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Diisononyl phthalate (DINP) is a high-molecular-weight phthalate, and has been recently introduced as di-(2-ethyl hexyl) phthalate (DEHP) substitute and commonly used in a large variety of plastic items. The fat tissue is an important target for DINP exposure, however, very little is understood about its toxicity and mechanism(s) in adipocyte cells. Therefore, the present work aimed to investigate the role of DINP in adipogenesis using 3T3-L1 preadipocytes. DINP exposure for 10 days extensively induced adipogenesis in 3T3-L1 preadipocytes to adipocytes as assessed by lipid accumulation and gene expression of adipogenic markers. The RT-qPCR results showed that DINP could upregulate the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα) and C/EBPβ, while the expression of sterol regulatory element binding transcription factor 1 (SREBF1) and C/EBPδ was not affected. The DINP-induced adipogenesis could be inhibited by using the selective PPARγ antagonist GW9662. The RNA-seq analysis was used to study the systemic toxicities of DINP on preadipocytes. A total of 1181 differently expressed genes (DEGs) (640 genes were up-regulated, 541 genes were down-regulated) were detected in 3T3-L1 preadipocytes under 50 μM DINP. The GO enrichment showed the GO term of "fat cell differentiation" was the most significantly affected metabolic functions, and the KEGG pathway enrichment showed the PPAR pathway was the top affected pathway. The interactive pathway (iPath) analysis showed that the changed metabolic pathways were focus on the lipid metabolism., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: To investigate the role of microRNA (miR)-27a and miR-27b in adipogenesis in an in vitro model of Graves' orbitopathy (GO)., Methods: Orbital fat tissues were harvested from GO and non-GO participants for primary orbital fibroblast cultures. The expression levels of miR-27a and miR-27b between GO and non-GO orbital fat tissues were compared. During adipogenesis of GO orbital fibroblasts, the expression levels of miR-27a and miR-27b were determined, and the effects of mimics of miR-27a and miR-27b transfection on adipogenesis of GO orbital fibroblast were investigated., Results: Real time-polymerase chain reaction showed significantly more decreases in miR-27a and miR-27b levels in orbital fat tissues in GO participants than in non-GO participants (p < 0.05). The expression of both miR-27a and miR-27b was highest in orbital fibroblasts at day 0 and declined gradually after the induction of adipogenic differentiation. The expression levels of PPARγ, CCAAT/enhancer binding protein (C/EBP)α and C/EBPβ were decreased and Oil Red O-stained lipid droplets were lower in GO orbital fibroblasts transfected with miR-27a and miR-27b mimics than in negative controls., Conclusions: Our results indicated that miR-27a and miR-27b inhibited adipogenesis in orbital fibroblasts from GO patients. Further studies are required to examine the potential of miR-27a and miR-27b as targets for therapeutic strategies., Competing Interests: The authors have declared that no competing interests exist.

CCAAT/enhancer binding protein ε (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe -knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe , we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6-kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6-kb enhancer, which suggests their role in regulating the expression of Cebpe In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation., (© 2019 by The American Society of Hematology.)

To unravel molecular mechanisms by which Runt-related transcription factor 1 (RUNX1) mutations contribute to leukemic transformation, we introduced the RUNX1-S291fs300X mutation in human CD34 + stem/progenitor cells and in human induced pluripotent stem cells (iPSCs). In both models, RUNX1mut overexpression strongly impaired myeloid commitment. Instead, self-renewal was enhanced, as shown, by increased long-term culture-initiating cell frequencies and enhanced colony-forming cell replating capacity. Long-term suspension cultures with RUNX1mut-transduced cord blood (CB) CD34 + cells continued for more than 100 days, during which the cells displayed an immature granulocyte-macrophage progenitor-like CD34 + /CD123 + /CD45RA + phenotype. The CD34 + /CD38 - hematopoietic stem cell (HSC) population most likely acted as cell of origin, as HSCs provided the best long-term proliferative potential on overexpression of RUNX1mut. CEBPA expression was reduced in RUNX1mut cells, and reexpression of CEBPA partly restored differentiation. RNA-seq analysis on CB/iPSC systems and on primary patient samples confirmed that RUNX1 mutations induce a myeloid differentiation block, and that a common set of RUNX1mut-upregulated target genes was strongly enriched for gene ontology terms associated with nucleosome assembly and chromatin structure. Interestingly, in comparison with AML1-ETO binding in acute myeloid leukemias (AMLs), we found significantly distinct genomic distribution and differential expression for RUNX1mut of genes such as TCF4 , MEIS1 , and HMGA2 that may potentially contribute to the underlying difference in clinical outcomes between RUNX1mut and AML1-ETO patients. In conclusion, RUNX1mut appears to induce a specific transcriptional program that contributes to leukemic transformation., (© 2019 by The American Society of Hematology.)

Background: The transcription factor CCAAT-enhancer-binding protein alpha (CEBPA) is a crucial regulator of cell proliferation and differentiation. Expression levels of CEBPA have been suggested to be prognostic in various tumor types., Methods: Here, we analyzed the immunohistochemical expression of CEBPA in a tissue microarray containing more than 17 000 prostate cancer specimens with annotated clinical and molecular data including for example TMPRSS2:ERG fusion and PTEN deletion status., Results: Normal prostate glands showed moderate to strong CEBPA staining, while CEBPA expression was frequently reduced (40%) or lost (30%) in prostate cancers. Absence of detectable CEBPA expression was markedly more frequent in ERG negative (45%) as compared to ERG positive cancers (20%, P < 0.0001). Reduced CEBPA expression was linked to unfavorable phenotype (P < 0.0001) and poor prognosis (P = 0.0008). Subgroup analyses revealed, that the prognostic value of CEBPA loss was entirely driven by tumors carrying both TMPRSS2:ERG fusions and PTEN deletions. In this subgroup, CEBPA loss was tightly linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), positive nodal stage (0.0003), and early biochemical recurrence (P = 0.0007), while these associations were absent or markedly diminished in tumors with normal PTEN copy numbers and/or absence of ERG fusion., Conclusions: CEBPA is down regulated in about one third of prostate cancers, but the clinical impact of CEBPA loss is strictly limited to the subset of about 10% prostate cancers carrying both ERG fusion and deletions of the PTEN tumor suppressor. Our findings challenge the concept that prognostic molecular markers may be generally applicable to all prostate cancers., (© 2018 Wiley Periodicals, Inc.)

3'-untranslated regions (UTRs) can vary through the use of alternative polyadenylation sites during pre-mRNA processing. Multiple publically available pipelines combining high profiling technologies and bioinformatics tools have been developed to catalog changes in 3'-UTR lengths. In our recent RNA-seq experiments using cells with hyper-activated mammalian target of rapamycin (mTOR), we found that cellular mTOR activation leads to transcriptome-wide alternative polyadenylation (APA), resulting in the activation of multiple cellular pathways. Here, we developed a novel bioinformatics algorithm, IntMAP, which integrates RNA-Seq and PolyA Site (PAS)-Seq data for a comprehensive characterization of APA events. By applying IntMAP to the datasets from cells with hyper-activated mTOR, we identified novel APA events that could otherwise not be identified by either profiling method alone. Several transcription factors including Cebpg (CCAAT/enhancer binding protein gamma) were among the newly discovered APA transcripts, indicating that diverse transcriptional networks may be regulated by mTOR-coordinated APA. The prevention of APA in Cebpg using the CRISPR/cas9-mediated genome editing tool showed that mTOR-driven 3'-UTR shortening in Cebpg is critical in protecting cells from endoplasmic reticulum (ER) stress. Taken together, we present IntMAP as a new bioinformatics algorithm for APA analysis by which we expand our understanding of the physiological role of mTOR-coordinated APA events to ER stress response. IntMAP toolbox is available at http://compbio.cs.umn.edu/IntMAP/.

