Immune Precipitation Assays Research Articles

Kappa Opioid Receptor Activation Induces Epigenetic Silencing of Brain-Derived Neurotropic Factor via HDAC5 in Depression.

Treatment-resistant depression (TRD) occurs in almost 50% of the depressed patients. Central kappa opioid receptor (KOR) agonism has been demonstrated to induce depression and anxiety, while KOR antagonism alleviates depression-like symptoms in rodent models and TRD in clinical studies. Previously, we have shown that sustained KOR activation leads to a TRD-like phenotype in mice, and modulation of brain-derived neurotrophic factor (BDNF) expression in the prefrontal cortex (PFC) appears to be one of the molecular determinants of the antidepressant response. In the present study, we observed that sustained KOR activation by a selective agonist, U50488, selectively reduced the levels of Bdnf transcripts II, IV, and Bdnf CDS (protein-coding Exon IX) in the PFC and cultured primary cortical neurons, which was blocked by selective KOR antagonist, norbinaltorphimine. Considering the crucial role of epigenetic pathways in BDNF expression, we further investigated the role of various epigenetic markers in KOR-induced BDNF downregulation in mice. We observed that treatment with U50488 resulted in selective and specific downregulation of acetylation at the ninth lysine residue of the histone H3 protein (H3K9ac) and upregulation of histone deacetylase 5 (HDAC5) expression in the PFC. Further, using anti-H3K9ac and anti-HDAC5 antibodies in the chromatin immune precipitation assay, we detected decreased enrichment of H3K9ac and increased HDAC5 binding at Bdnf II and IV transcripts after U50488 treatment, which were blocked by a selective KOR antagonist, norbinaltorphimine. Further mechanistic studies using HDAC5 selective inhibitor, LMK235, in primary cortical neurons and adeno-associated viral shRNA-mediated HDAC5-knockdown in the PFC of mice demonstrated an essential role of HDAC5 in KOR-mediated reduction of Bdnf expression in the PFC and in depression-like symptoms in mice. These results suggest that KOR engages multiple pathways to induce depression-like symptoms in mice and provide novel insights into the mechanisms by which activation of KOR regulates major depressive disorders.

Integrated genomic and proteomic analyses identify PYGL as a novel experimental therapeutic target for clear cell renal cell carcinoma

Sunitinib, the first-line targeted therapy for metastatic clear cell renal cell carcinoma (ccRCC), faces a significant challenge as most patients develop acquired resistance. Integrated genomic and proteomic analyses identified PYGL as a novel therapeutic target for ccRCC. PYGL knockdown inhibited cell proliferation, cloning capacity, migration, invasion, and tumorigenesis in ccRCC cell lines. PYGL expression was increased in sunitinib-resistant ccRCC cell lines, and CP-91149 targeting the PYGL could restore drug sensitivity in these cell lines. Moreover, chromatin immune-precipitation assays revealed that PYGL upregulation is induced by the transcription factor, hypoxia-inducible factor 1α. Overall, PYGL was identified as a novel diagnostic biomarker by combining genomic and proteomic approaches in ccRCC, and sunitinib resistance to ccRCC may be overcome by targeting PYGL.

M6A‑modified HOXC10 promotes HNSCC progression via co‑activation of ADAM17/EGFR and Wnt/β‑catenin signaling.

