HDAC1 mRNA Research Articles

Super-enhancer-driven ZFP36L1 promotes PD-L1 expression in infiltrative gastric cancer

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide. Despite the widespread recognition of tumor immunotherapy in treating unresectable GC, challenges, including ineffective immunotherapy and drug resistance, persist. Therefore, understanding the regulatory mechanisms of PD-L1, particularly in the context of super-enhancers (SEs) and zinc finger protein 36 ring finger protein-like 1 (ZFP36L1) RNA-binding protein, is crucial. In this study, we performed H3K27ac Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, investigated the heterogeneity of SEs between two GC subtypes with differential growth patterns, and revealed the immune escape signatures driven by ZFP36L1-SE in infiltrative GC through SEs inhibitors treatment. The regulation of ZFP36L1 to PD-L1 was evaluated by quantitative PCR, western blot, flow cytometry, and immunohistochemistry. Furthermore, we explored its regulatory mechanisms using a combination of molecular biology techniques, including luciferase reporter assay, GST/RNA pull-down, chromatin immunoprecipitation (ChIP)/RIP experiments, and in vivo functional assays. We demonstrated that ZFP36L1, driven by an SE, enhances IFN-γ-induced PD-L1 expression, with SPI1 identified as the specific transcription factor binding to ZFP36L1-SE. Mechanistically, ZFP36L1 binds to the adenylate uridylate-rich element in the 3ʹ untranslated region (3ʹUTR) of HDAC3 mRNA, exacerbating its mRNA decay, and thereby facilitating PD-L1 abnormal transcriptional activation. Collectively, our findings provide mechanistic insights into the role of the SPI1-ZFP36L1-HDAC3-PD-L1 signaling axis in orchestrating immune escape mechanisms in GC, thereby offering valuable insights into the potential targets for immune checkpoint therapy in GC management.

Super-enhancer-driven ZFP36L1 promotes PD-L1 expression in infiltrative gastric cancer.

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide. Despite the widespread recognition of tumor immunotherapy in treating unresectable GC, challenges, including ineffective immunotherapy and drug resistance, persist. Therefore, understanding the regulatory mechanisms of PD-L1, particularly in the context of super-enhancers (SEs) and zinc finger protein 36 ring finger protein-like 1 (ZFP36L1) RNA-binding protein, is crucial. In this study, we performed H3K27ac Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, investigated the heterogeneity of SEs between two GC subtypes with differential growth patterns, and revealed the immune escape signatures driven by ZFP36L1-SE in infiltrative GC through SEs inhibitors treatment. The regulation of ZFP36L1 to PD-L1 was evaluated by quantitative PCR, western blot, flow cytometry, and immunohistochemistry. Furthermore, we explored its regulatory mechanisms using a combination of molecular biology techniques, including luciferase reporter assay, GST/RNA pull-down, chromatin immunoprecipitation (ChIP)/RIP experiments, and in vivo functional assays. We demonstrated that ZFP36L1, driven by an SE, enhances IFN-γ-induced PD-L1 expression, with SPI1 identified as the specific transcription factor binding to ZFP36L1-SE. Mechanistically, ZFP36L1 binds to the adenylate uridylate-rich element in the 3' untranslated region (3'UTR) of HDAC3 mRNA, exacerbating its mRNA decay, and thereby facilitating PD-L1 abnormal transcriptional activation. Collectively, our findings provide mechanistic insights into the role of the SPI1-ZFP36L1-HDAC3-PD-L1 signaling axis in orchestrating immune escape mechanisms in GC, thereby offering valuable insights into the potential targets for immune checkpoint therapy in GC management.

Therapeutic role of histone deacetylase inhibition in an in vitro model of Graves' orbitopathy.

Graves' orbitopathy (GO), a manifestation of Graves' disease, is characterized by orbital fibroblast‑induced inflammation, leading to fibrosis or adipogenesis. Histone deacetylase (HDAC) serves a central role in autoimmune diseases and fibrosis. The present study investigated HDAC inhibition in orbital fibroblasts from patients with GO to evaluate its potential as a therapeutic agent. Primary cultured orbital fibroblasts were treated with an HDAC inhibitor, panobinostat, under the stimulation of IL‑1β, TGF‑β or adipogenic medium. Inflammatory cytokines, and fibrosis‑ and adipogenesis‑related proteins were analyzed using western blotting. The effects of panobinostat on HDAC mRNA expression were measured in GO orbital fibroblasts, and specific HDACs were inhibited using small interfering RNA transfection. Panobinostat significantly reduced the IL‑1β‑induced production of inflammatory cytokines and TGF‑β‑induced production of fibrosis‑related proteins. It also suppressed adipocyte differentiation and adipogenic transcription factor production. Furthermore, it significantly attenuated HDAC7 mRNA expression in GO orbital fibroblasts. In addition, the silencing of HDAC7 led to anti‑inflammatory and anti‑fibrotic effects. In conclusion, by inhibiting HDAC7 gene expression, panobinostat may suppress the production of inflammatory cytokines, profibrotic proteins and adipogenesis in GO orbital fibroblasts. The present in vitro study suggested that HDAC7 could be a potential therapeutic target for inhibiting the inflammatory, adipogenic and fibrotic mechanisms of GO.