CCAAT/enhancer binding protein α, β, δ (C/EBP α, β, δ) are essential transcriptional factors in regulating adipose development. However, information about their sequence characteristics and functions during adipocyte development still remains scarce in birds. In present study, we found that duck C/EBP α, β, δ differed in their phosphorylation sites and low complexity regions (LCRs) among their orthologs and paralogs. Phylogenetic analysis showed that C/EBP α, β, δ had different evolutionary patterns, and each of duck C/EBP α, β, δ was strikingly diverged from orthologs of other Aves. Results of quantitative real-time PCR exhibited that C/EBP α, β, δ were all highly expressed in duck adipose tissues. Indeed, investigations of changes in both their mRNA levels and lipid droplet content during duck adipocytes differentiation showed that their expression profiles were closely related to cellular lipid accumulation. Furthermore, hierarchical clustering analysis of the C/EBPs and lipid metabolism-related genes expression profiles showed that C/EBP α was clustered with genes related to lipolysis, lipogenesis and fatty acid desaturation, whereas C/EBP β, δ were clustered with genes related to de novo lipogenesis and fatty acid elongation, which were different from mammals. In summary, C/EBP α, β, δ of duck differ from other species in their structures and have different effects on lipid metabolism during adipocytes differentiation. This research serve as a foundation for further investigations about avian C/EBP α, β, δ in adipocytes differentiation and adipose development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Obesity is characterized by excess body fat accumulation due to an increase in the size and number of differentiated mature adipocytes. Adipocyte differentiation is regulated by genetic and environmental factors, and its inhibition could represent a strategy for obesity prevention and treatment. The current study was designed with two aims: (i) to evaluate the changes in the expression of adipogenic markers (C/EBP α , PPAR γ variant 1 and variant 2, and GLUT4) in 3T3-L1 murine preadipocytes at four stages of the differentiation process and (ii) to compare the effectiveness of sulforaphane, genistein, and docosahexaenoic acid in reducing lipid accumulation and modulating C/EBP α , PPAR γ 1, PPAR γ 2, and GLUT4 mRNA expression in mature adipocytes. All bioactive compounds were shown to suppress adipocyte differentiation, although with different effectiveness. These results set the stage for further studies considering natural food constituents as important agents in preventing or treating obesity.

Stratum corneum forms the UV barrier. The effect of ultraviolet B (UVB) on normal skin was extensively studied; however, its effect on barrier perturbed skin remains undefined. Both barrier perturbation and UVB irradiation induce endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in keratinocytes. Mild ER stress activates homeostatic UPR, while severe ER stress leads to abnormal UPR, promoting apoptosis and inflammation. Here, we investigated UV sensitivity and UVB-induced UPR in barrier-disrupted human skin and the effects of pseudoceramide-dominant emollient on UVB-induced skin responses. Tape-stripped skin of healthy volunteers showed enhanced susceptibility to erythema and augmented proinflammatory cytokines induction following suberythemal UVB irradiation. Suberythemal UVB activated XBP1 in normal skin, while increased CHOP transcription in barrier perturbed skin. After tape stripping, pseudoceramide-dominant emollient was applied for 3 days, and then, the areas were irradiated with suberythemal UVB. Pretreatment with topical pseudoceramide protected against UVB-induced upregulation of IL-1β, IL-6, and TNF-α transcription and reduced susceptibility to erythema following UVB. Topical pseudoceramide also suppressed suberythemal UVB-induced CHOP transcription in barrier-disrupted skin. Taken together, these data indicate that permeability barrier disruption increases UV sensitivity in human skin, partly via switch the UVB-induced UPR, from homeostatic signals to pro-apoptotic and proinflammatory signals. In addition, we conclude that pseudoceramide-dominant emollient suppresses excessive ER stress induction and CHOP activation following UVB in barrier damaged skin, providing evidence that pseudoceramide-dominant emollients can be promising strategies for photoprotection of the barrier damaged skin.

Esophageal adenocarcinoma (EAC) is one of the most common malignancies in the world which is associated the increased prevalence of obesity. In the context of obesity, leptin can directly contribute to progression of EAC. Adiponectin inhibits leptin-induced oncogenic signaling in EAC cells. However, the exact molecular mechanisms linking obesity, adipokines, and EAC remain far from completely understood. In the present study, we tested the role of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) in adiponectin-induced protective effects against leptin-induced EAC cell proliferation. We found that globular adiponectin (gAD) significantly inhibited leptin-induced increase of cell proliferation and decrease of apoptosis in OE 19 cells. Moreover, leptin-induced increase of UHRF1 expression was suppressed by gAD. Compared with normal controls, UHRF1 expression was markedly increased in EAC tissues and cell lines. Silence of UHRF1 increased the expression of cleaved caspase 3 and 9 and Bax, reduced the expression of Bcl-2, promoted apoptosis, and inhibited cell proliferation in OE 19 cells. Overexpression of UHRF1 significantly blocked gAD-induced decrease of cell proliferation and increase of apoptosis in leptin-treated cells. Silence of adiponectin receptor 1/2 (AdipoR1/2) could inhibit gAD-induced decrease of cell proliferation and increase of apoptosis in leptin-treated cells. Silence of AdipoR2, but not AdipoR1, suppressed gAD-induced decrease of UHRF1 expression in leptin-treated cells. The results indicated that gAD inhibited the prooncogenic effects of leptin via AdipoR2-mediated suppression of UHRF1. Our study provides novel insights into the role of UHRF1 in the development of EAC and the mechanism of antitumor effect of gAD.

DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells., (© The Author 2017. Published by Oxford University Press.)

As the second most common cancer in men around the world, prostate cancer is increasingly gaining more attention. Dihydroartemisinin (DHA) has been proven to be a promising anticancer agent in vitro as well as in vivo in accumulating data. However, the detailed mechanisms of how DHA action in human prostate cancer PC-3 cells remain elusive. This study aimed to investigate the effects of DHA, a novel anticancer agent, by inhibiting the expression of ubiquitin like containing PHD and ring finger 1 (UHRF1) in PC-3 cells. The apoptosis and cell-cycle distribution were detected by flow cytometry. Quantitative real-time PCR was performed to examine both UHRF1 and DNA methyltransferase 1 (DNMT1) expressions at mRNA levels, whereas the expressions of UHRF1, DNMT1, and p16 proteins at protein levels were detected by Western blotting. Methylation levels of p16 CpG islands were determined by bisulfite genomic sequencing. We showed that DHA induced the downregulation of UHRF1 and DNMT1, accompanied by an upregulation of p16 in PC-3 cells. Decreased p16 promoter methylation levels in DHA-treated groups were also observed in PC-3 cells. Furthermore, DHA significantly induced apoptosis and G1/S cell-cycle arrest in PC-3 cells. Our results suggested that downregulation of UHRF1/DNMT1 is upstream to many cellular events, including G1 cell arrest, demethylation of p16, and apoptosis. Together, our study provides new evidence that DHA may serve as a potential therapeutic agent in the treatment of prostate cancer.

Increasing energy expenditure through brown adipocyte recruitment is a promising approach to combat obesity. We report here the comprehensive profiling of the epigenome and transcriptome throughout the lineage commitment and differentiation of C3H10T1/2 mesenchymal stem cell line into brown adipocytes. Through direct comparison to datasets from differentiating white adipocytes, we systematically identify stage- and lineage-specific coding genes, lncRNAs and microRNAs. Utilizing chromatin state maps, we also define stage- and lineage-specific enhancers, including super-enhancers, and their associated transcription factor binding motifs and genes. Through these analyses, we found that in brown adipocytes, brown lineage-specific genes are pre-marked by both H3K4me1 and H3K27me3, and the removal of H3K27me3 at the late stage is necessary but not sufficient to promote brown gene expression, while the pre-deposition of H3K4me1 plays an essential role in poising the brown genes for expression in mature brown cells. Moreover, we identify SOX13 as part of a p38 MAPK dependent transcriptional response mediating early brown cell lineage commitment. We also identify and subsequently validate PIM1, SIX1 and RREB1 as novel regulators promoting brown adipogenesis. Finally, we show that SIX1 binds to adipogenic and brown marker genes and interacts with C/EBPα, C/EBPβ and EBF2, suggesting their functional cooperation during adipogenesis., Competing Interests: The authors have declared that no competing interests exist.

