Promoter Hypomethylation Research Articles

Increased expression of Rho-associated protein kinase 2 confers astroglial Stat3 pathway activation during epileptogenesis

Patients with TLE are prone to tolerance to antiepileptic drugs. Based on the perspective of molecular targets for drug resistance, it is necessary to explore effective drug resistant genes and signaling pathways for the treatment of TLE. We performed gene expression profiles in hippocampus of patients with drug-resistant TLE and identified ROCK2 as one of the 20 most significantly increased genes in hippocampus. In vitro and in vivo experiments were performed to identify the potential role of ROCK2 in epileptogenesis. In addition, the activity of Stat3 pathway was tested in rat hippocampal tissues and primary cultured astrocytes. The expression levels of ROCK2 in the hippocampus of TLE patients were significantly increased compared with the control group, which was due to the hypomethylation of ROCK2 promoter. Fasudil, a specific Rho-kinase inhibitor, alleviated epileptic seizures in the pilocarpine rat model of TLE. Furthermore, ROCK2 activated the Stat3 pathway in pilocarpine-treated epilepsy rats, and the spearman correlation method confirmed that ROCK2 is associated with Stat3 activation in TLE patients. In addition, ROCK2 was predominantly expressed in astrocytes during epileptogenesis, and induced epileptogenesis by activating astrocyte cell cycle progression via Stat3 pathway. The overexpressed ROCK2 plays an important role in the pathogenesis of drug-resistant epilepsy. ROCK2 accelerates astrocytes cell cycle progression via the activation of Stat3 pathway likely provides the key to explaining the process of epileptogenesis.

Dual role of allele-specific DNA hypermethylation within the TERT promoter in cancer.

Aberrant activation of telomerase in human cancer is achieved by various alterations within the TERT promoter, including cancer-specific DNA hypermethylation of the TERT hypermethylated oncological region (THOR). However, the impact of allele-specific DNA methylation within the TERT promoter on gene transcription remains incompletely understood. Using allele-specific next-generation sequencing, we screened a large cohort of normal and tumor tissues (n = 652) from 10 cancer types and identified that differential allelic methylation (DAM) of THOR is restricted to cancerous tissue and commonly observed in major cancer types. THOR-DAM was more common in adult cancers, which develop through multiple stages over time, than in childhood brain tumors. Furthermore, THOR-DAM was especially enriched in tumors harboring the activating TERT promoter mutations (TPMs). Functional studies revealed that allele-specific gene expression of TERT requires hypomethylation of the core promoter, both in TPM and TERT WT cancers. However, the expressing allele with hypomethylated core TERT promoter universally exhibits hypermethylation of THOR, while the nonexpressing alleles are either hypermethylated or hypomethylated throughout the promoter. Together, our findings suggest a dual role for allele-specific DNA methylation within the TERT promoter in the regulation of TERT expression in cancer.

Lower HDAC6 mRNA expression and promoter hypomethylation are associated with RA susceptibility

Recent studies showed that Histone deacetylases 6 (HDAC6) inhibitors could improve arthritis in rheumatoid arthritis (RA) rodent models, whereas lower HDAC6 expression was observed in RA patients' synovial fibroblasts, raising the concerns to use HDAC6 inhibitors to treat RA patients. In the present study, we investigated the involvement of HDAC6 mRNA expression and promoter methylation in RA. The DNA and RNAs were extracted from the peripheral blood mononuclear cells (PBMCs) from 138 RA patients and 102 healthy controls. The pyrosequencing technique was used for promoter methylation analysis. The quantitative real-time polymerase chain reaction was used to determine the HDAC6 mRNA expression. The patients' clinical characteristics and disease biomarkers were recorded when blood sampling. The HDAC6 mRNA expression was lower in the RA patients than controls (p=0.001). The RA patients had significant hypomethylation of the HDAC6 promoter (p<0.001). The HDAC6 promoter was hypo-methylated in the -229, -225, -144, and -142 CpG sites in RA patients (p<0.05). Unexpectedly, promoter methylation and mRNA expression of the HDAC6 gene were positively associated (p<0.001). The HDAC6 mRNA expression and promoter methylation status were associated with the risk of RA (p=0.006 and 0.002, respectively). The inflammatory cytokines, TNF-α and IL-6, were significantly increased after HDAC6 knockdown in PMA-stimulated THP1 cells and SW982cells (p<0.05). The HDAC6 mRNA expression and promoter methylation were lower in RA patients. Both HDAC6 mRNA expression level and promoter hypomethylation were associated the susceptibility of RA. HDAC6 inhibitors seem not proper for RA patients' treatment.

FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes.

Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

HIST1H1B Promotes Basal-Like Breast Cancer Progression by Modulating CSF2 Expression.

BackgroundBasal-like breast cancer (BLBC) is associated with a poor clinical outcome; however, the mechanism of BLBC aggressiveness is still unclear. It has been shown that a linker histone functions as either a positive or negative regulator of gene expression in tumors. Here, we aimed to investigate the possible involvement and mechanism of HIST1H1B in BLBC progression.Experimental designWe analyzed multiple gene expression datasets to determine the relevance of HIST1H1B expression with BLBC. We employed quantitative real-time PCR, transwell assay, colony formation assay, and mammosphere assay to dissect the molecular events associated with the expression of HIST1H1B in human breast cancer. We studied the association of HIST1H1B with CSF2 by ChIP assay. Using tumorigenesis assays, we determine the effect of HIST1H1B expression on tumorigenicity of BLBC cells.ResultsHere, we show that the linker histone HIST1H1B is dramatically elevated in BLBC due to HIST1H1B copy number amplification and promoter hypomethylation. HIST1H1B upregulates colony-stimulating factor 2 (CSF2) expression by binding the CSF2 promoter. HIST1H1B expression promotes, whereas knockdown of HIST1H1B expression suppresses tumorigenicity. In breast cancer patients, HIST1H1B expression is positively correlated with large tumor size, high grade, metastasis and poor survival.ConclusionHIST1H1B contributes to basal-like breast cancer progression by modulating CSF2 expression, indicating a potential prognostic marker and therapeutic target for this disease.

Association of CYP26C1 Promoter Hypomethylation with Small Vessel Occlusion in Korean Subjects.

The risk factors for stroke, a fatal disease, include type two diabetes, hypertension, and genetic influences. Small vessel occlusion (SVO) can be affected by epigenetic alterations, but an association between SVO and the methylation of cytochrome P450 family 26 subfamily C member 1 (CYP26C1) has not been identified. In this study, we measured the level of DNA methylation in the CYP26C1 promoter and the 5′ untranslated region of 115 normal subjects and 56 patients with SVO in Korea. The DNA methylation level of each subject was measured by bisulfite amplicon sequencing, and statistical analysis was performed using the general linear model or Pearson’s correlation. The average level of DNA methylation was markedly lower in patients with SVO than in normal subjects (20.4% vs. 17.5%). We found that the methylation of CYP26C1 has a significant positive correlation with blood parameters including white blood cells, hematocrit, lactate dehydrogenase, and Na+ in subjects with SVO. We predicted that binding of RXR-α and RAR-β might be affected by CYP26C1 methylation at CpG sites −246–237 and −294–285. These findings suggest that CYP26C1 methylation in the promoter region may be a predictor of SVO.

The DNMT1-associated lncRNA UCA1 was upregulated in TK6 cells transformed by long-term exposure to hydroquinone and benzene-exposed workers via DNA hypomethylation.