Alleviated diabetic osteoporosis and peripheral neuropathic pain by Rehmannia glutinosa Libosch polysaccharide via increasing regulatory T cells

Diabetic peripheral neuropathy (DPN) and diabetic osteoporosis (DOP) are conditions that significantly impact the quality of life of patients worldwide. Rehmanniae Radix Preparata, a component of traditional Chinese medicine with a history spanning thousands of years, has been utilized in the treatment of osteoporosis and diabetes. Specifically, Rehmannia glutinosa Libosch polysaccharide (RGP), a key bioactive compound of Rehmanniae Radix Preparata, has demonstrated immune-modulating properties and beneficial effects on hyperglycemia, hyperlipidemia, and vascular inflammation in diabetic mice. Despite these known actions, the precise mechanisms of RGP in addressing DOP and DPN remain unclear. Our study aimed to explore the impact of RGP on osteoporosis and peripheral neuropathic pain in diabetic mice induced by streptozotocin (STZ). The findings revealed that RGP not only improved hyperglycemia and osteoporosis in STZ-induced diabetic mice but also enhanced osteogenesis, insulin production, and nerve health. Specifically, RGP alleviated distal pain, improved nerve conduction velocity, nerve fiber integrity, and immune cell balance in the spleen. Mechanistically, RGP was found to upregulate HDAC6 mRNA expression in regulatory T cells, potentially shedding light on novel pathways for preventing DOP and DPN. These results offer promising insights for the development of new therapeutic approaches for diabetic complications.

HDAC6 modulates the cognitive behavioral function and hippocampal tissue pathological changes of APP/PS1 transgenic mice through HSP90-HSF1 pathway.

The aim of this study was to investigate histone deacetylase 6 (HDAC6) modifies the heat shock protein 90 (HSP90) and heat shock transcription factor 1 (HSF1) affect the levels of pathological markers such as Aβ oligomers (Aβo) and Tau phosphorylation (p-Tau) in APP/PS1 double transgenic mice hippocampal tissues or HT22 neurons as well as the changes in cognitive behavioral functions of mice. (1) APP/PS1 transgenic mice (6 months old, 25 ~ 30g) were randomly assigned to 5 experimental groups, C57BL/6J mice (6 months old, 25 ~ 30g) were used as 4 control groups, with 8 mice in each group. All mice underwent intracerebroventricular (i.c.v.) cannulation, and the experimental groups were administered with normal saline (APP + NS group), HDAC6 agonist tubastatin A hydrochloride (TSA) (APP + TSA group) or HDAC6 agonist theophylline (Theo) (APP + Theo group), HSP90 inhibitor Ganetespib (Gane) (APP + Gane group), or a combination of pre-injected Gane by TSA (APP + Gane + TSA group); the control group received i.c.v. injections of Gane (Gane group), TSA (TSA group), Theo (Theo group) or NS (NS group), respectively. (2) Mouse hippocampal neurons HT22 were randomly divided into a control group (Control) and an Aβ1-42 intervention group (Aβ). Within the Aβ group, further divisions were made for knockdown HSP90 (Aβ + siHSP90 group), overexpression HSP90 (Aβ + OE-HSP90 group), knockdown HSF1(Aβ + siHSF1 group) and knockdown HSF1 followed by overexpression HSP90 (Aβ + siHSF1 + OE-HSP90 group), resulting in a total of 6 groups. Morris water maze test was used to evaluate the cognitive behavior of the mice. Western blot and immunohistochemistry or immunofluorescence were performed to detect the levels of HDAC6, HSP90, HSF1, Aβ1-42, Tau protein, and p-Tau in the hippocampal tissue or HT22 cells. qRT-PCR was used to measure the levels of hdac6, hsp90, and hsf1 mRNA in the hippocampus or nerve cells. (1) The levels of HDAC6, Aβ1-42 and p-Tau were elevated, while HSP90 and HSF1 were decreased in the hippocampal tissue of APP/PS1 transgenic mice (all P < 0.01). Inhibiting HDAC6 upregulated the expressions of HSP90 and HSF1 in the hippocampal tissue of APP/PS1 mice, while decreasing the levels of Aβ1-42 and p-Tau as well as improving the spatial cognitive behavior in mice (P < 0.05 or P < 0.01). The opposite effects were observed upon HDAC6 activation. However, inhibiting HSP90 reduced the expression of HSF1 (P < 0.01) and increased the levels of Aβ1-42 and p-Tau (P < 0.05 or P < 0.01) but did not significantly affect the expression of HDAC6 (P > 0.05). No significant changes were observed in the aforementioned indicators in the 4 control groups (P > 0.05). (2) In the Aβ1-42 intervention group, HDAC6 and Aβ1-42, p-Tau expression levels were elevated, while HSP90 and HSF1 expressions were all decreased, and cell viability was reduced (P < 0.05 or P < 0.01). Overexpression of HSP90 upregulated HSF1 expression, decreased the levels of Aβ1-42 and p-Tau, and increased cell viability (P < 0.05 or P < 0.01). Knocking down HSP90 had the opposite effect; and knocking down HSF1 increased the levels of Aβ1-42 and p-Tau and decreased cells viability (all P < 0.01), but did not result in significant changes in the expression levels of HSP90 (P > 0.05). Inhibiting HDAC6 can upregulate the expressions of HSP90 and HSF1 but reduce the levels of Aβ1-42 and p-Tau in the hippocampus of APP/PS1 mice and improvement of cognitive behavioral function in mice; Overexpression of HSP90 can increase HSF1 but decrease Aβ1-42 and p-Tau levels in the hippocampal neurons and increase cell activity. It is suggested that HDAC6 may affect the formation of Aβ oligomers and the changes in Tau protein phosphorylation levels in the hippocampus of AD transgenic mouse as well as the alterations in cognitive behavioral functions by regulating the HSP90-HSF1 pathway.

