HDAC Research Articles

Investigation of the Effect of Zebularine in Comparison to and in Combination with Trichostatin A on p21Cip1/Waf1/ Sdi1, p27Kip1, p57Kip2, DNA Methyltransferases and Histone Deacetylases in Colon Cancer LS 180 Cell Line

Background:The heart of the cell cycle regulatory machine is a group of enzymes named cyclin-dependent kinases (Cdks). The active form of these enzymes includes a kinase and its partner, a cyclin. The regulation of cyclin-Cdk complexes is provided by Cdk inhibitors (CKIs) such as Cip/Kip family comprising p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2. The hypermethylation and deacetylation of Cip/Kip gene family seem to be frequent in numerous cancers. It has been indicated that increased expression of DNMTs and HDACs contributes to cancer induction. Previously, we reported the effect of DNA demethylating agents and histone deacetylase inhibitors on histone deacetylase 1, DNA methyltransferase 1, and CIP/KIP family in colon cancer. The current study was designed to evaluate the effect of zebularine in comparison to and in combination with trichostatin A (TSA) on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, DNA methyltransferases (DNMT1, 3a and 3b) and histone deacetylases (HDAC1, 2, and 3) genes expression, cell growth inhibition and apoptosis induction in colon cancer LS 180 cell line. Materials and Methods:The colon cancer LS 180 cell line was cultured and treated with zebularine and TSA. To determine cell viability, apoptosis, and the relative expression level of the genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively. Results:Both compounds significantly inhibited cell growth, and induced apoptosis. Furthermore, both compounds increased p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 significantly. Additionally, zebularine and TSA decreased DNMTs and HDACs gene expression respectively. Conclusion: The zebularine and TSA can reactivate the CIP/KIP family through inhibition of DNMTs and HDACs genes activity.

0892 Sleep Abnormalities in Cornelia De Lange Syndrome

Abstract Introduction Cornelia de lange is an autosomal dominant disorder (when associated with NIPBL, RAD21, or SMC3 genes) with an incidence of 1:10,000 to 1:50,000 live births, patients affected are known to have a wide variety of sleep disorders, those range from insomnia and abnormal circadian cycle to sleep disordered breathing and hypoventilation. The exact etiology of increased risk of sleep-disordered breathing in patients affected is not fully understood. It is possible that some facial features in these patients expose them to a higher risk (micrognathia, high arched palate, and short neck). We wanted to analyze the sleep related problems in CDLS. Methods We included 3 patients with the disorder, age range from 15 months to 16 years old. All patients met criteria for CDLS diagnosis, all had intellectual disability and behavioral associated symptoms. The somnology evaluation included questionnaires of diurnal behavior and sleep focused logs. We performed nocturnal polysomnography in only 2 patients due to inability to tolerate the test in one case. Results Sleep clinical information was abnormal in all the cases. Overnight behavioral video evaluation was done. The behavioral abnormalities were evident in all subjects and severe in one. Overnight polysomnography demonstrated a moderate to severe degree of OSA, delayed sleep onset suggestive of insomnia, sleep-wake transition disorder with elevated WASO time, and arousal disorder with elevated spontaneous arousal index. It is of interest the finding of sleep related hypoxemia with limited evidence of obstructive component in one patient. Conclusion The abnormalities in sleep are frequent in CDLS, there are wide and present in the sleep architecture and the sleep ventilation, sleep apnea syndromes are frequent but are not the only major sleep-related abnormalities. When CDLS is caused by mutations in the HDAC8 or SMC1A gene, the condition has an X-linked dominant pattern inheritance. Most cases result from new mutations in the HDAC8 or SMC1A gene and occur in people with no history of the condition in their family, likely our cases are related to this mode of transmission and potential different patters of sleep disruption are dependent on different genes involved. Support None

Cyclo-RGD Truncated Polymeric Nanoconstruct with Dendrimeric Templates for Targeted HDAC4 Gene Silencing in a Diabetic Nephropathy Mouse Model.