Studies have showed the involvement of ubiquitin-like with PHD and RING finger domains 1 (UHRF1) in tumorigenesis and progression. This study focused on the relationships between UHRF1 and medulloblastoma (MB). Immunostaining and western blotting demonstrated differential expression of UHRF1 in MB tissues and no UHRF1 expression in normal cerebellum tissues. Univariate survival analysis revealed MB patients with high UHRF1 expression had significantly shorter OS and PFS than patients with low UHRF1 (OS p = 0.009, PFS p = 0.003). Multivariate analysis illustrated that UHRF1 expression level is an independent prognostic factor influencing the OS and PFS (OS p = 0.038, PFS p = 0.014). UHRF1 expression levels were significantly different among molecular subgroups of MB (p = 0.003). Down-regulation of UHRF1 by RNAi inhibited proliferation and clonogenic ability of MB cell lines with cell cycle arrest in G1/G2-phase. Meanwhile, cells transfected with lenti-shUHRF1 showed increased p16 expression and location shift of CDK4 in MB cells. These findings indicate UHRF1 may promote cell proliferation and be a potential biomarker that can be used as a prognostic parameter and a therapeutic target for MB.

MEK/ERK signal pathway was required for the differentiation of granulocytes, megakaryocytes and erythrocytes. Recently, MEK/ERK cascade was reported to be involved in all-trans retinoic acid (ATRA) induced differentiation in acute promyelocytic leukemia (APL) cells. However, the upstream and downstream molecules of MEK/ERK signal pathway in this cell model remains to be elucidated. In this work, we showed that RAF-1 was activated and the blockade of RAF-1 activation attenuated MEK/ERK activation as well as ATRA-induced differentiation. ATRA-enhanced protein levels of C/EBPβ, C/EBPε and PU.1, which were required for differentiation in APL cells, were suppressed by the specific inhibitor of MEK. However, MEK inhibition had no effect on the degradation of PML-RARα fusion protein or the restoration of PML nuclear bodies by ATRA treatment. Taken together, our study suggested that RAF-1/MEK/ERK cascade was involved in ATRA-induced differentiation in APL cells through enhancing the protein level of C/EBPβ, C/EBPε and PU.1., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To investigate the association between the polymorphism of miR-146a and The ubiquitin-like with PHD and ring-finger domains 1 (UHRF1) expression in patients with gastric cancer., Methods: MiR-146a rs2910164 was genotyped in 130 patients with gastric cancer and 130 cancer-free individuals using polymerase chain reaction (PCR)-restriction fragment length polymorphism. UHRF1 expression was analyzed in 22 gastric cancer tissues and their adjacent normal tissues using quantitative real-time PCR., Results: No significant differences in genotype distributions of miR-146a rs2910164 were found between cases and controls, but we observed that grade II tumors were more frequently detected in patients with CG/CC genotype compared to those with CC genotype. UHRF1 expressions in cancerous tissues were significantly higher than in noncancerous tissues (1.89-fold). Patients with CC genotype showed a significant increase in UHRF1 expression in comparison to the carriers of GG/CG genotype. A higher UHRF1 expression was associated with cancer stage IV and grade III (P<0.05)., Conclusion: The overexpression of UHRF1 was correlated with the stage and grade of gastric cancer and is associated with the genotype distribution of rs2910164., (© 2016 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

Over the last few decades, research focusing on canola (Brassica napus L.) seed oil content and composition has expanded. Oil production and accumulation are influenced by genes participating in embryo and seed development. The Arabidopsis LEAFY COTYLEDON1 (LEC1) is a well characterized regulator of embryo development that also enhances the expression of genes involved in fatty acid (FA) synthesis. B. napus lines over-expressing or down-regulating BnLEC1 were successfully generated by Agrobacterium-mediated transformation. The constitutive expression of BnLEC1 in B. napus var. Polo, increased seed oil content by 7-16%, while the down-regulation of BnLEC1 in B. napus var. Topas reduced oil content by 9-12%. Experimental manipulation of BnLEC1 caused transcriptional changes in enzymes participating in sucrose metabolism, glycolysis, and FA biosynthesis, suggesting an enhanced carbon flux towards FA biosynthesis in tissues over-expressing BnLEC1. The increase in oil content induced by BnLEC1 was not accompanied by alterations in FA composition, oil nutritional value or glucosinolate (GLS) levels. Suppression of BnLEC1 reduced seed oil accumulation and elevated the level of GLS possibly through the transcriptional regulation of BnST5a (Sulphotransferase5a), the last GLS biosynthetic enzyme. Collectively, these findings demonstrate that experimental alterations of BnLEC1 expression can be used to influence oil production and quality in B. napus., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anticancer agent possessing the ability to induce apoptosis in various cancer cells but not in non‑malignant cells. However, certain type of cancer cells are resistant to TRAIL‑induced apoptosis and some acquire resistance after the first treatment. So development of an agent that can reduce or avoid resistance in TRAIL‑induced apoptosis has garnered significant attention. The present study evaluated the anticancer potential of hispolon in TRAIL‑induced apoptosis and indicated hispolon can sensitize cancer cells to TRAIL. As the mechanism of action was examined, hispolon was found to activate caspase‑3, caspase‑8 and caspase‑9, while downregulating the expression of cell survival proteins such as cFLIP, Bcl‑2 and Bcl‑xL and upregulating the expression of Bax and truncated Bid. We also found hispolon induced death receptors in a non‑cell type‑specific manner. Upregulation of death receptors by hispolon was found to be p53-independent but linked to the induction of CAAT enhancer binding protein homologous protein (CHOP). Overall, hispolon was demonstrated to potentiate the apoptotic effects of TRAIL through downregulation of anti‑apoptotic proteins and upregulation of death receptors linked with CHOP and pERK elevation.

Mammalian Sirtuin proteins (SIRTs) are homologs of yeast Sir2, and characterized as class III histone deacetylases of NAD(+) dependence. Unlike their lower counterparts that are directly involved in the extending of lifespan, mammalian SIRTs mainly function in metabolism and cellular homeostasis, among them, SIRT7 is the least understood. SIRT7 is localized in the nucleus and rich in nucleoli associated with RNA polymerase I, and correlated with cell proliferation. In contrast, SIRT7 has recently been demonstrated to specifically deacetylate H3K18ac in the chromatin, and in most cases represses proliferation. Although MicroRNA as miR-125b has been reported to down-regulate SIRT7 by binding to its 3'UTR, however, how SIRT7 gene is regulated remains unclear. Here, we identified the transcription initiation site of human SIRT7 gene at the upstream 23rd A nucleotide respective to the translational codon, and the SIRT7 is a TATA-less and initiator-less gene. The sequences in the upstream region between -256 and -129 bp are identical with important functions in the three species detected. A C/EBPα responding element is found that binds both C/EBPα and C/EBPβ in vitro. We showed TSA induced SIRT7 gene transcription and only the HDAC3, but not its catalytic domain depleted mutant, interacted with C/EBPα to occupy the C/EBPα element and repressed SIRT7 gene in the hepatocellular carcinoma cells. To our knowledge, this is the first report on the regulation mechanism of SIRT7 gene, in which, HDAC3 collaborated with C/EBPα to occupy its responding element in the upstream region of SIRT7 gene and repressed its expression in human cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background/aims: This study aimed to examine whether UHRF-1 and p53 overexpression is a prognostic marker for gastric cancer., Patients and Methods: Sixty-four patients with gastric cancer (study group) and 23 patients with gastritis (control group) were evaluated. Immunohistochemistry was used to examine expression of UHRF-1 and p53 in gastric cancers and a control group diagnosed with gastritis., Results: The median age was 63 years (18-83 years) in the study group. UHRF-1 was positive in 15 (23%) patients with gastric cancer and fi ve (21.7%) patients with gastritis (P = 0.559). UHRF1 expression level in gastric cancer is more powerful than in gastritis (P = 0.046). Thirty-seven (61%) patients with gastric cancer and only one patient with gastritis were p53 positive (P < 0.001). After a median follow-up of 12 months (1-110), the 2-year overall survival rates were 55% and 30% in negative and positive p53, respectively (P = 0.084). Also, the 2-year overall survival rates were 45% and 53% in negative and positive UHRF-1, respectively (P = 0.132)., Conclusion: According to this study, UHRF-1 and p53 were not prognostic factors for gastric cancer, whereas they may have a diagnostic value for differentiating between gastric cancer and gastritis.