Histone Deacetylase Inhibition Increases Glycerol Uptake in Normal and Hyperglycemic Conditions in Human Corneal Epithelial Cells

Delayed corneal wound healing, such as is seen in diabetes, can lead to visual impairment. Diabetes afflicts over 30 million Americans and leads to multiple comorbidities. Histone deacetylase (HDAC) expression and activity are increased in diabetes in correlation with an upregulation of inflammatory molecules and reactive oxygen species (ROS). HDACs have been previously found to suppress the expression of the glycerol channel aquaporin-3 (AQP3) in epithelial cells of the skin, the epidermal keratinocytes. Global deletion of AQP3 in mice has been found to delay corneal wound healing. We hypothesized that hyperglycemic conditions would decrease AQP3 levels and function while increasing the expression of HDAC(s), ROS scavenger proteins, and inflammatory mediators in human corneal epithelial cells (HCECs). HCECs were incubated for 48-hours in high glucose (25.5 mM) or normal glucose (~5.0 mM osmotically matched by mannitol) conditions. Under hyperglycemic conditions, the mRNA expression of interleukin-1α and -1β was significantly elevated, providing evidence for a pro-inflammatory effect of acute hyperglycemia in HCECs. Under these conditions, there was a significant increase in the mRNA expression of the ROS scavenger genes, superoxide dismutase-1 and -2 and peroxiredoxin-3 and -6, suggesting a compensatory response to an increase in ROS generation. Under acute hyperglycemic conditions, HDAC3 and HDAC8 mRNA expression also tended to increase while HDAC1 and HDAC2 mRNA levels remained unchanged. Although an upregulation of HDAC expression would be predicted to decrease AQP3 expression, AQP3 mRNA and protein expression levels were also unchanged. In addition, acute hyperglycemia (48 hours) did not alter glycerol transport into the HCECs, as measured by their uptake of radiolabeled [14C]-glycerol. However, HDAC inhibition using the broad-spectrum inhibitor SAHA for 24 hours increased [14C]-glycerol uptake under both normoglycemic and hyperglycemic conditions, suggesting that HDAC activity plays a role in AQP3 function. Overall, acute hyperglycemic conditions were pro-inflammatory and led to increased expression of ROS-scavenging genes; hyperglycemia also tended to upregulate HDAC expression, although without affecting AQP3 expression. On the other hand, HDAC inhibition increased glycerol uptake, an indicator of AQP3 function, suggesting an ability of HDAC activity to regulate AQP3 glycerol transport activity. National Institutes of Health/National Eye Institute award #R01EY030576 (to MW and WBB) VA Merit #BX005055 (to WBB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of HDAC9 and ARRDC4 as potential biomarkers and targets for treatment of type 2 diabetes

We aimed to identify the key potential insulin resistance (IR)-related genes and investigate their correlation with immune cell infiltration in type 2 diabetes (T2D). The GSE78721 dataset (68 diabetic patients and 62 controls) was downloaded from the Gene Expression Omnibus database and utilized for single-sample gene set enrichment analysis. IR-related genes were obtained from the Comparative Toxicology Genetics Database, and the final IR-differentially expressed genes (DEGs) were screened by intersecting with the DEGs obtained from the GSE78721 datasets. Functional enrichment analysis was performed, and the networks of the target gene with microRNA, transcription factor, and drug were constructed. Hub genes were identified based on a protein–protein interaction network. Least absolute shrinkage and selection operator regression and Random Forest and Boruta analysis were combined to screen diagnostic biomarkers in T2D, which were validated using the GSE76894 (19 diabetic patients and 84 controls) and GSE9006 (12 diabetic patients and 24 controls) datasets. Quantitative real-time polymerase chain reaction was performed to validate the biomarker expression in IR mice and control mice. In addition, infiltration of immune cells in T2D and their correlation with the identified markers were computed using CIBERSORT. We identified differential immune gene set regulatory T-cells in the GSE78721 dataset, and T2D samples were assigned into three clusters based on immune infiltration. A total of 2094 IR-DEGs were primarily enriched in response to endoplasmic reticulum stress. Importantly, HDAC9 and ARRDC4 were identified as markers of T2D and associated with different levels of immune cell infiltration. HDAC9 mRNA level were higher in the IR mice than in control mice, while ARRDC4 showed the opposite trend. In summary, we discovered potential vital biomarkers that contribute to immune cell infiltration associated with IR, which offers a new sight of immunotherapy for T2D.