Diabetic nephropathy (DN), a chronic progressive kidney disease, is a significant complication of diabetes mellitus. Dysregulation of the histone deacetylases (HDACs) gene has been implicated in the pathogenesis of DN. Hence, the HDAC-inhibitors have emerged as a critical class of therapeutic agents in DN; however, the currently available HDAC4-inhibitors are mostly nonselective in nature as well as inhibit multiple HDACs. RNA interference of HDAC4 (HDAC4 siRNA) has shown immense promise, but the clinical translation has been impeded due to lack of a targeted, specific, and in vivo applicable delivery modality. In the present investigation, we examined Cyclo(RGDfC) (cRGD) truncated polymeric nanoplex with dendrimeric templates for targeted HDAC4 Gene Silencing. The developed nanoplex exhibited enhanced encapsulation of siRNA and offered superior protection against serum RNase nucleases degradation. The nanoplex was tested on podocytes (in vitro), wherein it showed selective binding to the αvβ3 integrin receptor, active cellular uptake, and significant in vitro gene silencing. The in vivo experiments showed remarkable suppression of the HDAC4 and inhibition in the progression of renal fibrosis in the Streptozotocin (STZ) induced DN C57BL/6 mice model. Histopathological and toxicological studies revealed nonsignificant abnormality/toxicity with the nanoplex. Conclusively, nanoplex was found as a promising tactic for targeted therapy of podocytes and could be extended for other kidney-related ailments.

SUN-LB65 Circulating Micrornas Linked to Bone Metabolism Are Affected by Sequential Anti Osteoporotic Treatment in Postmenopausal Osteoporosis

Depending on osteoporosis severity a long-term treatment, often in the form of a sequential regimen, may be required. The expression of microRNAs (miRs) related to bone metabolism in the serum is potentially affected by anti-osteoporotic treatment. Here, we investigated the effect of sequential treatments on microRNA expression profile in the serum. Methods: This is an observational, open label, non-randomized clinical trial that included 37 postmenopausal women with osteoporosis who were treated with denosumab (Dmab) for 1 year. Patients had been previously treated for 2 years with either teriparatide (n=20), or zolendronate (n=6), or were treatment-naïve (n=11). We evaluated changes in the relative serum expression of selected miRs linked to bone metabolism at 3 and 12 months of Dmab treatment at each group separately. Results: In the group of patients who were previously treated with teriparatide, the relative expressions of miR-21a-5p, miR-29a, and miR-2861 were significantly decreased at both 3 months (fold change 0.13, p<0.001 for miR-21a-5p; fold change 0.68, p=0.044 for miR-29a; fold change 0.16, p<.0001 for miR-2861), and 12 months (fold change 0.09, p<0.001 for miR-21a-5p; fold change 0.65, p=0.044 for miR-29a; fold change 0.19, p<0.001 for miR-2861) of Dmab treatment. The relative expression of miR-23a-3p was also significantly decreased (fold change 0.65, p<0.001) at 12 months of Dmab treatment. The relative expression of miRs was not changed among patients previously treated with zolendronate. No change was also observed during Dmab treatment in previously treatment-naïve osteoporotic women. Conclusions: The expression of circulatings miRs linked to bone metabolism during Dmab treatment is depended on previous treatment status. Patients previously on teriparatide treatment present alterations of the relative expression of miRs related to the expression of key osteoblastic genes such as RUNX-2 (miR-23), collagen type 1 (miR-29a) and HDAC5 gene (miR-2861) during subsequent treatment with Dmab Our data suggest that teriparatide may influence the subsequent anti-resorptive effect of Dmab on bone metabolism at post-transcriptional level.

Identification of functional mutations associated with environmental variance of litter size in rabbits

BackgroundEnvironmental variance (VE) is partly under genetic control and has recently been proposed as a measure of resilience. Unravelling the genetic background of the VE of complex traits could help to improve resilience of livestock and stabilize their production across farming systems. The objective of this study was to identify genes and functional mutations associated with variation in VE of litter size (LS) in rabbits. To achieve this, we combined the results of a genome-wide association study (GWAS) and a whole-genome sequencing (WGS) analysis using data from two divergently selected rabbit lines for high and low VE of LS. These lines differ in terms of biomarkers of immune response and mortality. Moreover, rabbits with a lower VE of LS were found to be more resilient to infections than animals with a higher VE of LS.ResultsBy using two GWAS approaches (single-marker regression and Bayesian multiple-marker regression), we identified four genomic regions associated with VE of LS, on chromosomes 3, 7, 10, and 14. We detected 38 genes in the associated genomic regions and, using WGS, we identified 129 variants in the splicing, UTR, and coding (missense and frameshift effects) regions of 16 of these 38 genes. These genes were related to the immune system, the development of sensory structures, and stress responses. All of these variants (except one) segregated in one of the rabbit lines and were absent (n = 91) or fixed in the other one (n = 37). The fixed variants were in the HDAC9, ITGB8, MIS18A, ENSOCUG00000021276 and URB1 genes. We also identified a 1-bp deletion in the 3′UTR region of the HUNK gene that was fixed in the low VE line and absent in the high VE line.ConclusionsThis is the first study that combines GWAS and WGS analyses to study the genetic basis of VE. The new candidate genes and functional mutations identified in this study suggest that the VE of LS is under the control of functions related to the immune system, stress response, and the nervous system. These findings could also explain differences in resilience between rabbits with homogeneous and heterogeneous VE of litter size.