Exposure to benzene or its metabolite hydroquinone (HQ) is a risk factor for a series of myeloid malignancies, and long noncoding RNAs play an important role in the process of pathogenesis. Urothelial cancer-associated 1 (UCA1) functions as an oncogene in the development of acute myeloid leukemia. However, the association between DNMT1 and UCA1 with benzene or HQ exposure has not been explored. We characterized UCA1 expression in cells briefly exposed to HQ (HQ-ST cells) and HQ-induced malignantly transformed (TK6-HT cells) treated with 5-aza-2'-deoxycytidine (5-AzaC) or trichostatin A (TSA). Compared to that in control cells, UCA1 expression was increased, whereas DNMT1 was decreased in HQ-ST cells and TK6-HT cells treated with 5-AzaC or TSA. Moreover, UCA1 expression was also upregulated and positively correlated with benzene exposure time in benzene-exposed workers. Furthermore, the expression of UCA1 was negatively associated with the DNA methylation level of its promoter in benzene-exposed workers. DNMT1 rather than DNMT3b knockout in TK6-HT cells activated the expression of UCA1 by inducing its promoter hypomethylation. These results suggest that benzene or HQ exposure leads to UCA1 upregulation via DNA hypomethylation in the UCA1 promoter, which is mediated by DNMT1.

Epigenetic DNA Modifications Upregulate SPRY2 in Human Colorectal Cancers.

Conventional wisdom is that Sprouty2 (SPRY2), a suppressor of Receptor Tyrosine Kinase (RTK) signaling, functions as a tumor suppressor and is downregulated in many solid tumors. We reported, for the first time, that increased expression of SPRY2 augments cancer phenotype and Epithelial-Mesenchymal-Transition (EMT) in colorectal cancer (CRC). In this report, we assessed epigenetic DNA modifications that regulate SPRY2 expression in CRC. A total of 4 loci within SPRY2 were evaluated for 5mC using Combined Bisulfite Restriction Analysis (COBRA). Previously sequenced 5hmC nano-hmC seal data within SPRY2 promoter and gene body were evaluated in CRC. Combined bioinformatics analyses of SPRY2 CRC transcripts by RNA-seq/microarray and 450K methyl-array data archived in The Cancer Genome Atlas (TCGA) and GEO database were performed. SPRY2 protein in CRC tumors and cells was measured by Western blotting. Increased SPRY2 mRNA was observed across several CRC datasets and increased protein expression was observed among CRC patient samples. For the first time, SPRY2 hypomethylation was identified in adenocarcinomas in the promoter and gene body. We also revealed, for the first time, increases of 5hmC deposition in the promoter region of SPRY2 in CRC. SPRY2 promoter hypomethylation and increased 5hmC may play an influential role in upregulating SPRY2 in CRC.

MiR-105-5p regulates PD-L1 expression and tumor immunogenicity in gastric cancer.

Cancer immunotherapies targeting the interaction between Programmed death 1 (PD-1) and Programmed death ligand 1 (PD-L1) have recently been approved for the treatment of multiple cancer types, including gastric cancer. However, not all patients respond to these therapies, while some eventually acquire resistance. A partial predictive biomarker for positive response to PD-1/PD-L1 therapy is PD-L1 expression, which has been shown to be under strict post-transcriptional control in cancer. By fractionating the PD-L1 3' untranslated region (3'UTR) into multiple overlapping fragments, we identified a small 100-nucleotide-long cis-acting region as being necessary and sufficient for post-transcriptional repression of PD-L1 expression in gastric cancer. In parallel, we performed a correlation analysis between PD-L1 expression and all host miRNAs in stomach cancer patient samples. A single miRNA, miR-105-5p, was predicted to bind to the identified cis-acting 3'UTR region and to negatively correlate with PD-L1 expression. Overexpression of miR-105-5p in gastric cancer cell lines resulted in decreased expression of PD-L1, both at the total protein and surface expression levels, and induced CD8+ T cell activation in co-culture assays. Finally, we show that expression of miR-105-5p in gastric cancer is partly controlled by DNA methylation of a cancer- and germline-specific promoter of its host gene, GABRA3. Dysregulation of miR-105-5p is observed in many cancer types and this study shows the importance of this miRNA in controlling the immunogenicity of cancer cells, thus highlighting it as a potential biomarker for PD-1/PD-L1 therapy and target for combinatorial immunotherapy.