The homeobox (HOX) gene family plays a fundamental role in carcinogenesis. However, the oncogenic mechanism of HOXC10 in head and neck squamous cell carcinoma (HNSCC) remains unclear. In the present study, it was revealed that HOXC10 expression was significantly higher in HNSCC tissues than in adjacent tissues, and a high level of HOXC10 was closely associated with worse clinical outcomes. HOXC10 overexpression promoted HNSCC cell proliferation, migration, and invasion, both invitro and invivo. Mechanistically, chromatin immunoprecipitation sequencing revealed that HOXC10 drove the transcriptional activation of a disintegrin and metalloproteinase17 (ADAM17), and the ADAM17/epidermal growth factor receptor (EGFR)/ERK1/2 signaling pathway facilitating the proliferation of HNSCC. Furthermore, mass spectrometric analysis indicated that HOXC10 interacted with ribosomal proteinS15A (RPS15A) and enhanced RPS15A protein expression, activating the Wnt/β‑catenin pathway and contributing to invasion and metastasis of HNSCC. Additionally, the methylated RNA immune precipitation and RNA antisense purification assays showed that N6‑methyladenosine (m6A) writer, methyltransferase‑like 3, catalyzed m6A modification of the HOXC10 transcript, m6A reader insulin like growth factor2 mRNA binding protein (IGF2BP)1 and IGF2BP3 involved in recognizing and stabilizing m6A‑tagged HOXC10 mRNA. In summary, the present study identified HOXC10 as a promising candidate oncogene in HNSCC. The m6A modification‑mediated HOXC10 promoted proliferation, migration, and invasion of HNSCC through co‑activation of ADAM17/EGFR and Wnt/β‑catenin signaling, providing a novel diagnostic and prognostic biomarker and a potential therapeutic target for HNSCC.

SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters.

During skeletal myogenesis, the zinc-finger transcription factors SNAI1 and SNAI2, are expressed in proliferating myoblasts and regulate the transition to terminally differentiated myotubes while repressing pro-differentiation genes. Here, we demonstrate that SNAI1 is upregulated in vivo during the early phase of muscle regeneration induced by bupivacaine injury. Using shRNA-mediated gene silencing in C2C12 myoblasts and whole-transcriptome microarray analysis, we identified a collection of genes belonging to the endoplasmic reticulum (ER) stress pathway whose expression, induced by myogenic differentiation, was upregulated in absence of SNAI1. Among these, key ER stress genes, such as Atf3, Ddit3/Chop, Hspa5/Bip, and Fgf21, a myokine involved in muscle differentiation, were strongly upregulated. Furthermore, by promoter mutant analysis and Chromatin immune precipitation assay, we demonstrated that SNAI1 represses Fgf21 and Atf3 in proliferating myoblasts by directly binding to multiple E boxes in their respective promoter regions. Together, these data describe a new regulatory mechanism of myogenic differentiation involving the direct repressive action of SNAI1 on ER stress and Fgf21 expression, ultimately contributing to maintaining the proliferative and undifferentiated state of myoblasts.

Inhibition of histone demethylase KDM4 by ML324 induces apoptosis through the unfolded protein response and Bim upregulation in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is an extremely aggressive malignancy that ranks as the sixth-leading cause of cancer-associated death worldwide. Recently, various epigenetic mechanisms including gene methylation were reported to be potential next era HCC therapeutics and biomarkers. Although inhibition of epigenetic enzymes including histone lysine demethylase 4 (KDM4) enhanced cell death in HCC cells, the detailed mechanism of cell death machinery is poorly understood. In this study, we found that ML324, a small molecule KDM4-specific inhibitor, induced the death of HCC cells in a general cell culture system and 3D spheroid culture with increased cleavage of caspase-3. Mechanistically, we identified that unfolded protein responses (UPR) were involved in ML324-induced HCC cell death. Incubation of HCC cells with ML324 upregulated death receptor 5 (DR5) expression through the activation transcription factor 3 (ATF3)-C/EBP homologous protein (CHOP)-dependent pathway. Moreover, we identified BIM protein as a mediator of ML324-induced apoptosis using CRISPR/Cas9 knockout analysis. We showed that the loss of Bim suppressed ML324-induced apoptosis by flow cytometry analysis, colony formation assay, and caspase-3 activation assay. Interestingly, BIM protein expression by ML324 was regulated by ATF3, CHOP, and DR5 which are factors involved in UPR. Specifically, we confirmed the regulating roles of KDM4E in Bim and CHOP expression using a chromatin immune precipitation (ChIP) assay. Physical binding of KDM4E to Bim and CHOP promoters decreased the response to ML324. Our findings suggest that KDM4 inhibition is a potent anti-tumor therapeutic strategy for human HCC, and further studies of UPR-induced apoptosis and the associated epigenetic functional mechanisms may lead to the discovery of novel target for future cancer therapy.

LncRNA PCAT1 might coordinate ZNF217 to promote CRC adhesion and invasion through regulating MTA2/MTA3/Snai1/E-cadherin signaling.