Abstract 1712: Identifying a novel alternative promoter usage in HDAC9: Implications for LUAD prognosis and therapy

Abstract Promoters are key elements in gene regulation. It has been estimated that more than half of protein-coding genes in the human genome possess multiple promoters. Aberrant expression and dysregulated activity of alternative promoters have been correlated with numerous diseases, especially cancer. In this study, we utilized RNA-seq data from 608 lung adenocarcinoma (LUAD) patients and systematically analyzed promoter activities using the proActiv algorithm. Of the expressed genes in LUAD, approximately 57% have at least two distinct promoters. In addition, we identified 1852 tumor-specific alternative promoters that showed a significant shift (2-fold changes) in promoter activity without any notable changes in the expression of corresponding 1603 genes between tumor and adjacent non-tumor tissues. Functional enrichment analysis showed that these genes were associated with key cellular functions such as signaling pathways, cell cycle, and cell junctions. Unsupervised clustering of patients based on altered promoters revealed a significant prognostic value. Among them, we observed a major promoter switch in HDAC9, which mainly utilizes three promoters: prmt3280, prmt3283, and prmt3287 in our LUAD dataset. Notably, prmt3287 was exclusively active in tumor samples and functioned as the dominant promoter, while prmt3283 served as the main promoter in adjacent non-tumor samples. Intriguingly, even though the overall expression of HDAC9 mRNA lacked prognostic relevance both in our dataset and in the TCGA dataset, higher expression of the alternative promoter prmt3287 (using ≥50% activity level as a cutoff) was associated with adverse outcomes in LUAD patients. We further compared the DNA methylation level around the HDAC9 domain between paired tumor and non-tumor samples and found that positions with the most significant differences (p&amp;lt;0.001) overlapped with the prmt3287 region. Moreover, DNA methylation levels around prmt3287 significantly decreased in prmt3287 high-activity patient groups. Next, we investigated the ChIP-seq levels over the HDAC9 region, and ChIPseeker successfully annotated the alternative promoters of HDAC9. In addition, the alternative promoter prmt3287 exhibited the highest active enhancer signals. Furthermore, we performed PCR to determine the expression of six distinct transcript isoforms from alternative promoters in four different LUAD cell lines, and we observed high expression of isoform 10, which is possibly generated by prmt3287 in all cell lines. The impact of prmt3287 on cell proliferation, cell cycle, and cell migration will be further addressed. Our results indicate that alternative promoter usage can capture insights that might be missed at the overall gene expression level, and alternative promoter usage in HDAC9 could be a novel biomarker and a promising therapy target. Citation Format: Amina Bolatkan, Ken Asada, Syuzo Kaneko, Masaaki Komatsu, Ryuji Hamamoto. Identifying a novel alternative promoter usage in HDAC9: Implications for LUAD prognosis and therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1712.

MicroRNA-mediated epigenetic regulation of HDAC8 and HDAC6: Functional significance in cervical cancer

Cervical cancer, a leading global cause of female mortality, exhibits diverse molecular aberrations influencing gene expression and signaling pathways. Epigenetic factors, including histone deacetylases (HDACs) such as HDAC8 and HDAC6, along with microRNAs (miRNAs), play pivotal roles in cervical cancer progression. Recent investigations have unveiled miRNAs as potential regulators of HDACs, offering a promising therapeutic avenue. This study employed in-silico miRNA prediction, qRT-PCR co-expression studies, and Dual-Luciferase reporter assays to identify miRNAs governing HDAC8 and HDAC6 in HeLa, cervical cancer cells. Results pinpointed miR-497–3p and miR-324–3p as novel negative regulators of HDAC8 and HDAC6, respectively. Functional assays demonstrated that miR-497–3p overexpression in HeLa cells suppressed HDAC8, leading to increased acetylation of downstream targets p53 and α-tubulin. Similarly, miR-324–3p overexpression inhibited HDAC6 mRNA and protein expression, enhancing acetylation of Hsp90 and α-tubulin. Notably, inhibiting HDAC8 via miRNA overexpression correlated with reduced cell viability, diminished epithelial-to-mesenchymal transition (EMT), and increased microtubule bundle formation in HeLa cells. In conclusion, miR-497–3p and miR-324–3p emerge as novel negative regulators of HDAC8 and HDAC6, respectively, with potential therapeutic implications. Elevated expression of these miRNAs in cervical cancer cells holds promise for inhibiting metastasis, offering a targeted approach for intervention in cervical malignancy.