Co-expression network analysis identified cell cycle signaling hub genes associated with progression and prognosis of multiple myeloma

Abstract There is a gap in understanding the molecular mechanisms behind early multiple myeloma (MM) progression in the newly diagnosed patient on standard treatment, i.e., lenalidomide, bortezomib, and dexamethasone (RVD). As a result, the relatively high percentage of patients that progress after early treatment with RVD is significant. Therefore, identification of clinically relevant clusters of co-expressed genes or related biomarkers for MM progression, while on RVD, would help identify new molecular mechanisms and drug targets. The objective of this study is to use weighted gene co-expression network analysis (WGCNA) to identify gene-signaling networks associated with early MM progression. Therefore, we performed a novel WGCNA to determine module-trait correlations. We next examined the overrepresentation of upregulators and signal pathway networks from MM patients in the MMRF CoMMpass dataset (n = 175). WGCNA constructed 45 modules based on the correlations between patients on RVD and death using 30,598 gene transcripts. We are using death as a biomarker for MM progression and two years as cut off for treatment. One of the highest modules identified; Pink module has HDAC8, HSPG2, LILRB4, CDC and FOXM1 genes and other 595 genes that are co-expressed together, that are significantly and positively correlated (p &amp;lt; 3.6 × 10-8, R2 =0.38) with overall survival of &amp;lt; 2 years following initial treatment with RVD. These genes are associated with cell cycle signaling, and Jak-Stat signaling pathways and their biological process is largely significant for proliferation and tumorigenesis. In future, connectivity mapping will be used to identify drug signatures that can target any of the hub genes. This study is expected to improve MM’s relapse.

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration.

Sex control technology is of great significance in the production of domestic animals, especially for rapidly breeding water buffalo (bubalus bubalis), which served as a research model in the present study. We have confirmed that a fluorescence protein integrated into the Y chromosome is fit for sexing pre-implantation embryos in the mouse. Firstly, we optimized the efficiency of targeted integration of exogenous gene encoding enhanced green fluorescent protein (eGFP) and mCherry in Neuro-2a cells, mouse embryonic stem cells, mouse embryonic cells (NIH3T3), buffalo fetal fibroblast (BFF) cells. The results showed that a homology arm length of 800 bp on both sides of the target is more efficient that 300 bp or 300 bp/800 bp. Homology-directed repair (HDR)-mediated knock-in in BFF cells was also significantly improved when cells were supplemented with pifithrin-μ, which is a small molecule that inhibits the binding of p53 to mitochondria. Three pulses at 250 V resulted in the most efficient electroporation in BFF cells and 1.5 μg/mL puromycin was found to be the optimal concentration for screening. Moreover, Y-Chr-eGFP transgenic BFF cells and cloned buffalo embryos were successfully generated using CRISPR/Cas9-mediated gene editing combined with the somatic cell nuclear transfer (SCNT) technique. At passage numbers 6–8, the growth rate and cell proliferation rate were significantly lower in Y-Chr-eGFP transgenic than in non-transgenic BFF cells; the expression levels of the methylation-related genes DNMT1 and DNMT3a were similar; however, the expression levels of the acetylation-related genes HDAC1, HDAC2, and HDAC3 were significantly higher (p < 0.05) in Y-Chr-eGFP transgenic BFF cells compared with non-transgenic cells. Y-Chr-eGFP transgenic BFFs were used as donors for SCNT, the results showed that eGFP reporter is suitable for the visualization of the sex of embryos. The blastocyst rates of cloned buffalo embryos were similar; however, the cleavage rates of transgenic cloned embryos were significantly lower compared with control. In summary, we optimized the protocol for generating transgenic BFF cells and successfully generated Y-Chr-eGFP transgenic embryos using these cells as donors.