LncRNA prostate cancer-associated transcript 1 (PCAT1) is a well-known oncogene, but the mechanisms of exosomes PCAT1 in colorectal cancer (CRC) remain largely unknown. Thus, the mechanisms of exosomes lncRNA PCAT1 were investigated. The expressions of exosomes lncRNA PCAT1 in tissues from stage 0-I and stage II-III CRC patients, and intestinal epithelial cell line FHC and two CRC cell lines, HT29 and HCT8 were measured by real-time quantitative PCR. The effects of lncRNA PCAT1 on adhesion and invasion of two CRC cell lines were investigated by cell-matrix adhesion and transwell assays. In addition, the target of PCAT1 (ZNF217) was validated using an RNA immune precipitation assay. Finally, the protein levels of MTA2, MTA3, SNAI1, and E-cadherin in normal participants, stage 0-I and stage II-III CRC patients, as well as two cell lines with stable ZNF217 knockdown were investigated by western blotting. The plasma exosomal lncRNA PCAT1 was found to be significantly increased in the CRC tissues and cell lines. In addition, lncRNA PCAT1 knockdown significantly inhibited the adhesion and invasion of HT29 and HCT8 cells. RIP assay results showed lncRNA PCAT1 could target ZNF217, and downregulation of lncRNA PCAT1 could decrease the protein expressions of ZNF217 in two CRC cells lines. Moreover, ZNF217 knockdown significantly decreased MTA2, MTA3, and SNAI1 expressions, but increased E-cadherin expressions in both CRC cells lines. Exosomal lncRNA PCAT1 can promote the adhesion and invasion of CRC cells, and PCAT1 overexpression may lead to ZNF217 upregulation that regulates EMT-related MTA2/MTA3/Snai1/E-cadherin signaling.

Drug resistance to nelarabine in leukemia cell lines might be caused by reduced expression of deoxycytidine kinase through epigenetic mechanisms.

Drug resistance is a serious problem in leukemia therapy. A novel purine nucleoside analogue, nelarabine, is available for the treatment of children with T cell acute lymphoblastic leukemia. We investigated the mechanisms of drug resistance to nelarabine. Nelarabine-resistant cells were selected by stepwise and continuous exposure to nelarabine using the limiting dilution method in human B and T cell lymphoblastic leukemia cell lines. Expression analysis was performed using real-time polymerase chain reaction, and epigenetic analysis was performed using methylation-specific polymerase chain reaction and chromatin immunoprecipitation. The RNA expression level for deoxycytidine kinase (dCK) was decreased in nelarabine-resistant leukemia cells. There were no differences between the parental and nelarabine-resistant leukemia cells in the methylation status of the promoter region of the dCK gene. In the chromatin immune precipitation assay, decreased acetylation of histones H3 and H4 bound to the dCK promoter was seen in the nelarabine-resistant cells when compared to the parental cells. Furthermore, treatment with a novel histone deacetylase inhibitor, vorinostat, promoted the cytotoxic effect of nelarabine along with increased expression of the dCK gene, and it increased acetylation of both histones H3 and H4 bound to the dCK promoter in nelarabine-resistant leukemia cells. The combination index showed that the effect of nelarabine and vorinostat was synergistic. This study reports that nelarabine with vorinostat can promote cytotoxicity in nelarabine-resistant leukemia cells through epigenetic mechanisms.

To measure musk ABS in ACHR AB negative sera: RIPA is more specific, and SPR is more sensitive

Muscle-specific kinase (MuSK) myasthenia gravis (MG) is often a more severe form of MG that presents with a rapid onset and prominent bulbar symptoms. Today, the diagnosis of MuSK MG is heavily reliant on the antibody (Ab) status of the patient. In most laboratories worldwide, the Abs are measured by radio immune precipitation assay (RIPA). We have developed another testing platform using surface plasmon resonance (SPR) technology. Our aim was to measure the clinical sensitivity and specificity for two different MuSK Ab assays.

FKBP51 Affects TNF-Related Apoptosis Inducing Ligand Response in Melanoma.