One of the major obstacles in generating induced pluripotent stem (iPS) cells suitable for therapeutic application is the low efficiency of the process and the long time required, with many iPS lines acquiring genomic aberrations. In this chapter we describe a highly efficient iPS reprogramming system based on the transient expression in pre-B cells of the transcription factor C/EBPα, followed by the induction of the four Yamanaka factors (OSKM). In addition, the process is very rapid, yielding Oct4 positive cells within 2 days and Nanog-positive iPS cell colonies within a week.

Ubiquitin-like with PHD and ring finger domains 1 (UHRF1), known as ICB90 or Np95, has been found to be overexpressed in numerous cancers. In this study, we evaluated the expression level of UHRF1 in ovarian cancer. UHRF1 levels in paired ovarian cancer tissues and adjacent normal tissues from 80 ovarian cancer patients were detected using relative quantitatively PCR and Western blot. Small interfering RNA (siRNA) was introduced in two human ovarian cancer cell lines (SKOV-3 and OVCAR-3) to downregulate the expression of UHRF1. The proliferation of siRNA-treated cells was examined using cell counting kit-8 (CCK-8) assay. The growth of these cells showed a remarkable decrease. Moreover, flow cytometric and Hoechst 33342 assays were used to detect the apoptosis. The diagnostic value of UHRF1 messenger RNA (mRNA) expression in ovarian cancer was estimated by receiver-operator characteristic (ROC) curve. The correlation between UHRF1 mRNA expression and clinicopathologic features of ovarian cancer patients was evaluated by χ (2) test. Our results demonstrated that both UHRF1 mRNA and protein were highly expressed in ovarian cancer tissues and significantly higher than that in adjacent normal tissues. Moreover, the inhibition of UHRF1 may lead to cells to undergo apoptosis. Thus, UHRF1 could act as a new oncogenic factor in ovarian cancer and be a potential molecular target for ovarian cancer gene therapy.

The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. In Caenorhabditis elegans, the JNK and p38 MAPK pathways are important for axon regeneration. Axonal injury induces expression of the svh-2 gene encoding a receptor tyrosine kinase, stimulation of which by the SVH-1 growth factor leads to activation of the JNK pathway. Here, we identify ETS-4 and CEBP-1, related to mammalian Ets and C/EBP, respectively, as transcriptional activators of svh-2 expression following axon injury. ETS-4 and CEBP-1 function downstream of the cAMP and Ca2+-p38 MAPK pathways, respectively. We show that PKA-dependent phosphorylation of ETS-4 promotes its complex formation with CEBP-1. Furthermore, activation of both cAMP and Ca2+ signaling is required for activation of svh-2 expression. Thus, the cAMP/Ca2+ signaling pathways cooperatively activate the JNK pathway, which then promotes axon regeneration.

Cx43 (connexin43) is an enhancer of the metastasis of breast cancer cells. Our previous study identified miR-381 as an indirect suppressor of Cx43 gene expression, with the precise mechanism being not understood. In the present study, using a reporter gene assay, we found that miR-381 suppressed Cx43 gene expression via the promoter region -500/-250. With site-directed gene mutation, we demonstrated that miR-381 could directly bind with the sequences CACUUGUAU in the 3'-UTR so as to inhibit C/EBPα (CCAAT/enhancer-binding protein α) expression. C/EBPα was further identified as a novel transcription factor by binding to a canonic element (AATTGTC) locating at -459/-453 in the promoter region of the Cx43 gene. Functionally, we demonstrated that miR-381 suppressed C/EBPα- and Cx43-dependent migration and invasion of breast cancer cells. Finally, we revealed that decreased levels of miR-381 as well as increased expression of C/EBPα and Cx43 in the metastatic breast cancer cells and tissues. Therefore we are the first to identify that miR-381 suppresses C/EBPα-dependent Cx43 expression in breast cancer cells. The miR-381-C/EBPα-Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer., (© 2015 Authors.)

Mammalian target of rapamycin (mTOR) is frequently upregulated in hepatocellular carcinoma (HCC). Blockage of mTOR was found to induce marked reduction in HCC growth in preclinical models. In the present study, we tested a novel mTOR inhibitor, Torin-2, for its antitumor efficacy in HCC cell lines Hep G2, SNU-182 and Hep 3B2.1-7. The HCC cell lines were cultured in vitro. These cells were treated with Torin-2. Cell apoptosis was evaluated by Annexin V staining. Cell proliferation and cell cycle progression were determined by Ki67 staining and propidium iodide staining, respectively. mTOR signaling, autophagy induction and expression of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) were assessed by western blot analysis. The UHRF1 mRNA level was determined by real-time PCR. We found that Torin-2 effectively suppressed the growth and survival of HCC cell lines, demonstrated by reduced proliferation and a high rate of apoptosis. Further study elucidated that in addition to blocking mTOR complex 1 (mTORC1)-associated cell cycle progression and induction of autophagy, Torin-2 downregulated transcription of UHRF1, an essential regulator of DNA methylation that is highly expressed in HCC cell lines. Consistently, the level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) was higher after treatment of the HCC cell lines with Torin-2. The downregulation of UHRF1 by Torin-1 was partially due to a decrease in the UHRF1 mRNA level. Torin-2 effectively inhibited HCC cell proliferation through induction of autophagy. Torin‑2-induced downregulation of UHRF1 expression may also contribute to its antitumor effect. Our research provides new clues regarding the antitumor effects of Torin-2 and sheds light on a novel therapeutic approach for HCC.

Aims: Chronic inflammation might be associated with hepatic lipid deposition independent of overnutrition. However, the mechanism is not fully understood. In this study, we investigate if impaired adipogenesis in adipose tissue is associated with hepatic lipid deposition induced by chronic inflammation in mice with chew diet., Main Methods: Casein injection in C57BL/6J mice was given every other day to induce chronic inflammation. All mice were sacrificed after 18weeks of injections. The serum, liver and adipose tissue were collected for analysis. Real-time polymerase chain reaction and western blotting were used to examine the gene and protein expressions of molecules involved in hepatic lipid metabolism and adipose adipogenesis., Key Findings: Casein injection elevated serum levels of insulin, free fatty acid (FFA) and proinflammatory factors. The gene expression of proinflammatory factors of adipose tissue and the liver also increased in the casein group as compared with the control group. Chronic inflammation up-regulated the hepatic expression of fatty acid translocase (CD36) and down-regulated microsomal triacylglycerol transfer protein (MTP), carnitine palmitoyltransferase 1a (CPT1a) and acyl-coenzyme a oxidase 1 (ACOX1). Meanwhile, chronic inflammation not only diminished the size of adipocytes, but also down-regulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding proteinα (C/EBPα), both indicating an impaired adipogenesis., Significance: Besides disturbed lipid metabolism in the liver per se, impaired adipogenesis in the adipose tissue might also be associated with hepatic lipid deposition induced by chronic inflammation in mice with chew diet., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) usually lose their stemness during in vitro expansion as they are deprived of their niche environment. Cell-extracellular matrix (ECM) interaction is known to play important roles in preserving the stemness of the cells in their stem cell niche environment. Previously, coating with bone marrow MSC (BMSC)-derived ECM was found able to maintain the differentiation potential of in vitro cultured MSCs. This study aimed to determine if this ECM coating could also maintain the stemness of cultured murine adipose-derived stem cells (ASCs) using a regular culture flask as a control. Cells were expanded in ECM-coated and ECM-noncoated flasks for two and four passages and then harvested for various analyses. The results showed that ASCs exhibited fibroblast-like spindle morphology in ECM-coated flasks, whereas ASCs gradually spread and enlarged in the ECM-noncoated flasks. After three and five passages, both groups of cells exhibited similar cytokinetics in the MSC culture medium (MesenPRO RS™ Medium). However, when cultured in Dulbecco's modified Eagles medium (DMEM) plus 10% fetal bovine serum, coating group cells exhibited more potent proliferation than control group cells with a significant difference in both passages 3 and 5 (p<0.01). When seeded at low density (500 cells/10-cm dish), coating group cells formed significantly more and larger sized cell colonies than control group cells with significant difference in cell colony numbers between two groups (p<0.05). In addition, coated colony cells were much smaller and more compactly arranged compared to control colony cells. Furthermore, ASCs expanded in coated flasks exhibited greater potentials for adipogenic, osteogenic, and chondrogenic differentiations than the cells expanded in regular flasks. Quantitatively, the Oil Red O staining area, Alizarin staining area, and Toluidine Blue staining area were all significantly larger than the respective staining areas of control cells (p<0.05). Real-time polymerase chain reaction also revealed significantly higher gene expression levels of peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte protein 2 (aP2), CCAAT/enhancer-binding protein (C/EBP), Runx2, osteocalcin, Sox9, collagen II, and aggrecan in ECM-coated group cells than in control group cells (p<0.05). Collectively, these results suggest that human BMSC decellular ECM coating helps to preserve the stemness of cultured murine ASCs.

Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) has been reported as a marker for the differential diagnosis of pancreatic cancer and chronic pancreatitis. However, the expression pattern and biological functions of UHRF1 in the progression of pancreatic cancer are not fully understood. In this study, it was found that the expression of UHRF1 was significantly up-regulated in pancreatic cancer samples compared to their adjacent normal tissues. Meanwhile, the expression of UHRF1 was inversely correlated with the survival of pancreatic cancer patients. Moreover, in the biological function studies, UHRF1 was shown to promote the growth, migration, and metastasis of pancreatic cancer cells in vitro and in vivo. Mechanistically, the expression of UHRF1 was induced by oncogenic Ras in both pancreatic cancer mouse model and cultured cells. Taken together, our study demonstrated that UHRF1 played an oncogenic role in the progression of pancreatic cancer, and UHRF1 might be a promising target for the treatment of pancreatic cancer.

Resveratrol is a non-flavonoid polyphenol which belongs to the stilbenes group and is produced naturally in several plants in response to injury or fungal attack. Resveratrol has been recently reported as preventing obesity. The present review aims to compile the evidence concerning the potential mechanisms of action which underlie the anti-obesity effects of resveratrol, obtained either in cultured cells lines and animal models. Published studies demonstrate that resveratrol has an anti-adipogenic effect. A good consensus concerning the involvement of a down-regulation of C/EBPα and PPARγ in this effect has been reached. Also, in vitro studies have demonstrated that resveratrol can increase apoptosis in mature adipocytes. Furthermore, different metabolic pathways involved in triacylglycerol metabolism in white adipose tissue have been shown to be targets for resveratrol. Both the inhibition of de novo lipogenesis and adipose tissue fatty acid uptake mediated by lipoprotein lipase play a role in explaining the reduction in body fat which resveratrol induces. As far as lipolysis is concerned, although this compound per se seems to be unable to induce lipolysis, it increases lipid mobilization stimulated by β-adrenergic agents. The increase in brown adipose tissue thermogenesis, and consequently the associated energy dissipation, can contribute to explaining the body-fat lowering effect of resveratrol. In addition to its effects on adipose tissue, resveratrol can also acts on other organs and tissues. Thus, it increases mitochondriogenesis and consequently fatty acid oxidation in skeletal muscle and liver. This effect can also contribute to the body-fat lowering effect of this molecule.

Gene expression studies of subcutaneous adipose tissue may help to better understand the mechanisms behind body fat changes in HIV-infected patients who initiate antiretroviral therapy (ART). Here, we evaluated early changes in adipose tissue gene expression and their relationship to fat changes in ART-naive HIV-infected patients randomly assigned to initiate therapy with emtricitabine/tenofovir plus efavirenz (EFV) or ritonavir-boosted lopinavir (LPV/r). Patients had abdominal subcutaneous adipose tissue biopsies at baseline and week 16 and dual-energy-X-ray absorptiometry at baseline and weeks 16 and 48. mRNA changes of 11 genes involved in adipogenesis, lipid and glucose metabolism, mitochondrial energy, and inflammation were assessed through reverse transcription-quantitative PCR (RT-qPCR). Additionally, correlations between gene expression changes and fat changes were evaluated. Fat increased preferentially in the trunk with EFV and in the limbs with LPV/r (P < 0.05). After 16 weeks of exposure to the drug regimen, transcripts of CEBP/A, ADIPOQ, GLUT4, LPL, and COXIV were significantly down-regulated in the EFV arm compared to the LPV/r arm (P < 0.05). Significant correlations were observed between LPL expression change and trunk fat change at week 16 in both arms and between CEBP/A or COXIV change and trunk fat change at the same time point only in the EFV arm and not in the LPV/r arm. When combined with emtricitabine/tenofovir as standard backbone therapy, EFV and LPV/r induced differential early expression of genes involved in adipogenesis and energy metabolism. Moreover, these mRNA expression changes correlated with trunk fat change in the EFV arm. (This was a substudy of a randomized clinical trial [LIPOTAR study] registered at ClinicalTrials.gov under identifier NCT00759070.)., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Genetic mutations in pancreatic ductal adenocarcinoma (PDAC) with critical roles have been well examined. The recent discovery of alterations in genes encoding histone modifiers suggests their possible roles in the complexity of cancer development. We previously reported loss of heterozygosity of the KDM6B gene, which encodes a histone demethylase for trimethylated histone H3 lysine 27, a repressive chromatin mark, in PDAC cells. In this study, we demonstrated that loss of KDM6B enhanced aggressiveness of PDAC cells. KDM6B has been regarded as a tumor suppressor that mediates oncogenic KRAS-induced senescence. Consistently, KDM6B was highly expressed in pancreatic precancerous lesions (pancreatic intraepithelial neoplasms); then, the expression decreased as the malignant grade progressed. We found that knockdown of KDM6B in PDAC cells promoted tumor sphere formation and increased peritoneal dissemination and liver metastasis in vivo. Microarray and chromatin immunoprecipitation analysis implicated CEBPA for aggressiveness induced by KDM6B knockdown. CEBPA knockdown recapitulated the phenotypic change of PDAC cells after KDM6B knockdown, which was reversed by forced expression of C/EBPα. Moreover, similar protein expression patterns of KDM6B and C/EBPα in human PDAC emphasized their functional correlation. Notably, pharmacological inhibition of the H3K27 methylase EZH2 in PDAC cells inhibited tumor sphere formation along with the upregulation of CEBPA expression, and this effect was impaired in KDM6B knockdown cells, highlighting the role for KDM6B in the activation of CEBPA. Together, our results propose a significant role for the KDM6B-C/EBPα axis in the PDAC phenotype., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

MicroRNAs are endogenous, conserved, and non-coding small RNAs that function as post-transcriptional regulators of fat development and adipogenesis. Adipogenic marker genes, such as CCAAT/enhancer binding protein α (Cebpa), peroxisome proliferator-activated receptor γ (Pparg), adipocyte fatty acid binding protein (Ap2), and fatty acid synthase (Fas), are regarded as the essential transcriptional regulators of preadipocyte differentiation and lipid storage in mature adipocytes. Canonical Wnt/β-catenin signaling is recognized as a negative molecular switch during adipogenesis. In the present work we found that miR-135a-5p is markedly downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-135a-5p impairs the expressions of adipogenic marker genes as well as lipid droplet accumulation and triglyceride content, indicating the importance of miR-135a-5p for adipogenic differentiation and adipogenesis. Further studies show that miR-135a-5p directly targets adenomatous polyposis coli (Apc), contributes to the translocation of β-catenin from cytoplasm to nucleus, and then activates the expressions of cyclin D1 (Ccnd1) and Cmyc, indicating the induction of canonical Wnt/β-catenin signaling. In addition, inhibition of APC with siRNA exhibits the same effects as overexpression of miR-135a-5p. Our findings demonstrate that miR-135a-5p suppresses 3T3-L1 preadipocyte differentiation and adipogenesis through the activation of canonical Wnt/β-catenin signaling by directly targeting Apc. Taken together, these results offer profound insights into the adipogenesis mechanism and the development of adipose tissue., (© 2014 Society for Endocrinology.)