Association between rs2107595 HDAC9 gene polymorphism and advanced carotid atherosclerosis in the Slovenian cohort

BackgroundHistone deacetylase 9 (HDAC9) plays an important role in transcriptional regulation, cell cycle progression and developmental events; moreover, it has been investigated as a candidate gene in a number of conditions, including the onset and progression of atherosclerosis. We hypothesized that the rs2107595 HDAC9 gene polymorphism may be associated with advanced carotid artery disease in a Slovenian cohort. We also investigated the effect of this polymorphism on HDAC9 receptor expression in the internal carotid artery (ICA) specimens obtained by endarterectomy.MethodsThis case-control study enrolled 619 unrelated Slovenian patients: 311 patients with ICA stenosis > 75% as the study group and 308 patients with ICA stenosis < 50% as the control group. Patient laboratory and clinical data were obtained from the medical records. The rs2107595 polymorphisms were genotyped using TaqMan SNP Genotyping assay. HDAC9 expression was assessed by immunohistochemistry in 30 ICA specimens from patients with ICA atherosclerosis > 75%, and the numerical areal density of HDAC9 positive cells was calculated.ResultsThe occurrence of advanced ICA atherosclerosis in the Slovenian cohort was 3.81 times higher in the codominant genetic model (OR = 3.81, 95%CI = 1.06–13.77, p = 0.04), and 3.10 times higher in the recessive genetic model (OR = 3.10, 95%CI = 1.16–8.27, p = 0.02). In addition, the A allele of rs2107595 was associated with increased HDAC9 expression in the ICA specimens obtained by endarterectomy.ConclusionsWe observed a significant association between the AA genotype of rs2107595 with the advanced carotid artery disease in our Slovenian cohort, indicating that this polymorphism may be a genetic risk factor for ICA atherosclerosis.

Adipose tissue mRNA expression of HDAC1, HDAC3 and HDAC9 in obese women in relation to obesity indices and insulin resistance.

It is well-established that an impaired adipose tissue function and morphology caused by a dysregulated gene expression contribute substantially to obesity. Nowadays, animal model studies and in vitro surveys provide evidence for possible roles of HDACs as emerging epigenetic players in the pathogenesis of obesity. However, the clinical pertinence of HDACs in the field of obesity research in humans is not yet obvious. Here, we investigated mRNA expression of HDAC1, 3 and 9 in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) of obese female participants (n = 20) and normal-weight women (n = 19). We also evaluated the association of the afore-mentioned HDACs gene expression with obesity indices, insulin resistance parameters, and other obesity-related characteristics. Our data revealed the mRNA level of HDAC1 was significantly decreased in both VAT and SAT of obese women, compared to controls. Moreover, the SAT mRNA expression of HDAC3 and VAT mRNA levels of HDAC9 were significantly lower in obese subjects than those found in controls. We observed that HDAC1 and HDAC3 expression in adipose tissue from the whole population is inversely correlated with obesity indices; BMI, waist, hip and waist-to-height ratio (WHtR). Moreover, we found that HDAC3 expression in adipose tissue had an inverse correlation with HOMA-IR, insulin levels, and serum concentration of hs-CRP. Moreover, VAT HDAC9 mRNA level is inversely correlated with obesity indices; BMI, waist, hip and WHtR and with HOMA-IR, insulin levels, and serum concentration of hs-CRP. Hence, it seems that decreased HDAC1,3 and 9 mRNA expression in adipose tissue might be associated with obesity and related abnormalities. However, more studies are needed to establish this concept.