Melanoma is one of the most immunogenic tumors and has the highest potential to elicit specific adaptive antitumor immune responses. Immune cells induce apoptosis of cancer cells either by soluble factors or by triggering cell-death pathways. Melanoma cells exploit multiple mechanisms to escape immune system tumoricidal control. FKBP51 is a relevant pro-oncogenic factor of melanoma cells supporting NF-κB-mediated resistance and cancer stemness/invasion epigenetic programs. Herein, we show that FKBP51-silencing increases TNF-related apoptosis-inducing ligand (TRAIL)-R2 (DR5) expression and sensitizes melanoma cells to TRAIL-induced apoptosis. Consistent with the general increase in histone deacetylases, as by the proteomic profile, the immune precipitation assay showed decreased acetyl-Yin Yang 1 (YY1) after FKBP51 depletion, suggesting an impaired repressor activity of this transcription factor. ChIP assay supported this hypothesis. Compared with non-silenced cells, a reduced acetyl-YY1 was found on the DR5 promoter, resulting in increased DR5 transcript levels. Using Crispr/Cas9 knockout (KO) melanoma cells, we confirmed the negative regulation of DR5 by FKBP51. We also show that KO cells displayed reduced levels of acetyl-EP300 responsible for YY1 acetylation, along with reduced acetyl-YY1. Reconstituting FKBP51 levels contrasted the effects of KO on DR5, acetyl-YY1, and acetyl-EP300 levels. In conclusion, our finding shows that FKBP51 reduces DR5 expression at the transcriptional level by promoting YY1 repressor activity. Our study supports the conclusion that targeting FKBP51 increases the expression level of DR5 and sensitivity to TRAIL-induced cell death, which can improve the tumoricidal action of immune cells.

Curcumin Alleviates Aβ42-Induced Neuronal Metabolic Dysfunction via the Thrb/SIRT3 Axis and Improves Cognition in APPTG Mice.

Curcumin has been reported to have a therapeutic effect on Alzheimer's disease (AD), but the specific mechanism remains to be elucidated. In the present research, we aimed to investigate the effect and molecular mechanism of curcumin on AD. Mouse primary hippocampal neuron cells were treated with various concentrations of beta-amyloid 42 (Aβ42) and the results found that Aβ42 inhibited cell viability in a dose-dependent manner. Compared with 50ng/mL Aβ42, 500ng/mL Aβ42 could further promote cell apoptosis, reduce the ratio of Nicotinamide adenine dinucleotide (NAD(+))/Nicotinamide adenine diphosphate hydride (NADH) and Adenosine 5'-triphosphate (ATP) level, and inhibit Sirtuins 3 (SIRT3) deacetylation activity and protein expression of Thyroid hormone receptor beta (Thrb) and SIRT3. Hence, 500ng/mL Aβ42 was used to establish a cell model of AD. Curcumin significantly reversed the inhibitory effects of Aβ42 on cell viability, SIRT3 deacetylation activity, the ratio of NAD+/NADH, ATP level and the protein expression of Thrb and SIRT3, and the promotive effect on apoptosis. ChIPBase was used to predict the binding region of Thrb and SIRT3. Dual luciferase reporter gene and Chromatin immune precipitation (ChIP) assays were employed to verify the relationship between Thrb and promoter of SIRT3 mRNA. Overexpression of Thrb recovered Aβ42 induced metabolic dysfunction, while Thrb silence aggravated Aβ42 induced metabolic dysfunction. Moreover, Thrb silence or 3-TYP (a selective inhibitor of SIRT3) treatment abolished the amelioration of curcumin on Aβ42 induced metabolic dysfunction. Additionally, curcumin attenuated memory deficits in Amyloid precursor protein transgenic (APPTG) mice. Collectively, curcumin alleviated Aβ42-induced neuronal metabolic dysfunction through increasing Thrb expression and SIRT3 activity and improved cognition in APPTG mice.

Euchromatin histone methyltransferase II (EHMT2) regulates the expression of ras-related GTP binding C (RRAGC) protein

Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been impli-cated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5-Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells.