Epigenetics Modifications in Large-Artery Atherosclerosis: A Systematic Review

ObjectivesIn recent years, the evidence of the relationship between epigenetics and acute ischemic stroke (AIS) were accumulating, however, the epigenetic characteristics that directs specifically towards the aetiology of large-artery atherosclerosis (LAA) remain ambiguous. The aim of this study was to highlight the overall evidence concerning the epigenetic mechanisms associated with the occurrence of LAA. Materials and methodsStudies that involve investigations related to epigenetic markers (DNA methylation and RNA modifications) and LAA were retrieved from eleven scientific publication databases. The studies were screened through the pre-set inclusion and exclusion criteria prior to the NOS evaluation. ResultsEligible studies (n=25) were evaluated. Of which, six reported on DNA methylation and 19 studies assessed RNA modifications (16 on miRNAs, two on lncRNAs, and one study on circRNA). Hypomethylation of MTRNR2L8 and ERα promoters; microRNAs (miR-7-2-3p, miR-16, miR-34a-5p, miR-126, miR-143, miR-200b, miR-223, miR-503, miR-1908, miR-146a rs2910164 C/G, miR-149 rs2292832 T/C, miR-200b rs7549819 T/C, miR-34a rs2666433); lncRNA of ZFAS1; and circRNA of hsa_circRNA_102488 were associated with LAA significantly. ConclusionCurrent systematic review highlighted hypomethylation of miRNAs and lncRNA might be the potential biomarkers for LAA.

Oxysterol binding protein-like 3 (OSBPL3) is a novel driver gene that promotes tumor growth in part through R-Ras/Akt signaling in gastric cancer

Gastric cancer (GC) is one of the most lethal malignant tumors. To improve the prognosis of GC, the identification of novel driver genes as therapeutic targets is in urgent need. Here, we aimed to identify novel driver genes and clarify their roles in gastric cancer. OSBPL3 was identified as a candidate driver gene by in silico analysis of public genomic datasets. OSBPL3 expression was analyzed by RT-qPCR and immunohistochemistry in GC cells and tissues. The biological functions and mechanisms of OSBPL3 in GC were examined in vitro and in vivo using GC cells. The association between OSBPL3 expression and clinical outcome in GC patients was also evaluated. Overexpression of OSBPL3 was detected in GC cells with OSBPL3 DNA copy number gains and promoter hypomethylation. OSBPL3-knockdown reduced GC cell growth in vitro and in vivo by inhibiting cell cycle progression. Moreover, an active Ras pull-down assay and western blotting demonstrated that OSBPL3 activates the R-Ras/Akt signaling pathway in GC cells. In a clinical analysis of two GC datasets, high OSBPL3 expression was predictive of a poor prognosis. Our findings suggest that OSBPL3 is a novel driver gene stimulating the R-Ras/Akt signaling pathway and a potential therapeutic target in GC patients.

Lower p66Shc promoter methylation in subjects with chronic renal failure.

