DNA Methylation Inhibitor Research Articles

Relative expression of key genes involved in nucleic acids methylation in Anopheles gambiae sensu stricto.

In vertebrates, enzymes responsible for DNA methylation, one of the epigenetic mechanisms, are encoded by genes falling into the cytosine methyltransferases genes family (Dnmt1, Dnmt3a,b and Dnmt3L). However, in Diptera, only the methyltransferase Dnmt2 was found, suggesting that DNA methylation might act differently for species in this order. Moreover, genes involved in epigenetic dynamics, such as Ten-eleven Translocation dioxygenases (TET) and Methyl-CpG-binding domain (MBDs), present in vertebrates, might play a role in insects. This work aimed at investigating nucleic acids methylation in the malaria vector Anopheles gambiae (Diptera: Culicidae) by analysing the expression of Dnmt2, TET2 and MBDs genes using quantitative real-time polymerase chain reaction (qRT-PCR) at pre-immature stages and in reproductive tissues of adult mosquitoes. In addition, the effect of two DNA methylation inhibitors on larval survival was evaluated. The qPCR results showed an overall low expression of Dnmt2 at all developmental stages and in adult reproductive tissues. In contrast, MBD and TET2 showed an overall higher expression. In adult mosquito reproductive tissues, the expression level of the three genes in males' testes was significantly higher than that in females' ovaries. The chemical treatments did not affect larval survival. The findings suggest that mechanisms other than DNA methylation underlie epigenetic regulation in An. gambiae.

Multi-omic analysis of the extension of broccoli quality during storage by folic acid

IntroductionFolic acid (FA) is a critical metabolite in all living organisms and an important nutritional component of broccoli. Few studies have been conducted on the impact of an exogenous application of FA on the postharvest physiology of fruits and vegetables during storage. In this regard, the mechanism by which an exogenous application of FA extends the postharvest quality of broccoli is unclear. ObjectiveThis study utilized a multicomponent analysis to investigate how an exogenous application of FA effects the postharvest quality of broccoli. MethodsBroccoli was soaked in 5 mg/L FA for 10 min and the effect of the treatment on the appearance and nutritional quality of broccoli was evaluated. These data were combined with transcriptomic, metabolomic, and DNA methylation data to provide insight into the potential mechanism by which FA delays senescence. ResultsThe FA treatment inhibited the yellowing of broccoli during storage. CHH methylation was identified as the main type of methylation that occurs in broccoli and the FA treatment was found to inhibit DNA methylation, promote the accumulation of endogenous FA and chlorophyl, and inhibit ethylene biosynthesis in stored broccoli. The FA treatment also prevented the formation of off-odors by inhibiting the degradation of glucosinolate. ConclusionsFA treatment inhibited the loss of nutrients during the storage of broccoli, delayed its yellowing, and inhibited the generation of off-odors. Our study provides deeper insight into the mechanism by which the postharvest application of FA delays postharvest senescence in broccoli and provides the foundation for further studies of postharvest metabolism in broccoli.

Allele-Specific Epigenetic Regulation of FURIN Expression at a Coronary Artery Disease Susceptibility Locus.

Genome-wide association studies have revealed an association between the genetic variant rs17514846 in the FURIN gene and coronary artery disease. We investigated the mechanism through which rs17514846 modulates FURIN expression. An analysis of isogenic monocytic cell lines showed that the cells of the rs17514846 A/A genotype expressed higher levels of FURIN than cells of the C/C genotype. Pyrosequencing showed that the cytosine (in a CpG motif) at the rs17514846 position on the C allele was methylated. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine increased FURIN expression. An electrophoretic mobility super-shift assay with a probe corresponding to the DNA sequence at and around the rs17514846 position of the C allele detected DNA-protein complex bands that were altered by an anti-MeCP2 antibody. A chromatin immunoprecipitation assay with the anti-MeCP2 antibody showed an enrichment of the DNA sequence containing the rs17514846 site. siRNA-mediated knockdown of MeCP2 caused an increase in FURIN expression. Furthermore, MeCP2 knockdown increased monocyte migration and proliferation, and this effect was diminished by a FURIN inhibitor. The results of our study suggest that DNA methylation inhibits FURIN expression and that the coronary artery disease-predisposing variant rs17514846 modulates FURIN expression and monocyte migration via an allele-specific effect on DNA methylation.