Impaired Sars-Cov-2 Specific Antibody Responses in Patients Treated with Anti-CD20 Antibodies

It has been proposed that patients with hematologic malignancy and autoimmune diseases receiving anti-CD20 monoclonal antibody (mAb) therapy are particularly at risk of severe Coronavirus disease (COVID-19) because the profound and long-lasting B-cell depletion induced by anti-CD20 mAb may impair virus clearance and may also contribute to reactivation of latent viruses, especially hepatitis B and JC viruses.As of July 20, 2020, the total number of COVID-19 cases reported by the Italian authorities reached 245,000. The north of the country was mostly hit, and Milan and Brescia were among the Italian provinces that registered the highest number of COVID-19 cases. Consistent with this, a high number of COVID-19 patients affected with multiple types of hematological disorders (n. 137) and with multiple sclerosis (MS, n. 114) were referred to ASST Spedali Civili di Brescia. Antibodies to SARS-CoV-2 were analyzed in 70 patients with hematological disease, and in few patients with MS. Among these, 10 patients (7 with hematologic disease and 3 with MS) had received treatment with rituximab or ocrelizumab, two anti-CD20 mAbs, within 3 months prior to COVID-19 onset. Clinical indication to CD20-depleting treatment for patients with hematological disorders included Diffuse Large B Cell Lymphoma (DLBCL) or Follicular Non Hodgkin Lymphoma (NHL).Anti-spike protein (anti-S) and anti-nucleocapsid (anti-N) antibodies to SARS-CoV-2 were analyzed during the acute phase of infection and up to 3 months since the onset of symptoms by quantitative measurements of plasma or serum antibodies with luciferase immune precipitation assay systems (LIPS). With this technique, production of anti-S and anti-N antibodies has been demonstrated between day 8 and day 14 after onset of symptoms in immunocompetent individuals, whereas specific antibody production was delayed by few days in immunocompromised patients (Burbelo PD et al, medRxiv. 2020 Apr 24:2020.04.20.20071423).All 10 patients remained seronegative to SARS-CoV-2 for the first 20 days since onset of symptoms. One patient with DLBCL secondary to Follicular NHL had detectable anti-S and anti-N antibodies at day +25, and one patient with MS developed anti-N antibodies by day +23. Two patients, one with DLBCL secondary to Follicular NHL and one with Follicular NHL were still seronegative for both anti-S and anti-N antibodies at 133 and 74 days since onset of symptoms. Two MS patients were seronegative at the last examination, and one other MS patient was anti-S seronegative at day +74.Three of the 10 patients have died; all three were SARS-CoV-2 RT-qPCR+ and seronegative at the time of death.While it has been reported that SARS-CoV-2 is cleared without significant problems by the majority of people with MS or other autoimmune diseases on immunotherapy, these data indicate that treatment with anti-CD20 mAb may significantly alter humoral responses to the virus. Until a vaccine to SARS-CoV-2 is available, the risk-benefit ratio of anti-CD20 mAb therapy in areas with high rates of SARS-CoV-2 infection should be carefully weighed. Moreover, for patients with B-cell malignancies or autoimmune diseases, transient discontinuation of this therapy, or use of alternative therapeutic approaches, should be considered once an efficacious vaccine becomes available.This study was performed according to protocol NP-4000 (Comitato Etico Provinciale), and supported by Regione Lombardia and by the Division of Intramural Research, NIAID.Figure 1DisclosuresImberti:Biogen: Honoraria; Genzyme-Sanofi: Honoraria; Meck-Serono: Honoraria; Novartis: Honoraria; Biogen: Other: Advisory board; FISM (Fondazione Italiana Sclerosi Multipla): Research Funding; Regione Lombardia: Research Funding. Capra:Biogen: Other: travel grants, Speakers Bureau; Roche: Other: travel grants, Speakers Bureau; Celgene: Other: travel grants, Speakers Bureau; Merck: Other: travel grants, Speakers Bureau; Novartis: Other: travel grants, Speakers Bureau. Rossi:Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Notarangelo:NIAID, NIH: Research Funding. Cohen:NIAID, NIH: Research Funding.

Antimigraine Drug Avitriptan Is a Ligand and Agonist of Human Aryl Hydrocarbon Receptor That Induces CYP1A1 in Hepatic and Intestinal Cells.