Association of changes in expression of HDAC and SIRT genes after drug treatment with cancer cell line sensitivity to kinase inhibitors

ABSTRACT Histone deacetylases (HDACs) and sirtuins (SIRTs) are important epigenetic regulators of cancer pathways. There is a limited understanding of how transcriptional regulation of their genes is affected by chemotherapeutic agents, and how such transcriptional changes affect tumour sensitivity to drug treatment. We investigated the concerted transcriptional response of HDAC and SIRT genes to 15 approved antitumor agents in the NCI-60 cancer cell line panel. Antitumor agents with diverse mechanisms of action induced upregulation or downregulation of multiple HDAC and SIRT genes. HDAC5 was upregulated by dasatinib and erlotinib in the majority of the cell lines. Tumour cell line sensitivity to kinase inhibitors was associated with upregulation of HDAC5, HDAC1, and several SIRT genes. We confirmed changes in HDAC and SIRT expression in independent datasets. We also experimentally validated the upregulation of HDAC5 mRNA and protein expression by dasatinib in the highly sensitive IGROV1 cell line. HDAC5 was not upregulated in the UACC-257 cell line resistant to dasatinib. The effects of cancer drug treatment on expression of HDAC and SIRT genes may influence chemosensitivity and may need to be considered during chemotherapy.

Peripheral neutrophils and oxidative stress-associated molecules for predicting the severity of asthma: a cross-sectional study based on multidimensional assessment.

This study aims to explore the relationship between the severity of asthma and neutrophils and related oxidative stress-associated molecules in peripheral blood and induced sputum. A total of 67 subjects were included in this study, namely, 25 patients with severe asthma and 42 patients with non-severe asthma. Clinical data, induced sputum and peripheral blood were collected. Lung function and molecules related to oxidative stress in induced sputum and peripheral blood of asthma patients were detected. The relationship between neutrophils and asthma severity was analyzed. HDAC2 mRNA and protein expression levels and HDAC2 activity were also analyzed. Multivariate logistic regression was performed to select statistically significant variables. The absolute value of neutrophils and percentage of neutrophils were higher in the severe asthma patients. These two values were used to predict the severity of asthma by ROC analysis, with the best cutoff values being 4.55 × 109/L (sensitivity 83.3%, specificity 64.0%) and 55.15% (sensitivity 54.8%, specificity 88.0%). The ROS concentration of neutrophils in the induced sputum samples and the 8-iso-PGF2α concentration in the peripheral blood samples were higher in the severe asthma group (P = 0.012; P = 0.044), whereas there was reduced HDAC2 protein activity in PBMCs (P < 0.001). A logistic equation and a nomogram were created to give a precise prediction of disease severity. Oxidative stress is increased in severe asthma patients. Peripheral blood neutrophils and 8-iso-PGF2α can be used as biomarkers to predict the severity of asthma. A prediction model was created for evaluating asthma severity.

Deficiency of circ_0103809 Attenuates Non-small Cell Lung Cancer Malignant Progression by Controlling miR-153-3p/HDAC1 Network.

Circular RNAs are vital players in tumorigenesis. We held the purpose to investigate the role and mechanism of circ_0103809 in non-small cell lung cancer (NSCLC).The expressions of circ_0103809, miR-153-3p and HDAC1 mRNA were determined using quantitative real-time PCR assay, and HDAC1 protein was quantified using western blot analysis. MTT, EdU, flow cytometry, tube-formation, wound healing and tube-formation assays were conducted for functional analysis. The predicted relationship among circ_0103809, miR-153-3p and HDAC1 was ascertained using dual-luciferase analysis, RIP assay and pull-down analysis. Animal models were further constructed to realize circ_0103809's role in vivo.Circ_0103809 was upregulated NSCLC specimens, cells and serum-derived exosomes. Serum exosomal circ_0103809 had the potency to be a diagnostic biomarker for NSCLC. Circ_0103809 silencing inhibited NSCLC cell growth, metastasis and angiogenesis and triggered cell cycle arrest and apoptosis. Circ_0103809 deficiency also suppressed the growth of transplanted tumors. Circ_0103809 acted as the miR-153-3p sponge, and the biological effects of circ_0103809 knockdown were relieved by miR-153-3p inhibition. HDAC1 was directly targeted by miR-153-3p, and miR-153-3p enrichment inhibited NSCLC cell malignant phenotypes by sequestering HDAC1.Circ_0103809 knockdown repressed NSCLC malignant progression partly by regulating miR-153-3p/HDAC1 signaling.