ObjectiveTo determine the correlation between DNA methylation of p66Shc promoter and some markers of inflammatory and oxidative stress in chronic renal failure (CRF) patients compared with healthy subjects.MethodsAn observational cross-sectional study was conducted in the nephrology department at Sidi Bouzid Regional Hospital (Tunisia). In total, 39 patients with CRF and 37 healthy subjects were included. Several biochemical parameters were measured. Furthermore, markers of the oxidative and inflammatory status (MDA, TAS, SOD, and CRP) were evaluated. The p66Shc methylation status was determined using the methylation-specific PCR.ResultsOur results showed that levels of blood glucose, urea, creatinine, uric acid, ChT, TG, albuminuria, CRP and MDA were significantly elevated in CRF patients compared to controls. Furthermore, p66Shc promoter region was highly demethylated in CRF patients compared to healthy controls (84% vs 4%). Our data showed a positive correlation between p66Shc hypomethylation and levels of MDA (r = 0.93; p<0, 05) and CRP (r = 0.89; P <0, 05), as well as a significant negative correlation between p66Shc hypomethylation, TAS (r = -0.76; P <0, 05) and SOD (r = -0.77; p<0, 05) levels. Similarly, there was a positive correlation between p66Shc hypomethylation and the disease stages. Importantly, multiple regression analysis showed that p66shc DNA hypomethylation remains strongly correlated with MDA, CRP and stages of CRF.ConclusionThis study indicates that the DNA hypomethylation of p66shc promoter was correlated with oxidative and inflammatory stress and the disease stages in CRF patients.

KIF18b-dependent hypomethylation of PARPBP gene promoter enhances oxaliplatin resistance in colorectal cancer

As the new platinum drug oxaliplatin has been widely used in clinical treatment of colorectal cancer (CRC), oxaliplatin resistance has become a burning problem. In this study, higher expression of PARP-1 binding protein (PARPBP) was detected in oxaliplatin-resistant CRC (OR-CRC) cells than in non-resistant cells. Further research showed that kinesin family member 18 b (KIF18b) induced the overexpression of PARPBP, sustaining oxaliplatin resistance in OR-CRC cells. Through exploring the PARPBP gene promoter, we found that SP1-recruited DNMT3b methylated PARPBP promoter to suppress transcription in CRC cells, and increased KIF18b attenuated the recruitment of DNMT3b to PARPBP promoter by directly interacting with SP1 in OR-CRC cells. Clinical analysis suggested a positive relationship between KIF18b and PARPBP in CRC tissues and indicated poor prognosis in CRC patients with high level of KIF18b or PARPBP. In summary, KIF18b-induced PARPBP contributes to the resistant phenotype of OR-CRC.

Genomic and epigenomic evolution of acquired resistance to combination therapy in esophageal squamous cell carcinoma.

BACKGROUNDTargeted arterial infusion of verapamil combined with chemotherapy (TVCC) is an effective clinical interventional therapy for esophageal squamous cell carcinoma (ESCC), but multidrug resistance (MDR) remains the major cause of relapse or poor prognosis, and the underlying molecular mechanisms of MDR, temporal intratumoral heterogeneity, and clonal evolutionary processes of resistance have not been determined.METHODSTo elucidate the roles of genetic and epigenetic alterations in the evolution of acquired resistance during therapies, we performed whole-exome sequencing on 16 serial specimens from 7 patients with ESCC at every cycle of therapeutic intervention from 3 groups, complete response, partial response, and progressive disease, and we performed whole-genome bisulfite sequencing for 3 of these 7 patients, 1 patient from each group.RESULTSPatients with progressive disease exhibited a substantially higher genomic and epigenomic temporal heterogeneity. Subclonal expansions driven by the beneficial new mutations were observed during combined therapies, which explained the emergence of MDR. Notably, SLC7A8 was identified as a potentially novel MDR gene, and functional assays demonstrated that mutant SLC7A8 promoted the resistance phenotypes of ESCC cell lines. Promoter methylation dynamics during treatments revealed 8 drug resistance protein-coding genes characterized by hypomethylation in promoter regions. Intriguingly, promoter hypomethylation of SLC8A3 and mutant SLC7A8 were enriched in an identical pathway, protein digestion and absorption, indicating a potentially novel MDR mechanism during treatments.CONCLUSIONOur integrated multiomics investigations revealed the dynamics of temporal genetic and epigenetic inter- and intratumoral heterogeneity, clonal evolutionary processes, and epigenomic changes, providing potential MDR therapeutic targets in treatment-resistant patients with ESCC during combined therapies.FUNDINGNational Natural Science Foundation of China, Science Foundation of Peking University Cancer Hospital, CAMS Innovation Fund for Medical Sciences, Major Program of Shenzhen Bay Laboratory, Guangdong Basic and Applied Basic Research Foundation, and the third round of public welfare development and reform pilot projects of Beijing Municipal Medical Research Institutes.