The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.

Long noncoding RNA TCL6 binds to miR-106a-5p to regulate hepatocellular carcinoma cells through PI3K/AKT signaling pathway.

Long noncoding RNAs (lncRNAs) have been reported to dysregulate and involve in the pathology of hepatocellular carcinoma (HCC). Nonetheless, the functional role of lncRNA T cell leukemia/lymphoma 6 (TCL6) and its underlying mechanism in HCC remain unclear. Herein, we analyzed the expression of TCL6 and elucidated its mechanistic involvement in HCC. Bioinformatics analyses indicated TCL6 was evidently downregulated in HCC tissues compared with normal controls. TCL6 was downregulated while microRNA-106a-5p (miR-106a-5p) was upregulated in HCC cell lines. Moreover, knockdown or overexpression of TCL6 significantly raised or diminished the expression level of miR-106a-5p in HCC cells, similar to the effect of miR-106a-5p on TCL6 expression. Functionally, TCL6 inhibited the proliferative, migratory, and invasive potentials of HCC cells as analyzed by cell counting kit-8, scratch wound healing, and transwell assays, respectively. Conversely, miR-106a-5p exerted an opposite effect on the proliferative, migratory, and invasive potentials of HCC. RNA immune precipitation and luciferase reporter assays revealed TCL6 directly bound to miR-106a-5p and luciferase reporter assay verified phosphatase and tensin homolog (PTEN) was a target gene of miR-106a-5p. Mechanistically, TCL6 knockdown evidently reduced PTEN expression at both messenger RNA and protein levels, and miR-106a-5p inhibitor partially rescued this reduction effect in HCC cells. Additionally, western blot assays demonstrated miR-106a-5p downregulation or TCL6 overexpression promoted the protein level of PTEN, and suppressed the phosphorylation level of AKT, the protein level of phosphatidylinositol 3-kinase (PI3K). Collectively, these results revealed TCL6 as a tumor-suppressive lncRNA regulates PI3K/AKT signaling pathway via directly binding to miR-106a-5p in HCC. This mechanism provides a theoretical basis for HCC pathogenesis and a potential therapeutic strategy for HCC treatment.

BES1 directly binds to the promoter of the ACC oxidase 1 gene to regulate gravitropic response in the roots of Arabidopsis thaliana

ABSTRACTBrassinosteroids (BRs) are known to be endogenous regulators of ethylene production, suggesting that some BR activity in plant growth and development is associated with ethylene. Here, we demonstrated that ethylene production in Arabidopsis thaliana roots is increased by BR signaling via the ethylene biosynthetic gene for ACC oxidase 1 (ACO1). Electrophoretic mobility shift and chromatin immune-precipitation assays showed that the BR transcription factor BES1 directly binds to two E-box sequences located in the intergenic region of ACO1. GUS expression using site mutations of the E-box sequences verified that ACO1 is normally expressed only when BES1 binds to the E-boxes in the putative promoter of ACO1, indicating that this binding is essential for ACO1 expression and the subsequent production of ethylene in A. thaliana roots. BR exogenously applied to A. thaliana roots enhanced the gravitropic response. Additionally, bes1-D exhibited a greater gravitropic response than did the wild-type specimens, proving that BR is a positive regulator of the gravitropic response in A. thaliana roots. The knock-down mutant aco1-1 showed a slightly lower gravitropic response than did the wild-type specimens, while bes1-D X aco1-1 exhibited a lower gravitropic response than did bes1-D. Therefore, ACO1 is a direct downstream target for BR transcription factor BES1, which controls ethylene production for gravitropism in A. thaliana roots.

Downregulation of LncRNA GAS5 promotes liver cancer proliferation and drug resistance by decreasing PTEN expression.