Recent findings from several groups have identified distinct classes of hematopoietic stem cells (HSCs) in the bone marrow, each with inherent functional biases in terms of their differentiation, self-renewal, proliferation, and lifespan. It has previously been demonstrated that myeloid- and lymphoid-biased HSCs can be prospectively enriched based on their degree of Hoechst dye efflux. In the present study, we used differential Hoechst efflux to enrich lineage-biased HSC subtypes and analyzed their functional potentials. Despite similar outputs in vitro, bone marrow transplantation assays revealed contrasting lineage differentiation in vivo. To stratify the molecular differences underlying these contrasting functional potentials at the clonal level, single-cell gene expression analysis was performed using the Fluidigm BioMark system and revealed dynamic expression of genes including Meis1, CEBP/α, Sfpi1, and Dnmt3a. Finally, single-cell gene expression analysis was used to unravel the opposing proliferative responses of lineage-biased HSCs to the growth factor TGF-β1, revealing a potential role for the cell cycle inhibitor Cdkn1c as molecular mediator. This work lends further credence to the concept of HSC heterogeneity, and it presents unprecedented molecular resolution of the HSC response to trophic factors using single-cell gene expression analysis., (Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The effects of all-trans retinoic acid (ATRA) and valproic acid (VPA), alone and in combination, on the human acute promyelocytic leukemia (APL) cell line NB4 were investigated in view of differentiation induction and growth inhibition. After 48 h of treatment, not only ATRA but also VPA induced differentiation in NB4 cells, and their combination further augmented the differentiation activity. Furthermore, the upregulation of transcription factors including CCAAT/enhancer-binding proteins (CEBPα, β, ε) and PU.1, which are known to be critical factors for normal myelopoiesis, granulocytic maturation and being repressed in APL, concurred with the differentiation induction. A significant cell growth inhibition was observed after the treatment with VPA, which was further strengthened by the addition of ATRA. Given the importance of C/EBPs and PU.1 in myeloid development, these results, thus, suggest that restoration of the normal function of the myeloid cell transcriptional machinery is a major molecular mechanism underlying the differentiation induction in NB4. Therefore, these results may provide novel insights into a possible combinational therapeutic approach for APL patients.

The statins competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and consequently the synthesis of mevalonate. The use of statins is associated with insulin resistance, presumably due to the impaired differentiation and diminished glucose utilization of adipocytes. We hypothesize that mevalonate is essential to adipocyte differentiation and adipogenic gene expression. Adipo-Red assay and Oil Red O staining showed that an eight-day incubation with 0-2.5 µmol/L lovastatin dose-dependently reduced the intracellular triglyceride content of murine 3T3-F442A adipocytes. Concomitantly, lovastatin downregulated the expression of peroxisome proliferator-activated receptor γ (Pparγ), leptin (Lep), fatty acid binding protein 4 (Fabp4), and adiponectin (AdipoQ) as measured by quantitative real-time polymerase chain reaction (real-time qPCR). The expression of sterol regulatory element binding protein 1 (Srebp-1), a transcriptional regulator of Pparγ and Lep genes, was also suppressed by lovastatin. Western-blot showed that lovastatin reduced the level of CCAAT/enhancer binding protein α (C/EBPα) while inducing a compensatory over-expression of HMG CoA reductase. The impact of lovastatin on intracellular triglyceride content and expression of the adipogenic genes was reversed by supplemental mevalonate. Mevalonate-derived metabolites have essential roles in promoting adipogenic gene expression and adipocyte differentiation.

In the seed, a fundamental transition between embryo and vegetative phases of plant development is coordinated by the interaction between the AFL and VAL sub-clades of the plant specific B3 domain transcription factor family. The AFL B3 factors together with LEC1-type HAP3 transcription factors promote embryo maturation; whereas the VAL B3 factors repress the LEC1/AFL (LAFL) network during seed germination. Recent advances reveal that genes in key developmental programs and hormone signaling pathways are downstream targets of the LAFL network highlighting the central role of the LAFL network in integration of intrinsic developmental and hormonal signals during plant development. The VAL B3 proteins are proposed to mediate repression by recruiting a histone deacetylase complex (HDAC) to LAFL genes that contain the Sph/RY cis-element recognized by AFL and VAL B3-DNA-binding domains. In addition to VAL B3 factors, epigenetic mechanisms are implicated in maintaining repression of LAFL network during vegetative development., (© 2013 Wiley Periodicals, Inc.)

Δ(9)-Tetrahydrocannabinol (THC), the major bioactive component of marijuana, has been shown to induce functional myeloid-derived suppressor cells (MDSCs) in vivo. Here, we studied the involvement of microRNA (miRNA) in this process. CD11b(+)Gr-1(+) MDSCs were purified from peritoneal exudates of mice administered with THC and used for genome-wide miRNA profiling. Expression of CD31 and Ki-67 confirmed that the THC-MDSCs were immature and proliferating. THC-induced MDSCs exhibited distinct miRNA expression signature relative to various myeloid cells and BM precursors. We identified 13 differentially expressed (>2-fold) miRNA in THC-MDSCs relative to control BM precursors. In silico target prediction for these miRNA and pathway analysis using multiple bioinformatics tools revealed significant overrepresentation of Gene Ontology clusters within hematopoiesis, myeloid cell differentiation, and regulation categories. Insulin-like growth factor 1 signaling involved in cell growth and proliferation, and myeloid differentiation pathways were among the most significantly enriched canonical pathways. Among the differentially expressed, miRNA-690 was highly overexpressed in THC-MDSCs (∼16-fold). Transcription factor CCAAT/enhancer-binding protein α (C/EBPα) was identified as a potential functional target of miR-690. Supporting this, C/EBPα expression was attenuated in THC-MDSCs as compared with BM precursors and exhibited an inverse relation with miR-690. miR-690 knockdown using peptide nucleic acid-antagomiR was able to unblock and significantly increase C/EBPα expression establishing the functional link. Further, CD11b(+)Ly6G(+)Ly6C(+) and CD11b(+)Ly6G(-)Ly6C(+) purified subtypes showed high levels of miR-690 with attenuated C/EBPα expression. Moreover, EL-4 tumor-elicited MDSCs showed increased miR-690 expression. In conclusion, miRNA are significantly altered during the generation of functional MDSC from BM. Select miRNA such as miR-690 targeting genes involved in myeloid expansion and differentiation likely play crucial roles in this process and therefore in cannabinoid-induced immunosuppression.

The ubiquitin-like with PHD and ring finger domains 1 (UHRF1) has been reported to be essential for cell proliferation and plays a critical role in the development and progression of many human carcinomas. However, its clinical and prognostic significance in laryngeal squamous cell carcinoma (LSCC) remains unclear. In the current study, 60 patients with LSCC were studied. UHRF1 expression at mRNA and protein levels was detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunohistochemistry, respectively, in both tissues of LSCC and corresponding adjacent normal larynx tissues. Statistical analyses were conducted to test the correlations between UHRF1 expression, clinicopathological parameters, and prognosis. qRT-PCR showed that the expression of UHRF1 mRNA in LSCC tissues was significantly higher than that in adjacent normal larynx tissues (P < 0.001). By immunohistochemistry, overexpression of UHRF1 protein was found in 78.3% (47/60) of the LSCC tissues, while there was negative expression in adjacent normal larynx tissues. Furthermore, increased expression of UHRF1 had remarkably positive relationships with smoking (P < 0.001), advanced T stage (P = 0.005) and clinical stage (P = 0.044), poor histological differentiation (P = 0.048), while there was no correlations between UHRF1, and sex, age and lymph node metastasis (P > 0.05). Overexpression of UHRF1 was also associated with worse overall survival examined by Kaplan-Meier method (P = 0.036). Multivariate analysis showed that increased expression of UHRF1 was an independent prognostic factor for LSCC (P = 0.013). Therefore, overexpression of UHRF1 may play an important role in the development and progression of LSCC, and UHRF1 might be a useful biomarker for the prognosis of LSCC.