HDAC9 over‐expression increases vascular ET‐1 and TLR4 mRNA abundance

Histone deactylases (HDACs) are classical epigenetic modifiers that deacetylate lysine residues on histones but also deacetylate non‐histone proteins. Previous studies suggest HDAC9 is a causative and disease‐relevant gene in stroke and coronary artery disease. Single nucleotide polymorphisms in the HDAC9 gene are associated with an increased risk of cardiovascular disease. As such, inhibitors of HDAC9 are suggested to be vasoprotective. However, the HDAC9 dependent mechanisms leading to vascular dysfunction remain elusive. Endothelial dysfunction is an early initiator of vascular disease. We previously found that vascular tissues with increased HDAC9 expression also showed increased endothelin‐1 (ET‐1) and toll‐like receptor 4 (TLR4). Thus, we hypothesized that HDAC9 directly increases ET‐1 and/or TLR4 expression in vascular tissue. Thoracic aortae and renal vessels were isolated from adult (12 weeks, male and female) HDAC9 over‐expressing mice (HDAC9‐OE) to measure ET‐1 and TLR4 expression. In general, HDAC9‐OE mice showed a 4‐fold increase in HDAC9 expression in the aorta and a 3‐fold upregulation in the renal vessels. RNA expression studies from aortic tissue revealed that both ET‐1 and TLR4 were increased in HDAC9‐OE mice compared to controls (relative mRNA; ET‐1: control 1.00 ±0.99 vs HDAC9‐OE 1.89 ±0.31; TLR4: control 1.00 ±0.78 vs HDAC9‐OE 10.89 ±4.59, n=2–10). Further, renal vessels showed a 5‐fold increase in ET‐1 expression (relative mRNA 1.00 ±0.87 vs 5.53 ±0.64, n=2–10) whereas TLR4 expression was unchanged in HDAC9‐OE mice compared to controls (relative mRNA 1.00 ±0.43 vs 0.96 ±0.18, n=2–10). Previous in vitro studies showed that over‐expression of HDAC9 in transfected mouse aortic endothelial cells upregulates ET‐1 expression supporting our in vivo results. In contrast, plasma of HDAC9‐OE mice showed no difference in circulating ET‐1 levels (control 0.51 ±0.1065 pg/ml vs HDAC9‐OE 0.40 ±0.02655 pg/ml, n=2–10). Therefore, our results indicate that HDAC9 over‐expression in the aorta induces expression of ET‐1 and TLR4 however in renal vessels HDAC9 over‐expression appears to only regulate ET‐1 and not TLR4. The HDAC9 dependent molecular mechanisms leading to ET‐1 expression may be tissue specific and will be investigated further.

Effects of Dexamethasone on HDAC4 Expression and Cellular Localization in C2C12 Cell‐Derived Myotubes: Implications for Skeletal Muscle Atrophy

Skeletal muscle can rapidly decrease its mass and force to various conditions such as denervation, mechanical unload, kidney disease, sepsis and hormonal stimuli such as corticoids. Although it is well known that such conditions can trigger a set of genes related to atrophy (mainly Atrogin‐1 and MuRF1), the specific cellular processes that control atrophy under different conditions are not well understood. Accordingly, histone deacetylases (HDACs) can be important modulators of skeletal muscle atrophy and it has been shown that HDAC4 can trigger Atrogin‐1, and MuRF‐1 expression in denervation model. Nonetheless, it is not known whether HDCA4 is modulated in models involving hormonal stimulation such as corticoids. Thus, our objective was to investigate HDAC4 expression and localization in C2C12 cell‐derived myotubes submitted to dexamethasone treatment. We examined HDAC4 localization, gene expression and protein content on a in vivo, and in vitro models. C2C12 cells, treated with dexamethasone (10 nM) for 12h, 24h and 48h and the results are expressed by means ± SD, and the statistical analysis was performed using Student’s t test (p ≤ 0.05). Initially we confirmed the responsiveness of HDCA4 to atrophy mediated mechanical unload. As expected, we found a marked increase in HDCA4 mRNA expression (~3 fold, p&lt;0.05) after 7 days of hind limb immobilization. Other marker of atrophy was also induced (Atrogin‐1 1.7 fold; p &lt;0.05). In C2C12 cell‐derived myotubes, we confirmed decreased in myotube diameter induced by dexamethasone (~25% p&lt;0.05). In addition, we observed increased HDAC4 mRNA levels after 24 h of dexamethasone treatment (~1.8 fold, p&lt;0.05). Atrogin‐1 mRNA level, as expected, was also elevated (~1.6 fold, p&lt;0.05). Concerning cell location, our preliminary findings suggest a nuclear accumulation of HDAC4 in myotubes treated with dexamethasone. As time course and dose response experiments are ongoing, overall, these results provide further insight into molecular mechanisms involving HDAC4 responses in skeletal muscle. This project was approved by the Ethics Committee on the Use of Animals by the University of São Paulo (Protocol No. 2034200718).Support or Funding InformationFAPESP; CAPES; CNPq