Food and medicinal plants from Nigeria with anti-Helicobacter pylori activities induce apoptosis in colon and gastric cancer cell lines

Background: Food and medicinal plants are used traditionally in Nigeria to treat gastrointestinal (GI) disorders such as gastritis, peptic ulcer disease and GI-related cancers. We have previously reported that specific extracts of Nigerian food and medicinal plants inhibited the growth of Helicobacter pylori, a bacterium known to cause peptic ulcer disease, as well as gastric and colon cancer. Objective: To determine the effects of three food plants, namely Anogeissus leiocarpus (DC.) Guill. &amp; Perr. (African birch, Combretaceae), Terminalia glaucescens Planch ex Benth. (Nigerian chewing sticks, Combretaceae) and Dillenia indica L. (Elephant apple, Dilleniaceae) used in Nigeria for the treatment of GI disorders and cancer in six colon and gastric cancer cell lines, and two non-cancerous cell lines. Methods: Cancer cells were grown in appropriate media and CellTiter-Glo® 2.0 and ApoTox-Glo™ Triplex assays were used to measure cell growth and apoptosis in SW480, SW620, HCT116, Caco2 cell lines, as well as AGS and NCI-N87 gastric cancer cells. Caspase-Glo® 3/7, and Caspase-Glo® 8 were used to determine caspase activities and apoptosis. Gene expression was measured using quantitative PCR.Results: The methanol extract of A. leiocarpus roots inhibited the growth of HCT-116, SW480 and SW620 colon cancer cells (IC50 of 15.8, 10.0, and 20.8 µg/mL, respectively), but weakly active in the AGS and NCI-N87 cells (IC50 77.0 and &gt;100 µg/mL respectively). The T. glaucescens extract was weakly active in HCT-116 cells (IC50 64.9 µg/ml) and AGS cells (52.2 µg/ml). The D. indica extract was active in SW480 (IC50 15.8 µg/mL), and weakly active in Caco2 (IC50 35.2 µg/mL) and HCT-116 (IC50 53.2 µg/mL). In HCT-116 cells, A. leiocarpus root extract and its aqueous partition increased caspase 8 activity, as well as mRNA expression of p53, while expression of Bcl-2 and HDAC1 mRNA was reduced. Bioassay guided fractionation of the aqueous partition led to the isolation and identification of the known compound methyl gallate.Conclusions: These data suggest that the Nigerian food plants with anti-Helicobacter pylori activities increase the expression of the tumor suppressor p53 and inhibit HDAC1, thereby inducing apoptosis in HCT-116 colon cancer cells.Running Title: Inhibitory Effects of Nigerian food plants on GIT CancersKeywords: African birch, AGS, Apoptosis, Anogeissus leiocarpus, Dillenia indica, Elephant apple, Terminalia glaucescens, Nigerian chewing sticks, HCT-116, SW480, SW620

High-intensity interval training ameliorates chronic unpredictable mild stress-induced depressive behaviors via HDAC2-BDNF signaling in the ventral hippocampus

Major depressive disorder (MDD) is a devastating psychiatric disease, and current therapies could not well meet the demand for MDD treatment. Exercise benefits mental illness, and notably, exercise has been recommended as an alternative option for MDD treatment in some countries. However, the paradigm and intensity of exercise for MDD treatment has yet to be determined. High-intensity interval training (HIIT) is a potent and time-efficient type of exercise training and has gained popularity in recent years. In this study, we exposed the mice to chronic unpredictable mild stress (CUMS) and found HIIT exerted substantial antidepressant effect. Moreover, HIIT further enhanced the antidepressant effect of fluoxetine, a classic antidepressant in the clinic, confirming the antidepressant role of HIIT. HIIT significantly reversed the CUMS-induced upregulations in HDAC2 mRNA and protein level in the ventral hippocampus. We also found HIIT rescued the CUMS-induced downregulation in the expression of brain-derived neurotrophic factor (BDNF) and HDAC2 overexpression counteracted the HIIT-induced increase in BDNF level. More importantly, both virus-mediated HDAC2 overexpression and microinfusion of TrkB-Fc, a BDNF scavenger, in the ventral hippocampus abolished the antidepressant effect of HIIT. Together, our results strongly demonstrate that HIIT attenuates depressive behaviors, probably via HDAC2-BDNF signaling pathway and reveal that HIIT may serve as an alternative option for MDD treatment.

Targeting histone deacetylase 9 represses fibrogenic phenotypes in buccal mucosal fibroblasts with arecoline stimulation