Accumulating evidence has shown that the long noncoding RNAs (lncRNAs) play a crucial role in the regulation of hepatocellular carcinoma (HCC) progression and drug resistance. In this study, we aimed to investigate the biological function roles of lncRNAs growth arrest-specific 5 (GAS5) and its underlying molecular mechanism in the development of HCC. qRT-PCR was used to detect GAS5, miR-21, and PTEN levels. MTT, cell counting assays, and xenograft mouse model were applied to measure cell proliferation rate in vitro and in vivo. The luciferase reporter assay and RNA immune-precipitation assay were introduced to evaluate the relationship between GAS5 and miR-21. We found that GAS5 was downregulated in HCC cell lines and tumor tissues. Knockdown of GAS5 enhanced HCC cell proliferation in vitro and in vivo and increased HCC cell resistance to doxorubicin. GAS5 acted as a sponge for miR-21 silencing and consequently led to the elevation of PTEN expression. Our data demonstrated that GAS5 functioned as a tumor suppressor role in HCC through regulation of miR-21-PTEN singling pathways, suggesting a potential application of GAS5 in HCC therapy.

Abstract 2616: A novel iron-chelating agent reduces MYC transcription via E2F gene family regulation

Abstract Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tumour of infancy. Amplification of the MYCN oncogene, which occurs in approximately 25% of patients with neuroblastoma, is one of the most powerful independent prognostic markers for this disease. MYCN belongs to the Myc gene family that codes for transcription factors often aberrantly expressed in both solid and liquid malignancies. However, despite their importance in adult and childhood malignancies, Myc oncoproteins have to date proven to be ‘undruggable’. Using a chemical library of 34,000 compounds we aimed to identify novel MYCN inhibitors, and among a number of molecules identified, M606 was found to markedly reduce MYCN protein levels as well as its downstream targets in MYCN-amplified SK-N-BE(2)-C neuroblastoma cells. Bioinformatic analysis of signalling pathways affected by M606 using FACTORIAL™ technology (Attagene Inc) demonstrated that this compound inhibits Myc-mediated transcription and simultaneously activates the HIF1α pathway. MYCN/MYC protein and mRNA stability were unaltered whereas their mRNA levels were significantly decreased. Metabolomics analysis suggested that the mechanism by which M606 inhibits MYCN transcription involves its capacity to chelate iron and, in this regard, M606-mediated inhibition of MYCN was reversed by the addition of iron. Progressive deletions of the MYCN promoter in luciferase reporter assays identified a minimal promoter region responsive to M606 that contains two E2F sites. Microarray assays performed on SK-N-BE(2)-C cells after M606 treatment showed significant changes in the expression level of the E2F gene family. Chromatin immune precipitation (ChIP) and gel shift (EMSA) assays revealed a reduction in E2F binding at the MYCN promoter region after M606 treatment. Moreover, western blot analysis showed that the RB protein, which is involved in E2F binding and transcriptional activation, becomes inactive due to hypophosphorylation following M606 treatment. On the other hand, M606 lost its inhibitory activity on a pRB-deficient osteosarcoma cell line SAOS-2, underlying the importance of the RB/E2F axis for M606 function. In conclusion, M606 is a novel Myc inhibitor and iron chelator that directly downregulates MYCN/MYC transcription via the E2F signalling pathway, providing a potentially valuable therapeutic approach in the treatment of cancers overexpressing Myc oncoproteins. Citation Format: Emanuele Valli, Chengyuan Xue, Leanna Cheung, Laura Gamble, Ruby Pandher, Simone Di Giacomo, Catherine Burkhart, Natalia Fedtsova, Francesca Ferrucci, Sergei Makarov, Thomas Telfer, Rachel Codd, David Scott, Giovanni Perini, Andrei Osterman, Andrei Gudkov, Murray Norris, Michelle Haber. A novel iron-chelating agent reduces MYC transcription via E2F gene family regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2616.

DHAV-1 Inhibits Type I Interferon Signaling to Assist Viral Adaption by Increasing the Expression of SOCS3.