Aberrant expression of lncRNAs SNHG6, TRPM2-AS1, MIR4435-2HG, and hypomethylation of TRPM2-AS1 promoter in colorectal cancer.

Accumulating evidence has indicated that deregulation of lncRNAs plays essential roles in colorectal cancer (CRC) carcinogenesis. The goal of this study was to analyze the expression of lncRNAs in colorectal cancer and their association with clinicopathological variables. Bioinformatics analysis of published CRC microarray data was performed to identify the important lncRNAs. The expression levels of candidate genes were assessed in the human colon cancer/normal cell lines, CRC, adenomatous colorectal polyps, and their marginal tissues by qRT-PCR. Moreover, the methylation status of the TRPM2-AS1 promoter was studied using qMSP assay. Furthermore, we investigated the molecular mechanisms of these lncRNAs in CRC progression using in silico analysis. Microarray analysis revealed that lncRNAs SNHG6, MIR4435-2HG, and TRPM2-AS1 were upregulated in CRC. These results were validated in colon cell lines. Moreover, qRT-PCR showed that the expression levels of SNHG6 and TRPM2-AS1 were upregulated in the colorectal tumor tissues compared with their paired tissues. Nonetheless, there was no significant increase in MIR4435-2HG expression in CRC samples. Furthermore, we observed a significant hypomethylation of TRPM2-AS1 promoter and its activation in CRC tissues. By in silico analysis, we found that the lncRNAs upregulation could promote proliferation and drug resistance of colorectal cancer cells via miRNAs sponging and modulation of their targets expression. In conclusion, based on our results upregulation of SNHG6 and TRPM2-AS1, and hypomethylation of TRPM2-AS1 promoter might be considered as potential diagnostic biomarkers for CRC initiation and development.

DZIP1 Expression as a Prognostic Marker in Gastric Cancer: A Bioinformatics-Based Analysis.

PurposeGastric cancer (GC) is a common type of cancer worldwide. It can relapse and metastasize even after standard treatment; therefore, it has a poor prognosis. Moreover, sensitive biomarkers for prognosis prediction in GC are lacking. In this study, using a bioinformatics approach, we aimed to examine the value of DAZ Interacting Protein 1 (DZIP1) as a prognostic predictor and therapeutic target in GC.MethodsWe explored the clinical relevance, function, and molecular role of DZIP1 in GC using MethSurv, cBioPortal, TIMER, Gene Expression Profiling Interactive Analysis, IMEx, ONCOMINE, MEXPRESS, and EWAS Atlas databases. The GSE118919 dataset was used to plot receiver operating characteristic curves. Using The Cancer Genome Atlas, we developed a Cox regression model and assessed the clinical significance of DZIPs. In addition, we used the “xCELL” algorithm to make reliable immune infiltration estimations. Western blot and immunohistochemistry were used to examine protein expression. The results were visualized with the ‘ggplot2ʹ and “circlize” packages.ResultsIn GC patients, DZIP1 was over-expressed at both the mRNA and protein levels. High levels of DZIP1 were found to be associated with poor survival in patients with GC. Our results indicated that DZIP1 could be involved in multiple cancer-related pathways such as the PI3K-Akt signaling pathway, WNT signaling pathway, and RAS signaling pathway, and its expression was correlated with the infiltration of activated myeloid dendritic cells, naive CD4+ T cells, and naive CD8+ T cells. Furthermore, we found that mutations in DZIP1 were correlated with a good prognosis in GC patients. Finally, we demonstrated a correlation between hypomethylation of the DZIP1 gene promoter and a poor prognosis in GC.ConclusionThis study is the first to demonstrate a significant correlation between high levels of DZIP1 and a poor prognosis in GC patients. Our results clarify multiple potential mechanisms that could contribute to this correlation and may thus provide novel insights into the clinical diagnosis and treatment of GC.