Background/purposeOral submucosal fibrosis (OSF) is a premalignant disorder positively associated with betel nut chewing. Recent studies supported the promising benefits of histone deacetylase (HDAC) inhibitors for fibrosis treatment. Here we aim to clarify the pro-fibrogenic role of HDAC9 in regulating OSF. Materials and methodsHealthy and OSF specimens were collected to investigate the clinical significance of HDAC9. Chronic arecoline treatment process was used to induce arecoline-mediated myofibroblasts-related activation of primary buccal mucosa fibroblasts (BMFs). Functional analysis of collagen gel contraction, transwell migration, and wound-healing assays were performed to assess the change in pro-fibrogenic properties of BMFs and fibrotic BMFs (fBMFs). Lentiviral-mediated HDAC9 knockdown was used to verify the role of HDAC9 in the pro-fibrogenic process. ResultsWe found that arecoline significantly increased the mRNA and protein expression of HDAC9 of BMFs in a dose-dependent manner. Knockdown of HDAC9 in BMFs reversed the strengthened effects of arecoline on collagen gel contraction, cell migration, and wound-healing ability. We further demonstrated that knockdown of HDAC9 in fBMFs significantly attenuated its inherent pro-fibrogenic properties. Furthermore, we confirmed a significantly increased expression of HDAC9 mRNA in OSF compared to normal tissues, which suggested a positive correlation between the up-regulation of HDAC9 and OSF. ConclusionWe demonstrated that silencing of HDAC9 inhibited arecoline-induced activation and inherent pro-fibrogenic properties, suggesting potential therapeutics by targeting HDAC9 in the OSF treatment.

The RNA Binding Protein HuR Promotes Neuronal Apoptosis in Rats with Spinal Cord Injury via the HDAC1/RAD21 Axis

The current research aims to study the regulation of the RNA binding protein HuR on neuronal apoptosis during spinal cord injury (SCI) and its underlying mechanism. SCI rat models were injected with HuR shRNA and/or pcDNA3.1-RAD21, followed by the evaluation of motor function, the degree of SCI, the expression of HuR and RAD21, and neuronal-like apoptosis. The co-localization of HuR-RAD21, RAD21-NeuN, and NeuN-cleaved caspase 3 was measured by immunofluorescence. Additionally, targeting relationships among HuR, HDAC1, and RAD21 were verified by chromatin immunoprecipitation and RNA immunoprecipitation. After transfection, apoptosis of PC12 cells was tested by flow cytometry. Results showed that silencing HuR or up-regulating RAD21 could alleviate SCI and reduce neuronal apoptosis. HuR could combine HDAC1 mRNA, and HDAC1 combined the promoter of RAD21. Further experiments revealed that HuR enhanced HDAC1 expression and reduced RAD21 promoter region acetylation. Overexpression of RAD21 reversed the enhancement in apoptosis of PC12 cells caused by overexpression of HuR. The injection of HuR shRNA in tail vein of SCI rats increased basso, beattie, and bresnahan score, relieved SCI, reduced HuR and HDAC1 expression, elevated RAD21 expression, and decreased neuronal-like apoptosis. However, this result was reversed by co-injection of pcDNA3.1-HDAC1. In conclusion, down-regulation of HuR alleviated SCI and neuronal apoptosis in rats by suppressing HDAC1 expression and promoting RAD21 expression.

Analysis of global DNA methylation and epigenetic modifiers (DNMTs and HDACs) in human foetal endothelium exposed to gestational and type 2 diabetes

ABSTRACT Foetuses exposed to maternal gestational diabetes (GDM) and type 2 diabetes (T2D) have an increased risk of adverse perinatal outcomes. Epigenetic mechanisms, including DNA methylation and histone modifications, may act as mediators of persistent metabolic memory in endothelial cells (ECs) exposed to hyperglycaemia, even after glucose normalization. Therefore, we investigated alterations in global DNA methylation and epigenetic modifier expression (DNMT1, DNMT3a, DNMT3b, HDAC1, and HDAC2) in human umbilical vein ECs (HUVECs) from the umbilical cords of mothers with GDM (n = 8) and T2D (n = 3) compared to that of healthy mothers (n = 6). Global DNA alteration was measured using a 5-methylation cytosine colorimetric assay, followed by quantitative real-time polymerase chain reaction to measure DNA methyltransferase and histone acetylase transcript expression. We revealed that DNA hypermethylation occurs in both GDM- and T2D-HUVECs compared to that in Control-HUVECs. Furthermore, there was a significant increase in HDAC2 mRNA levels in GDM-HUVECs and increase in DNMT3b mRNA levels in T2D-HUVECs. Overall, our results suggest that GDM and T2D are associated with global DNA hypermethylation in foetal endothelial cells under normoglycemic conditions and the aberrant mRNA expression of HDAC2 and DNMT3b could play a role in this dysregulation.

Role of miR-124-3p in regulatory mechanisms of Gpm6a expression in the hippocampus of chronically stressed rats.