Dense module searching for gene networks associated with multiple sclerosis

BackgroundMultiple sclerosis (MS) is a complex disease in which the immune system attacks the central nervous system. The molecular mechanisms contributing to the etiology of MS remain poorly understood. Genome-wide association studies (GWAS) of MS have identified a small number of genetic loci significant at the genome level, but they are mainly non-coding variants. Network-assisted analysis may help better interpret the functional roles of the variants with association signals and potential translational medicine application. The Dense Module Searching of GWAS tool (dmGWAS version 2.4) developed in our team is applied to 2 MS GWAS datasets (GeneMSA and IMSGC GWAS) using the human protein interactome as the reference network. A dual evaluation strategy is used to generate results with reproducibility.ResultsApproximately 7500 significant network modules were identified for each independent GWAS dataset, and 20 significant modules were identified from the dual evaluation. The top modules included GRB2, HDAC1, JAK2, MAPK1, and STAT3 as central genes. Top module genes were enriched with functional terms such as “regulation of glial cell differentiation” (adjusted p-value = 2.58 × 10− 3), “T-cell costimulation” (adjusted p-value = 2.11 × 10− 6) and “virus receptor activity” (adjusted p-value = 1.67 × 10− 3). Interestingly, top gene networks included several MS FDA approved drug target genes HDAC1, IL2RA, KEAP1, and RELA,ConclusionsOur dmGWAS network analyses highlighted several genes (GRB2, HDAC1, IL2RA, JAK2, KEAP1, MAPK1, RELA and STAT3) in top modules that are promising to interpret GWAS signals and link to MS drug targets. The genes enriched with glial cell differentiation are important for understanding neurodegenerative processes in MS and for remyelination therapy investigation. Importantly, our identified genetic signals enriched in T cell costimulation and viral receptor activity supported the viral infection onset hypothesis for MS.

Cornelia de Lange syndrome: A rare genetic disorder

Cornelia de Lange Syndrome (CdLS) was first reported by Vrolik in 1849 and Brachmann in 1916, followed by Cornelia de Lange in 1933, after whom the syndrome is named. This disorder has a varied presentation but is mainly characterized by distinctive facial features, growth retardation, microcephaly, hirsutism, psychomotor delay, intellectual disability, and malformations of the upper limbs. Initial diagnosis is usually based on clinical features following specific diagnostic scoring systems. The precise prevalence of the disease is unknown but is estimated to be 1–10:100,000. Depending on the mutated gene, Cornelia de Lange syndrome (CdLS) can be inherited in an autosomal dominant manner, when it is caused by variations in the NIPBL, SMC2, or RAD21 genes, or it can have an X-linked inheritance when it is caused by variations in the SMC1A or HDAC8 genes. However, most cases (more than 99%) result from new (de novo) mutations, which means that are not inherited from the parents and occur in people with no family history of the conditionabout 30% of the people affected by the syndrome do not have any known cause. Many studies focused on the importance of neurologic findings and reported an incidence of epilepsy in CdLS ranging from 14% to 25%, especially in the classic and more severe form of the syndrome, but there is no data about its electroclinical features and long-term outcome. Life expectancy is relatively normal for people with CdLS and most affected children live well into adulthood. However, certain features of this condition, particularly severe malformations of the heart or throat, may decrease life expectancy in some affected people. The diagnosis is suspected clinically and later confirmed by clinical exome sequencing.

An Antisense Oligonucleotide Leads to Suppressed Transcription of Hdac2 and Long-Term Memory Enhancement

Knockout of the memory suppressor gene histone deacetylase 2 (Hdac2) in mice elicits cognitive enhancement, and drugs that block HDAC2 have potential as therapeutics for disorders affecting memory. Currently available HDAC2 catalytic activity inhibitors are not fully isoform specific and have short half-lives. Antisense oligonucleotides (ASOs) are drugs that elicit extremely long-lasting, specific inhibition through base pairing with RNA targets. We utilized an ASO to reduce Hdac2 messenger RNA (mRNA) in mice and determined its longevity, specificity, and mechanism of repression. A single injection of the Hdac2-targeted ASO in the central nervous system produced persistent reduction in HDAC2 protein and Hdac2 mRNA levels for 16 weeks. It enhanced object location memory for 8 weeks. RNA sequencing (RNA-seq) analysis of brain tissues revealed that the repression was specific to Hdac2 relative to related Hdac isoforms, and Hdac2 reduction caused alterations in the expression of genes involved in extracellular signal-regulated kinase (ERK) and memory-associated immune signaling pathways. Hdac2-targeted ASOs also suppress a nonpolyadenylated Hdac2 regulatory RNA and elicit direct transcriptional suppression of the Hdac2 gene through stalling RNA polymerase II. These findings identify transcriptional suppression of the target gene as a novel mechanism of action of ASOs.

