatibility. The aim of this work was to develop silk fibroin nanoparticles (SFNs) derived from the fibrous protein, which is a novel vector for enzyme modification in food processing. Silk fibroin was dissolved in highly concentrated CaCl2 and subjected to lengthy desalting in water. The resulting liquid silk, which contained water-soluble polypeptides with molecular mass ranging from 10 to 200 kDa, and ß-glucosidase were added rapidly into acetone. The ß-glucosidase molecules were embedded into silk fibroin nanoparticles, forming ß-glucosidase-silk fibroin nanoparticles (ßG-SFNs) with a diameter of 50-150 nm. The enzyme activity of the ßG-SFN bioconjugates was determined with p-nitrophenyl-ß-D-glucoside as the substrate, and the optimum conditions for the preparation of ßG-SFNs were investigated. The enzyme activity recovery of ßG-SFNs was 59.2 % compared to the free enzyme (specific activity was 1 U mg(-1)). The kinetic parameters of the ßG-SFNs and the free ß-glucosidase were the same. The ßG-SFNs had good operational stability and could be used repeatedly. These results confirmed that silk protein nanoparticles were good carriers as bioconjugates for the modification of enzymes with potential value for research and development. The method used in this study has potential applications in food processing and the production of flavour agents.

ß3 integrin promotes TGF-ß1/H2O2/HOCl-mediated induction of metastatic phenotype of hepatocellular carcinoma cells by enhancing TGF-ß1 signaling.

In addition to being an important mediator of migration and invasion of tumor cells, ß3 integrin can also enhance TGF-ß1 signaling. However, it is not known whether ß3 might influence the induction of metastatic phenotype of tumor cells, especially non-metastatic tumor cells which express low level of ß3. Here we report that H2O2 and HOCl, the reactive oxygen species produced by neutrophils, could cooperate with TGF-ß1 to induce metastatic phenotype of non-metastatic hepatocellular carcinoma (HCC) cells. TGF-ß1/H2O2/HOCl, but not TGF-ß1 or H2O2/HOCl, induced ß3 expression by triggering the enhanced activation of p38 MAPK. Intriguingly, ß3 in turn promoted TGF-ß1/H2O2/HOCl-mediated induction of metastatic phenotype of HCC cells by enhancing TGF-ß1 signaling. ß3 promoted TGF-ß1/H2O2/HOCl-induced expression of itself via positive feed-back effect on p38 MAPK activation, and also promoted TGF-ß1/H2O2/HOCl-induced expression of α3 and SNAI2 by enhancing the activation of ERK pathway, thus resulting in higher invasive capacity of HCC cells. By enhancing MAPK activation, ß3 enabled TGF-ß1 to augment the promoting effect of H2O2/HOCl on anoikis-resistance of HCC cells. TGF-ß1/H2O2/HOCl-induced metastatic phenotype was sufficient for HCC cells to extravasate from circulation and form metastatic foci in an experimental metastasis model in nude mice. Inhibiting the function of ß3 could suppress or abrogate the promoting effects of TGF-ß1/H2O2/HOCl on invasive capacity, anoikis-resistance, and extravasation of HCC cells. These results suggest that ß3 could function as a modulator to promote TGF-ß1/H2O2/HOCl-mediated induction of metastatic phenotype of non-metastatic tumor cells, and that targeting ß3 might be a potential approach in preventing the induction of metastatic phenotype of non-metastatic tumor cells.

Down-regulated KLF17 expression is associated with tumor invasion and poor prognosis in hepatocellular carcinoma.

Although the role of Krüppel-like factor 17 (KLF17) in regulating epithelial-mesenchymal transition (EMT) has been explored in breast cancer, its influence on primary hepatocellular carcinoma (HCC) remains unclear. This study aims to investigate the expression status of KLF17 in hepatocellular carcinoma (HCC) and the correlation between KLF17 expression and metastatic potential of HCC. KLF17 expression in HCC and adjacent liver tissues was studied by real-time PCR and Western blot, and the relationship between KLF17 expression and the clinicopathological features of HCC was evaluated in 60 patients. By using RNA interference technique, the correlation of KLF17 expression and metastatic potential was investigated by down-regulating KLF17 expression in HepG2 cells, and the effects of KLF17 down-regulation on cell migration, and invasion were then analyzed. Furthermore, the correlation between KLF17 expression and the surgical outcomes of a cohort of HCC patients was analyzed. Reduced expression of KLF17 is associated with a short survival time in clinical patients (P = 0.034). Low KLF17 expression is related to tumor T stage (P = 0.045), tumor size (P = 0.027), lymph node stage (P = 0.030), M stage (P = 0.048), and portal vein tumor thrombosis significantly in HCC. Reduced expression of KLF17 promoted motility and invasion ability of HepG2 cells and changed the expression of E-cadherin, ZO-1, Snai1, and vimentin (genes are associated with EMT). Overall, these findings suggest a repressing role of KLF17 in tumor invasion and a new prognostic indicator in directing therapy. It deserves further exploration.

HBx protein induces expression of MIG and increases migration of leukocytes through activation of NF-kappaB.

Elevated expression of monokine induced by the interferon-gamma (MIG) has been shown in HBV carriers, and it is involved in the infiltration of inflammatory cells and liver damage after HBV infection. However, the molecular mechanisms by which HBV-induced MIG expression have not been characterized. Our results indicated that HBx protein induced MIG expression in a dose-dependent manner. Such increase was due to the direct binding of NF-kappaB to the MIG promoter. By luciferase, chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that the NF-kappaB binding site at positions -147 was essential for transcriptional activation of MIG promoter by HBx protein. Chemotaxis assay showed that the up-regulation of MIG protein levels enhanced the migration of peripheral blood lymphocytes (PBLs), and inhibition of NF-kappaB significantly decreased the chemotaxis activity. Our findings provide a new insight into how leukocytes migrate to liver, and disclose a new regulatory mechanism of MIG expression after HBV infection.