Duck hepatitis A virus type 1 (DHAV-1) is one of the most lethal pathogens in the duck industry. The attenuated vaccine (the CH60 strain) is cultivated through serial passage in chicken embryos and is widely used for the prevention and control of the disease. However, the specific mechanism underlying its adaptation in chicken embryos has not been fully elucidated. In this study, we first infected chicken embryo fibroblasts (CEFs) with the DHAV-1 CH60 strain. The peak of viral proliferation occurred within 36–48 h post-infection. The different DHAV-1 strains significantly induced the expression of IFNα, IFNγ, and Suppressor of cytokine signaling 3 (SOCS3) in CEFs, and we found that SOCS3 overexpression significantly promoted viral replication. Furthermore, SOCS3 overexpression significantly inhibited the expression of IFNα but promoted the expression of IFNγ. In addition, SOCS3 overexpression clearly decreased the mRNA levels of STAT1 and STAT3 in the Janus kinase (JAK)-STAT signaling pathway and inhibited the expression of the antiviral proteins MX1 and OASL. Immune-precipitation assays indicated that SOCS3 and IFNα do not physically interact. Subcellular localization of SOCS3 and IFNα revealed that SOCS3 was mainly located in the nucleus and cytoplasm, while IFNα was located only in the cytoplasm. Co-localization of these two proteins was not observed in the cytoplasm. In conclusion, the DHAV-1 CH60 strain may inhibit the expression of IFNα by increasing the SOCS3 protein and SOCS3 can in turn decrease STAT1 and STAT3 mRNA levels, thereby inhibiting the antiviral protein MX1 and ultimately promoting viral proliferation, indirectly assisting in viral adaptation in chicken embryos.

Inhibition of the deubiquitinase USP9x induces pre-B cell homeobox 1 (PBX1) degradation and thereby stimulates prostate cancer cell apoptosis

Chemoresistance is a leading obstacle in effective management of advanced prostate cancer (PCa). A better understanding of the molecular mechanisms involved in PCa chemoresistance could improve treatment of patients with PCa. In the present study, using immune histochemical, chemistry, and precipitation assays with cells from individuals with benign or malignant prostate cancer or established PCa cell lines, we found that the oncogenic transcription factor pre-B cell leukemia homeobox-1 (PBX1) promotes PCa cell proliferation and confers to resistance against common anti-cancer drugs such as doxorubicin and cisplatin. We observed that genetic PBX1 knockdown abrogates this resistance, and further experiments revealed that PBX1 stability was modulated by the ubiquitin-proteasomal pathway. To directly probe the impact of this pathway on PBX1 activity, we screened for PBX1-specific deubiquitinases (Dubs) and found that ubiquitin-specific peptidase 9 X-linked (USP9x) interacted with and stabilized the PBX1 protein by attenuating its Lys-48-linked polyubiquitination. Moreover, the USP9x inhibitor WP1130 markedly induced PBX1 degradation and promoted PCa cell apoptosis. The results in this study indicate that PBX1 confers to PCa chemoresistance and identify USP9x as a Dub of PBX1. We concluded that targeting the USP9x/PBX1 axis could be a potential therapeutic strategy for managing advanced prostate cancer.

Promoter methylation and Hoxd4 regulate UII mRNA tissue-specific expression in olive flounder (paralichthys olivaceus)

The peptide urotensin II (UII) mediates multiple physiology effects in mammals and fishes, and UII expression shows a tissue-specific pattern. However the mechanism is still unknown. In the present study high level of UII mRNA was detected in the caudal neurosecretory system (CNSS) of the olive flounder when compared to other tissues. We examined whether epigenetic mechanisms of DNA methylation are involved in UII gene expression. Methylation DNA immune precipitation (MeDIP) assay showed low methylation of UII promoter in CNSS tissue compared with muscle and spinal cord. Methylation of UII promoter was further assessed through bisulphate sequencing analysis. Low level methylation (31%) in CpG island of UII promoter was detected in CNSS tissue, while methylation status in muscle and spinal cord was 89% and 91%, respectively. In addition, high conserved sites of Hoxd4 in UII promoter were found. Activation of Hoxd4 mRNA using transretinoic acid (RA) resulted in 18-fold increase of UII mRNA expression in CNSS and high locomotor activity in medaka, confirming that Hoxd4 is also involved in UII gene transcriptional regulation. Taken together, our data provide the first evidence of the epigenetic mechanism of promoter methylation in transcriptional regulation of UII expression in a tissue-specific manner, and Hoxd4 may also participate in UII gene transcription in flounder.