NF-κB/HDAC1/SREBP1c pathway mediates the inflammation signal in progression of hepatic steatosis

The transcription factor nuclear factor kappa B (NF-κB) is activated in hepatocytes in the pathogenesis of hepatic steatosis. However, the action mechanism of NF-κB remains to be established in the hepatic steatosis. In this study, the P50 subunit of NF-κB was found to promote the hepatic steatosis through regulation of histone deacetylase 1 (HDAC1) in hepatocytes. The activity was supported by the phenotypes of P50 knockout (P50-KO) mice and P65 knockout (P65-KO) mice. Hepatic steatosis was reduced in the P50-KO mice, but not in the P65-KO mice. The reduction was a result of inhibition of HDAC1 activity in the P50-KO cells. Knockdown of Hdac1 gene led to suppression of hepatocyte steatosis in HepG2 cells. A decrease in sterol-regulatory element binding protein 1c (SREBP1c) protein was observed in the liver of P50-KO mice and in cell with Hdac1 knockdown. The decrease was associated with an increase in succinylation of SREBP1c protein. The study suggests that P50 stabilizes HDAC1 to support the SREBP1c activity in hepatic steatosis in the pathophysiological condition. Interruption of this novel pathway in the P50-KO, but not the P65-KO mice, may account for the difference in hepatic phenotypes in the two lines of transgenic mice.

Valproic acid administration exerts protective effects against stress-related anhedonia in rats

Anhedonia or inability to experience pleasure is the sign of various neuropsychiatric conditions. Current treatment options do not provide adequate control of anhedonia. The present study was conducted to evaluate the protective effects of valproic acid (VPA) as a nonspecific histone deacetylase (HDAC) inhibitor to reverse the effects of stress on induction of anhedonia and explore possible mechanisms.To induce anhedonia, a rat model of chronic unpredictable mild stress (CUMS) was established. Animals were assigned into no stress, stress (6 weeks of CUMS) and two treatment groups. VPA treatment was carried out for 4 continuous weeks (200 mg/kg/day). Behavioral assessments were performed using sucrose consumption (SCT) and new object recognition (NOR) tests. The expression of genes was evaluated using qRT-PCR. The cell density was determined using Nissl staining.Rats with CUMS showed depressive-like behaviors and impaired memory performance compared with the non-stressed group (p < 0.01). Moreover, they had significantly higher levels of HDAC3 and MC4R expression in the nucleus accumbens (NAc) compared to the non-stressed group (p < 0.01). The NAc cell density was significantly higher in the non-stressed rats (p < 0.05). Corticosterone plasma level was increased in the CUMS compared to the non-stressed group (p < 0.05). In the CUMS + VPA subgroup, the corticosterone (CORT) plasma level was lower compared with the CUMS + Saline and/or the CUMS groups (p < 0.05).These findings suggest that VPA can improve anhedonia and stress. Although the protective effect of VPA might link to decreasing HDAC3 and MC4R genes expression in NAc.

Valproic acid enhances in vitro developmental competence of porcine handmade cloned embryos

Histone deacetylase inhibitors (HDACi) can change the histone acetylation and significantly enhance the developmental competence of the pre-implantation SCNT embryo. To find a suitable histone deacetylase inhibitor to improve the success rate and potentially developmental ability of handmade cloning (HMC) embryos of porcine, we preferred the histone deacetylase inhibitors VPA to study the effect on HMC embryo development, their histone acetylation level and the expression level of relevant genes. The blastocyst rate and a number of blastocyst cells of HMC embryos treated with VPA (HMC-V) were significantly higher than that of control (HMC-C). In addition, the acetylation level (acH3K14) of HMC-V embryos at 2-cell and blastocyst stage was significantly higher that of PA HMC-C embryos. However, the acetylation H3K14 of embryos at 1-cell, 2-cell and blastocyst stage had no significant difference between PA and HMC-C embryos. The expression level of Hat1, Oct4 and Nanog were higher in HMC-V blastocysts, compared to PA and HMC-C blastocysts. In contrast, the Hdac2 expression level was lower in HMC-V blastocysts compared to those of the PA and HMC-C blastocysts. Our results demonstrated that HMC embryos treated with VPA that could promote the pre-implantation development and increase the levels of histone H3K14 acetylation and expression of the OCT4 gene, while reduced the Hdac2 gene expression with a comparable level of PA and HMC-C embryos. Our results pointed out that VPA could effectively correct the abnormal histone acetylation during the HMC embryo development and subsequently improve the full-term developmental potential of the HMC embryos after embryo transplantation.