Ginsenoside Rh2 upregulates long noncoding RNA STXBP5-AS1 to sponge microRNA-4425 in suppressing breast cancer cell proliferation

BackgroundGinsenoside Rh2, a major saponin derivative in ginseng extract, is recognized for its anticancer activities. Compared to coding genes, studies on long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) that are regulated by Rh2 in cancer cells, especially on competitive endogenous RNA (ceRNA) are sparse. MethodsLncRNAs whose promoter DNA methylation level was significantly altered by Rh2 were screened from methylation array data. The effect of STXBP5-AS1, miR-4425, and RNF217 on the proliferation and apoptosis of MCF-7 breast cancer cells was monitored in the presence of Rh2 after deregulating the corresponding gene. The ceRNA relationship between STXBP5-AS1 and miR-4425 was examined by measuring the luciferase activity of a recombinant luciferase/STXBP5-AS1 plasmid construct in the presence of mimic miR-4425. ResultsInhibition of STXBP5-AS1 decreased apoptosis but stimulated growth of the MCF-7 cells, suggesting tumor-suppressive activity of the lncRNA. MiR-4425 was identified to have a binding site on STXBP5-AS1 and proven to be downregulated by STXBP5-AS1 as well as by Rh2. In contrast to STXBP5-AS1, miR-4425 showed pro-proliferation activity by inducing a decrease in apoptosis but increased growth of the MCF-7 cells. MiR-4425 decreased luciferase activity from the luciferase/STXBP5-AS1 construct by 26%. Screening the target genes of miR-4425 and Rh2 revealed that Rh2, STXBP5-AS1, and miR-4425 consistently regulated tumor suppressor RNF217 at both the RNA and protein level. ConclusionLncRNA STXBP5-AS1 is upregulated by Rh2 via promoter hypomethylation and acts as a ceRNA, sponging the oncogenic miR-4425. Therefore, Rh2 controls the STXBP5-AS1/miR-4425/RNF217 axis to suppress breast cancer cell growth.

Circular RNA 0025984 Ameliorates Ischemic Stroke Injury and Protects Astrocytes Through miR-143-3p/TET1/ORP150 Pathway.

MiR-143-3p is aberrantly expressed in patients with ischemic stroke and associated with ischemic brain injury. However, the underlying mechanisms are largely unknown. Here, we confirmed circ_0025984 and TET1 as a sponge and target of miR-143-3p, respectively, by luciferase reporter assay. In astrocytes, OGD significantly decreased circ_0025984 and TET1 levels but increased miR-143-3p levels, which was also observed in brains of mice with MCAO. Treatment with miR-143-3p inhibitor or circ_0025984 significantly decreased astrocyte apoptosis and autophagy, as well as cerebral injury and neuron loss in mice with MCAO. Notably, TET1 overexpression decreased astrocyte apoptosis and autophagy and induced promoter hypomethylation and expression of ORP150. Our results demonstrated for the first time that circ_0025984 protects astrocytes from ischemia-induced autophagy and apoptosis by targeting the miR-143-3p/TET1 pathway and might inhibit cerebral injury induced by ischemic stroke. Furthermore, our data revealed the important positive regulation of ORP150 by TET1, which could be associated with its neuroprotective role. Graphical abstract Model for signaling pathway of circ_0025984/miR-143-3p/TET1 inastrocytes cultured under OGD. In astrocytes, circ_0025984 acts as a sponge of miR-143-3p, which directly targets TET1 and decreases its expression (A). After translocatinginto the nucleus, TET1 binds to the promoter of ORP150, converts 5mC into 5hmC,leading to DNA demethylation and increased expression of ORP150 (B). In astrocytescultured under OGD, ER stress is induced and eventually leads to apoptosis andautophagy mediated by ATG7, which is regulated by circ_0025984 via ORP150 andGRP78 (C).