In response to aggravation by activated microglia, IL-13 can significantly enhance ER stress induction, apoptosis, and death via reciprocal signaling through CCAAT/enhancer-binding protein alpha (C/EBP-α) and C/EBP-beta (C/EBP-β). This reciprocal signaling promotes neuronal survival. Since the induction of cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptor gamma/heme oxygenase 1 (PPAR-γ/HO-1) by IL-13 plays a crucial role in the promotion of and protection from activated microglia, respectively; here, we investigated the role of IL-13 in regulating C/EBPs in activated microglia and determined its correlation with neuronal function. The results revealed that IL-13 significantly enhanced C/EBP-α/COX-2 expression and PGE2 production in LPS-treated microglial cells. Paradoxically, IL-13 abolished C/EBP-β/PPAR-γ/HO-1 expression. IL-13 also enhanced ER stress-evoked calpain activation by promoting the association of C/EBP-β and PPAR-γ. SiRNA-C/EBP-α effectively reversed the combined LPS-activated caspase-12 activation and IL-13-induced apoptosis. In contrast, siRNA-C/EBP-β partially increased microglial cell apoptosis. By NeuN immunochemistry and CD11b staining, there was improvement in the loss of CA3 neuronal cells after intrahippocampal injection of IL-13. This suggests that IL-13-enhanced PLA2 activity regulates COX-2/PGE2 expression through C/EBP-α activation. In parallel, ER stress-related calpain downregulates the PPAR-γ/HO-1 pathway via C/EBP-β and leads to aggravated death of activated microglia via IL-13, thereby preventing cerebral inflammation and neuronal injury., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We previously revealed that endogenous bone morphogenetic protein (Bmp) activity is required for lipid accumulation in 3T3-L1 adipocytes. The present study characterized the role of endogenous Bmp activity in preadipocytes. Endogenous Bmp activity was monitored by analyzing the level of phosphorylation of Smad1/5/8, downstream molecules in the Bmp pathway. Higher levels of phosphorylated Smad1/5/8 were detected in adipogenic cells but not in non-adipogenic cells prior to differentiation induction. The inhibition of the Bmp pathway during this period decreased the expression of Pparγ2 and C/ebpα, which are transcription factors responsible for adipocyte differentiation. The expression of these transcription factors were also down-regulated by Bmp4 knockdown. In addition, endogenous Bmp4 was required for the repression of Intrleukin-11 expression. Endogenous Bmp4 in preadipocytes is indispensable for the onset of the adipogenic program, and may help to maintain the preadipocytic state during adipocyte differentiation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

β-Arrestins were identified as scaffold-proteins that have the capacity to desensitize G protein-coupled receptors. However, it has been found that β-arrestins activate signaling pathways independent of G protein activation. The diversity of these signaling pathways has also been recognized for receptor tyrosine kinase. The aim of the present study was to validate the β-arrestin-dependent signaling mechanism(s) responsible for regulation of adipogenesis. Two signal models were selected, ghrelin and insulin, based on its β-arrestin-associated Akt activity. Herein, we found that β-arrestin 1 and 2 were essential molecules for adipocyte differentiation. More specifically, the role of these scaffolding proteins was demonstrated by depletion of β-arrestin 1 and 2 during ghrelin-induced adipogenesis in 3T3-L1 cells, which decreased the adipocyte differentiation and the expression levels of master regulators of early, the CCAAT/enhancer-binding protein β (C/EBPβ) and the CCAAT/enhancer-binding protein δ (C/EBPδ), and terminal, the peroxisome proliferator-activated receptor (PPARγ) and the CCAAT/enhancer-binding protein α (C/EBPα), adipogenesis. Accordingly ghrelin-induced Akt activity and its downstream targets, the mammalian target of rapamycin complex 1 (mTORC1) and the ribosomal protein S6 kinase beta-1 (S6K1), were inhibited by β-arrestin 1 and 2 siRNAs. By contrast, assays performed during insulin-activated adipogenesis showed an intensifying effect on the adipocyte differentiation as well as on the expression of C/EBPβ, C/EBPδ, PPARγ and C/EBPα. The increase in insulin-induced adipogenesis by β-arrestin knock-down was concomitant to a decrease in the insulin receptor susbtrate-1 (IRS-1) serine phosphorylation, proving the loss of the negative feedback loop on IRS-1/phosphoinositide 3-kinase (PI3K)/Akt. Therefore, β-arrestins control the extent and intensity of the lipogenic and adipogenic factors associated to Akt signaling, although the mechanistic and functional principles that underlie the connection between signaling and β-arrestins are specifically associated to each receptor type., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The non-invasive treatment of tumors with preserved fertility holds great promise. The application of pulsed electric field (PEF) is a new biomedical engineering technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely; however, research of the biological effects of psPEF on cells is limited. Electric theory predicts that when the pulse duration decreases to nanoseconds and picoseconds, it will mainly affect organelles and lead to intracellular electromanipulations. Previous studies have shown that psPEF targets the mitochondria and induces apoptosis through a mitochondrial-mediated pathway in HeLa cells. The endoplasmic reticulum is also involved in the intrinsic pathways of apoptosis. In the present study, HeLa cells were exposed to psPEF to investigate the underlying mechanisms of apoptosis. MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. Treatment with psPEF led to marked cell apoptosis and cell cycle arrest at the G2/M phase. In addition, psPEF affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). These changes were accompanied by the elevation of intracellular Ca2+ concentrations. Furthermore, the activation of caspase-12, -9 and -3, led to the release of cytochrome c, as well as the upregulation of Bax and the downregulation of Bcl-2, as observed in the HeLa cells. Taken together, these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

Obesity, inflammation, and insulin resistance are closely linked. Substance P (SP), via its neurokinin 1 receptor (NK1R), mediates inflammatory and, possibly, neuroendocrine processes. We examined SP effects on lipid storage and cytokine production in 3T3-L1 adipocytes and adipose tissues. 3T3-L1 adipocytes and preadipocytes express NK1R, and 8 days of SP supplementation during differentiation to 3T3-L1 preadipocytes decreased lipid droplet accumulation. SP (10 nM, 24 h) increased lipolysis in primary adipocytes (138 ± 7%, P < 0.05) and reduced fatty acid uptake (-31 ± 7%, P < 0.05) and mRNA expression of the differentiation-related transcription factors peroxisome proliferator-activated receptor-γ type 2 (-64 ± 2%, P < 0.001) and CCAAT enhancer-binding protein (CEBP)-α (-65 ± 2%, P < 0.001) and the lipid storage genes fatty acid-binding protein type 4 (-59 ± 2%, P < 0.001) and diacylglycerol O-acyltransferase-1 (-45 ± 2%, P < 0.01) in 3T3-L1 adipocytes, while CD36, a fatty acid transporter (+82 ± 19%, P < 0.01), was augmented. SP increased secretion of complement C3 (148 ± 15%, P < 0.04), monocyte chemoattractant protein-1 (156 ± 16%, P < 0.03), and keratinocyte-derived chemokine (148 ± 18%, P = 0.045) in 3T3-L1 adipocytes and monocyte chemoattractant protein-1 (496 ± 142%, P < 0.02) and complement C3 (152 ± 25%, P < 0.04) in adipose tissue and primary adipocytes, respectively. These SP effects were accompanied by downregulation of insulin receptor substrate 1 (-82 ± 2%, P < 0.01) and GLUT4 (-76 ± 2%, P < 0.01) mRNA expression, and SP acutely blocked insulin-mediated stimulation of fatty acid uptake and Akt phosphorylation. Although adiponectin secretion was unchanged, mRNA expression was decreased (-86 ± 8%, P < 0.001). In humans, NK1R expression correlates positively with plasma insulin, fatty acid, and complement C3 and negatively with adiponectin, CEBPα, CEBPβ, and peroxisome proliferator-activated receptor-γ mRNA expression in omental, but not subcutaneous, adipose tissue. Our results suggest that, beyond its neuroendocrine and inflammatory effects, SP could also be involved in targeting adipose tissue and influencing insulin resistance.

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.