Suppressing migration and invasion of H1299 lung cancer cells by honokiol through disrupting expression of an HDAC6-mediated matrix metalloproteinase 9.

Metastasis is the crucial mechanism to cause high mortality in lung cancer. Degradation of extracellular matrix (ECM) by proteolytic enzymes, especially matrix metalloproteinases (MMPs), is a key process for promoting cancer cell migration and invasion. Therefore, targeting MMPs might be a strategy for lung cancer metastasis suppression. Honokiol, a biological active component of Magnolia officinalis, has been indicated to suppress lung cancer tumorigenesis through epigenetic regulation. However, the regulation of MMPs‐mediated migration and invasion by honokiol through epigenetic regulation in lung cancer is still a mystery. In the present study, the migration and invasion ability of H1299 lung cancer was suppressed by noncytotoxic concentrations of honokiol treatment. The proteolytic activity of MMP‐9, rather than MMP‐2, was inhibited in honokiol‐treated H1299 cells. Honokiol‐inhibited MMP‐9 expression was through promoting MMP‐9 protein degradation rather than suppressing transcription mechanism. Furthermore, the expression of specific histone deacetylases 6 (HDAC6) substrate, acetyl‐α‐tubulin, was accumulated after honokiol incubation. The disassociation of MMP‐9 with hyper‐acetylated heat shock protein 90 (Hsp90) was observed resulting in MMP‐9 degradation after honokiol treatment. Meanwhile, honokiol‐suppressed MMP‐9 expression and invasion ability of H1299 lung cancer cells was rescued by HDAC6 overexpression. Accordingly, the results suggested that the suppression of migration and invasion activities by honokiol was through inhibiting HDAC6‐mediated Hsp90/MMP‐9 interaction and followed by MMP‐9 degradation in lung cancer.

Down-regulation of HDAC6 Expression Can Influence KG1α Cell Proliferation by Inhibiting ERK Pathway

To study the inhibitory effect of HDAC6 on proliferation of human leukemia KG1α and to explore its mechanism by ERK signaling pathways. .The siRNA interference technology was used to inhibit the HDAC6 gene expression; the expression of HDAC6 and prateins of ERK signal pathway was detected by Western blot; the cell proliferation ability was detected by colony forming experiment and trypan blue staining; cell cycle was detected by FCM; and the expression of Ki67 was detected by immunofluorescence. Western blot showed that HDAC6 expression was up-regulated in leukemia cell lines in comparison with the healthy volunteers and bone marrow stromal cells (P＜0.05). Knockdown of HDAC6 significantly inhibited the proliferation and colony formation ability of leukemia cells, promoted cell arrest at G0/G1 phase. The Western blot and immunefluorescence showed that knockdown of HDAC6 suppressed the expression level of Ki67, CDK4, Cyclin D1 and enhanced the expression level of p16, p21, p-ERK (P＜0.05). Knockdown of HDAC6 significantly inhibits the proliferation, arrest the cell cycle at G0/G1 phase, and its mechanism probably relates with the activation of ERK signaling pathway.

Down-regulation of HDAC6 Expression Can Influence KG1α Cell Proliferation by Inhibiting ERK Pathway

To study the inhibitory effect of HDAC6 on proliferation of human leukemia KG1α and to explore its mechanism by ERK signaling pathways. .The siRNA interference technology was used to inhibit the HDAC6 gene expression; the expression of HDAC6 and prateins of ERK signal pathway was detected by Western blot; the cell proliferation ability was detected by colony forming experiment and trypan blue staining; cell cycle was detected by FCM; and the expression of Ki67 was detected by immunofluorescence. Western blot showed that HDAC6 expression was up-regulated in leukemia cell lines in comparison with the healthy volunteers and bone marrow stromal cells (P＜0.05). Knockdown of HDAC6 significantly inhibited the proliferation and colony formation ability of leukemia cells, promoted cell arrest at G0/G1 phase. The Western blot and immunefluorescence showed that knockdown of HDAC6 suppressed the expression level of Ki67, CDK4, Cyclin D1 and enhanced the expression level of p16, p21, p-ERK (P＜0.05). Knockdown of HDAC6 significantly inhibits the proliferation, arrest the cell cycle at G0/G1 phase, and its mechanism probably relates with the activation of ERK signaling pathway.