The neuronal membrane glycoprotein M6a (GPM6A) belongs to the family of myelin proteolipid protein and plays a role in neuronal remodeling and plasticity. Decreased expression of GPM6A mRNA is observed in the hippocampal tissue of suicide victims who suffered from depression and after chronic stress exposure in animals. The regulatory mechanisms that impact expression of GPM6A under chronic stress or in pathological conditions are not well understood. Previously, miRNAs miR-133b, miR-124-3p, and miR-9-5p have been shown to regulate the expression of Gpm6a mRNA under normal conditions. Here, we employed the paradigm of chronic-restraint stress in rats and using quantitative polymerase chain reaction (qPCR) showed down-regulation of expression of Gpm6a and the brain-derived neurotrophic factor (Bdnf) genes at mRNA level as well as miR-133b, and miR-124-3p, but not miR-9-5p in the hippocampus of chronically stressed rats. Furthermore, we observed alterations in the expression of histone deacetylase (Hdac5) and myocyte enhancer factor 2C (Mef2c) mRNAs. Our data suggest that chronic stress influences Gpm6a expression by miR-124-mediated impact on the expression of Hdac5 and Mef2c. Upon miR-124 over-expression in hippocampal neurons cultured in vitro, we observed enhanced neuronal arborization as evaluated by Sholl analysis, increased Gpm6a and Mef2c expression, and decreased Hdac5 expression. Moreover, treatment of hippocampal neurons cultured in vitro with BDNF resulted in an elevation in the miR-124-3p expression, a decrease in the miR-9-5p expression but did not affect miR-133b. This was accompanied by augmented expression of Gpm6a and Mef2c mRNAs and significantly lower levels of Hdac5 mRNA. Altogether, these results indicate that the regulatory mechanism that influence expression of Gpm6a under chronic stress involves miR-124-mediated impact on the expression of Hdac5 and Mef2c and a role of BDNF in the activation of Gpm6a expression.

Identification of HDAC4 as a potential therapeutic target and prognostic biomarker for ZFTA-fused ependymomas.

Members of the HDAC family are predictive biomarkers and regulate the tumorigenesis in several cancers. However, the role of these genes in the biology of intracranial ependymomas (EPNs) remains unexplored. Here, an analysis of eighteen HDACs genes in an EPN transcriptomic dataset, revealed significantly higher levels of HDAC4 in supratentorial ZFTA fusion (ST-ZFTA) compared with ST-YAP1 fusion and posterior fossa EPNs, while HDAC7 and SIRT2 were downregulated in ST-ZFTA. HDAC4 was also overexpressed in ST-ZFTA as measured by single-cell RNA-Seq, quantitative real time-polymerase chain reaction, and immunohistochemistry. Survival analyses showed a significantly worse outcome for EPNs with higher HDAC4 and SIRT1 mRNA levels. Ontology enrichment analysis showed an HDAC4-high signature consistent with viral processes while collagen-containing extracellular matrix and cell-cell junction were enriched in those with an HDAC4-low signature. Immune gene analysis demonstrated a correlation between HDAC4 expression and low levels of NK resting cells. Several small molecules compounds targeting HDAC4 and ABCG2, were predicted by in silico analysis to be effective against HDAC4-high ZFTA. Our results provide novel insights into the biology of the HDAC family in intracranial ependymomas and reveal HDAC4 as a prognostic marker and potential therapeutic target in ST-ZFTA.

MicroRNA-30e-3p reduces coronary microembolism-induced cardiomyocyte pyroptosis and inflammation by sequestering HDAC2 from the SMAD7 promoter.

This study investigates the mechanism by which microRNA (miR)-30e-3p reduces coronary microembolism (CME)-induced cardiomyocyte pyroptosis and inflammation. Cardiac function tests, histological staining, and transmission electron microscopy were performed on CME-model rats injected with adeno-associated viral vectors. Cardiomyocytes were transfected 24 h before a cellular model of pyroptosis was established via treatment with 1 μg/mL lipopolysaccharide (LPS) for 4 h and 5 mM ATP for 30 min. Pyroptosis, inflammation, and Wnt/β-catenin signaling in cardiomyocytes were detected. Dual-luciferase reporter assays and/or RNA pull-down assays were performed to verify the binding of miR-30e-3p to HDAC2 mRNA or HDAC2 to the SMAD7 promoter. Chromatin immunoprecipitation was used to assess the level of H3K27 acetylation at the SMAD7 promoter. miR-30e-3p and SMAD7 expression levels were downregulated and HDAC2 expression was upregulated with CME. The overexpression of miR-30e-3p restored cardiac functions in CME-model rats and reduced serum cTnI, IL-18, and IL-1β levels, microinfarcts, inflammatory cell infiltration, apoptosis, collagen content, and GSDMD-N, cleaved caspase-1, and NLRP3 expression in the myocardium, but these effects were reversed by SMAD7 knockdown. The overexpression of miR-30e-3p or knockdown of HDAC2 reduced LDH, IL-18, and IL-1β secretion, propidium iodide intake, and GSDMD-N, NLRP3, cleaved caspase-1, Wnt3a, Wnt5a, and β-catenin expression in the cardiomyocyte model. miR-30e-3p inhibited the expression of HDAC2 by binding HDAC2 mRNA. HDAC2 repressed the expression of SMAD7 by catalyzing H3K27 deacetylation at the SMAD7 promoter. miR-30e-3p, by binding HDAC2 to promote SMAD7 expression, reduces CME-induced cardiomyocyte pyroptosis and inflammation.