Ubiquitin-like with PHD and ring-finger domain 1 (UHRF1) binds to methylated promoters of a number of tumor-suppressor genes, including p16INK4A and p14ARF, by forming complexes with DNA methyltransferases and HDAC1, resulting in the induction of carcinogenesis. Altered UHRF1 expression has been demonstrated in various types of cancers. Previous reports indicate that UHRF1 expression is regulated by E2F-1 expression. We investigated UHRF1 expression using immunohistochemical staining in 231 colorectal cancer and 40 adenoma specimens, analyzed the relationship between UHRF1 expression and clinicopathological findings and the association between UHRF1 and E2F-1 expression. To better understand the biological function of UHRF1 in colorectal cancer, knockdown of UHRF1 expression was performed using siRNA methods. High UHRF1 expression was observed in 152 of 231 (65.8%) colorectal cancer patients, and was detected in 35 of 40 adenoma specimens samples (87.5%). UHRF1 staining was detected in the nucleus of cancer cells, while it was not detected in colonic normal mucosa. High UHRF1 expression was significantly observed in right compared with left hemicolon cancer (p=0.008). Moreover, high UHRF1 expression tended to be associated with depth of invasion (p=0.051). UHRF1 expression was significantly associated with E2F-1 expression (p<0.0001). Knockdown of UHRF1 expression suppressed cellular growth in colon cancer cell lines, HCT116 and SW620. In conclusion, we demonstrated that UHRF1 expression was upregulated in approximately two-thirds of colorectal cancer specimens and was particularly expressed in right compared with left hemicolon cancer. Moreover, knockdown of UHRF1 expression induced growth inhibition in colon cancer cell lines. UHRF1 may be involved in cellular proliferation and molecular pathogenesis of colorectal cancer in the right hemicolon.

Background: In physiological conditions, it is postulated that neurons control microglial reactivity through a series of inhibitory mechanisms, involving either cell contact-dependent, soluble-factor-dependent or neurotransmitter-associated pathways. In the current study, we focus on CD200R1, a microglial receptor involved in one of these cell contact-dependent mechanisms. CD200R1 activation by its ligand, CD200 (mainly expressed by neurons in the central nervous system),is postulated to inhibit the pro-inflammatory phenotype of microglial cells, while alterations in CD200-CD200R1 signalling potentiate this phenotype. Little is known about the regulation of CD200R1 expression in microglia or possible alterations in the presence of pro-inflammatory stimuli., Methods: Murine primary microglial cultures, mixed glial cultures from wild-type and CCAAT/enhancer binding protein β (C/EBPβ)-deficient mice, and the BV2 murine cell line overexpressing C/EBPβ were used to study the involvement of C/EBPβ transcription factor in the regulation of CD200R1 expression in response to a proinflammatory stimulus (lipopolysaccharide (LPS)). Binding of C/EBPβ to the CD200R1 promoter was determined by quantitative chromatin immunoprecipitation (qChIP). The involvement of histone deacetylase 1 in the control of CD200R1 expression by C/EBPβ was also determined by co-immunoprecipitation and qChIP., Results: LPS treatment induced a decrease in CD200R1 mRNA and protein expression in microglial cells, an effect that was not observed in the absence of C/EBPβ. C/EBPβ overexpression in BV2 cells resulted in a decrease in basal CD200R1 mRNA and protein expression. In addition, C/EBPβ binding to the CD200R1 promoter was observed in LPS-treated but not in control glial cells, and also in control BV2 cells overexpressing C/EBPβ. Finally, we observed that histone deacetylase 1 co-immunoprecipitated with C/EBPβ and showed binding to a C/EBPβ consensus sequence of the CD200R1 promoter in LPS-treated glial cells. Moreover, histone deacetylase 1 inhibitors reversed the decrease in CD200R1 expression induced by LPS treatment., Conclusions: CD200R1 expression decreases in microglial cells in the presence of a pro-inflammatory stimulus, an effect that is regulated, at least in part, by C/EBPβ. Histone deacetylase 1 may mediate C/EBPβ inhibition of CD200R1 expression, through a direct effect on C/EBPβ transcriptional activity and/or on chromatin structure.

We investigated the expression and promoter methylation of dbpA in human hepatocellular carcinoma (HCC) and examined their correlation with clinicopathological features. In 96 paired samples of HCC and adjacent non-tumorous liver, and 10 normal liver specimens, dbpA mRNA was quantified by real-time RT-PCR, and promoter methylation was examined by methylation-specific polymerase chain reaction and bisulfite sequencing. The results showed that dbpA mRNA expression levels were higher in HCC compared to corresponding non-tumor tissues (P<0.01) and higher in non-virus-associated HCC compared to virus-associated cases (P<0.01). dbpA promoter was methylated in 37.7% of HCC samples and the promoter methylation was significantly correlated with the low expression of dbpA in non-virus-associated HCC (P<0.01), but not in virus-associated HCC. Surprisingly, poor prognosis was more significantly associated with high dbpA expression in non-tumorous liver (P=0.018) but not with that in HCC. Non-tumorous tissues consist of chronic hepatitis or liver cirrhosis, and these conditions are the background of hepatocarcinogenesis, defined as the hypercarcinogenic state. Our results suggest that the high expression of dbpA in the hypercarcinogenic state is an indicator of poor prognosis.

Impairment of redox homeostasis has been extensively associated with obesity, as a consequence of the chronic inflammatory state present in overweight subjects. Deregulation of glutathione (GSH), the most important non-enzymatic intracellular anti-oxidant, induces insulin resistance in mature adipocytes, but data are lacking about its effects on adipogenesis. In this report we demonstrate that during adipogenesis of 3T3-L1 cells the GSH/GSSG ratio decreases, shifting redox status towards oxidizing conditions. Moreover, we demonstrate that inhibition of GSH synthesis, obtained by treatment with L-buthionine-sulfoximine (BSO), enhances C/EBPβ LAP/LIP ratio and PPARγ expression during mitotic clonal expansion (MCE) stimulating adipogenesis. On the contrary, GSH ethyl ester (GSHest) supplementation completely abrogates this process also in the presence of BSO. GSH decrement during the first 24 h of adipogenesis is sufficient to induce higher triglyceride accumulation in differentiated adipocytes with respect to control, whereas GSHest treatment inhibits lipid droplets formation. We further demonstrate that Resveratrol (RV) could exert anti-adipogenic properties also by increasing GSH content through γ-glutamyl-cysteine ligase (GCL) induction. Overall data indicate that in pre-adipocytes the decrease of GSH accelerates adipogenesis, suggesting that the use of agents able to maintain GSH redox status in adipose tissue, such as RV, could be promising in stopping the lipogenic loop of obesity., (Copyright © 2010 Wiley-Liss, Inc.)

Previously, we had reported that overexpression of miR-23a~27a~24-2 cluster induces caspase-dependent and -independent apoptosis via JNK in human embryonic kidney (HEK293T) cells. Herein, we describe the molecular mechanism(s) responsible for miR-23a~27a~24-2 cluster induced apoptosis. Gene expression profiling was used to characterize the transcriptional response to miR-23a~27a~24-2 cluster overexpression in HEK293T cells. The microarray analysis gave 1,025 differentially expressed genes and analysis of the gene expression data with Ingenuity Pathway Analysis (IPA) software revealed p53 signaling, oxidative stress response and mitochondrial dysfunction among the top processes being affected. This data substantiates our previous study where we had reported increase of ROS and the release of proapoptotic factors such as cytochrome c (cyt c) and apoptosis-inducing factor (AIF) from the mitochondria to cytosol. Additionally, components of ER stress-mediated apoptosis pathway i.e., C/EBP homologous protein (CHOP/DDIT3/GADD153) and TRIB3, an Akt inhibitor were found to be significantly enriched. Also, the enhanced expression of ATF3 and ATF4 was observed at RNA as well as protein level in miR-23a~27a~24-2 cluster overexpressed HEK293T cells. Induction of BIM appeared to be specific, because ER stress caused only a minor change in the expression of the related BH3-only proteins BID or PUMA. The fact that miR-23a~27a~24-2 cluster triggered endoplasmic reticulum stress (ER stress) was further established by the increase in cytosolic calcium levels after overexpression of this cluster in HEK293T cells. These findings were also supported by PANTHER analysis wherein biological process categories of apoptosis and stress response were enriched. Taken together, this work underlines the role of ER stress in miR-23a~27a~24-2 cluster mediated apoptosis in HEK293T cells. Since the detailed knowledge of this cluster induced apoptosis has now been elucidated, the in vivo study of this cluster would help evaluate the prospect of its use as a therapeutic in diseases known to occur because of deregulation of apoptosis.