Adipose Tissue Hypoxia Correlates with Adipokine Hypomethylation and Vascular Dysfunction.

Obesity is characterized by the accumulation of dysfunctional adipose tissues, which predisposes to cardiometabolic diseases. Our previous in vitro studies demonstrated a role of hypoxia in inducing adipokine hypomethylation in adipocytes. We sought to examine this mechanism in visceral adipose tissues (VATs) from obese individuals and its correlation with cardiometabolic risk factors. We propose an involvement of the hypoxia-inducible factor, HIF1α, and the DNA hydroxymethylase, TET1. Blood samples and VAT biopsies were obtained from obese and non-obese subjects (n = 60 each) having bariatric and elective surgeries, respectively. The analyses of VAT showed lower vascularity, and higher levels of HIF1α and TET1 proteins in the obese subjects than controls. Global hypomethylation and hydroxymethylation were observed in VAT from obese subjects along with promoter hypomethylation of several pro-inflammatory adipokines. TET1 protein was enriched near the promotor of the hypomethylated adipokines. The average levels of adipokine methylation correlated positively with vascularity and arteriolar vasoreactivity and negatively with protein levels of HIF1α and TET1 in corresponding VAT samples, serum and tissue inflammatory markers, and other cardiometabolic risk factors. These findings suggest a role for adipose tissue hypoxia in causing epigenetic alterations, which could explain the increased production of adipocytokines and ultimately, vascular dysfunction in obesity.

Long-Term Effects of Maternal Low-Protein Diet and Post-weaning High-Fat Feeding on Glucose Metabolism and Hypothalamic POMC Promoter Methylation in Offspring Mice.

Substantial evidence indicated that maternal malnutrition could increase the susceptibility to obesity, insulin resistance, and type 2 diabetes in adulthood. It is increasingly apparent that the brain, especially the hypothalamus, plays a critical role in glucose homeostasis. However, little information is known about the mechanisms linking maternal protein restriction combined with post-weaning high-fat (HF) feeding with altered expression of brain neurotransmitters, and investigations into the epigenetic modifications of hypothalamus in offspring have not been fully elucidated. Our objective was to explore the effects of maternal protein restriction combined with post-weaning HF feeding on glucose metabolism and hypothalamic POMC methylation in male offspring mice. C57/BL6 mice were fed on either low-protein (LP) or normal chow (NC) diet throughout gestation and lactation. Then, the male offspring were randomly weaned to either NC or high-fat (HF) diet until 32 weeks of age. Gene expressions and DNA methylation of hypothalamic proopiomelanocortin (POMC) and melanocortin receptor 4 (MC4R) were determined in male offspring. The results showed that birth weights and body weights at weaning were both significantly lower in male offspring mice of the dams fed with a LP diet. Maternal protein restriction combined with post-weaning high-fat feeding, predisposes higher body weight, persistent glucose intolerance (from weaning to 32 weeks of age), hyperinsulinemia, and hyperleptinemia in male offspring mice. POMC and MC4R expressions were significantly increased in offspring mice fed with maternal LP and postnatal high-fat diet (P < 0.05). Furthermore, maternal protein restriction combined with post-weaning high-fat feeding induced hypomethylation of POMC promoter in the hypothalamus (P < 0.05) and POMC-specific methylation (%) was negatively correlated with the glucose response to a glucose load in male offspring mice (r = −0.42, P = 0.039). In conclusion, maternal LP diet combined with post-weaning high-fat feeding predisposed the male offspring to impaired glucose metabolism and hypothalamic POMC hypomethylation. These findings can advance our thinking about hypothalamic POMC gene methylation between maternal LP diet combined with post-weaning high-fat feeding and metabolic health in offspring.