Halting aberrant DNA methylation via in silico Identification of potent inhibitors of DNMT3B enzyme: Atomistic insights

To date, Cancer remains a global threat due to its impact on growing life expectancy. With the many efforts and methods of combating the disease, complete success remains a challenge owing to several limitations including cancer cells developing resistance through mutations, off-target effect of some cancer drugs resulting in toxicities, among many others. Aberrant DNA methylation is understood to be the primary reason for improper gene silence, which can result in neoplastic transformation, carcinogenesis, and tumour progression. DNA methyltransferase B (DNMT3B) enzyme is considered a potential target for the treatment of several cancers due to its important role in DNA methylation. However, only a few DNMT3B inhibitors have been reported to date. Herein, in silico molecular recognition techniques such as Molecular docking, Pharmacophore-based virtual screen and MD simulation were employed to identify potential inhibitors of DNMT3B that can halt aberrancy in DNA methylation. Findings initially identified 878 hit compounds based on a designed pharmacophore model from the reference compound Hypericin. Molecular docking was used to rank the hits by testing their efficiency when bound to the target enzyme and the top three (3) selected. All three (3) of the top hits showed excellent pharmacokinetic properties but two (2) (Zinc33330198 and Zinc77235130) were identified to be non-toxic. Molecular dynamic simulation of the final two hits showed good stability, flexibility, and structural rigidity of the compounds on DNMT3B. Finally, thermodynamic energy estimations show both compounds had favourable free energies comprising − 26.04 kcal/mol for Zinc77235130 and − 15.73 kcal/mol for Zinc33330198. Amongst the final two hits, Zinc77235130 showed consistency in favourable results across all the tested parameters and was thus selected as the lead compound for further experimental validation. The identification of this lead compound will form important basis for the inhibition of aberrant DNA methylation in cancer therapy.

1683-P: High-Fat Diet Downregulates CREG1 in the Liver via DNA Methylation

CREG1 is an endosomal-lysosomal protein which is highly expressed in the liver. Hepatocyte-specific deletion of Creg1 in mice causes insulin resistance and elevated glycemia. To test if CREG1 is nutritionally regulated, we fed mice for 10 weeks with a high-fat diet (HFD) which progressively downregulated CREG1 in the liver at both protein and mRNA levels. The CREG1 expression was restored after switch back to a standard chow diet for two weeks. The diet-induced CREG1 changes negatively correlated with blood glucose levels. Sequence analysis revealed enrichment of CpG islands in exon 1 of both human and mouse CREG1 genes. To test if methylation of these CpG islands contributes to HFD-induced CREG1 reduction, we fed mice with a HFD for 10 weeks and treated them with the DNA methyltransferase inhibitor 5-aza-deoxycytidine, which reversed HFD-induced CREG1 downregulation in the liver. At the cellular level, overload of cultured human hepatocytes with fatty acids reduced CREG1 expression, and this was reversed by inhibiting DNA methylation. Furthermore, removal of all CpG islands in the first exon with sense mutations abolished lipid overload-induced CREG1 downregulation. These results suggest that HFD downregulates CREG1 via DNA methylation of the CpG islands in the first exon. It would be interesting to test if CREG1 reduction mediates HFD-induced insulin resistance. Disclosure Y.Qi: None. J.Liu: None. J.C.Chao: None. S.A.Rahimi: None. L.Y.Lee: Speaker's Bureau; Abbott. S.Li: None. Funding Rutgers Biomedical and Health Sciences

Less Severe Polymicrobial Sepsis in Conditional mgmt-Deleted Mice Using LysM-Cre System, Impacts of DNA Methylation and MGMT Inhibitor in Sepsis.

The O6-methylguanine-DNA methyltransferase (MGMT) is a DNA suicide repair enzyme that might be important during sepsis but has never been explored. Then, the proteomic analysis of lipopolysaccharide (LPS)-stimulated wild-type (WT) macrophages increased proteasome proteins and reduced oxidative phosphorylation proteins compared with control, possibly related to cell injury. With LPS stimulation, mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated less profound inflammation; supernatant cytokines (TNF-α, IL-6, and IL-10) and pro-inflammatory genes (iNOS and IL-1β), with higher DNA break (phosphohistone H2AX) and cell-free DNA, but not malondialdehyde (the oxidative stress), compared with the littermate control (mgmtflox/flox; LysM-Cre-/-). In parallel, mgmt null mice (MGMT loss only in the myeloid cells) demonstrated less severe sepsis in the cecal ligation and puncture (CLP) model (with antibiotics), as indicated by survival and other parameters compared with sepsis in the littermate control. The mgmt null protective effect was lost in CLP mice without antibiotics, highlighting the importance of microbial control during sepsis immune modulation. However, an MGMT inhibitor in CLP with antibiotics in WT mice attenuated serum cytokines but not mortality, requiring further studies. In conclusion, an absence of mgmt in macrophages resulted in less severe CLP sepsis, implying a possible influence of guanine DNA methylation and repair in macrophages during sepsis.

Synergistic treatment of genetic kidney disease with DNA methylation inhibitors

A combination of drugs reduces the growth of cysts related to autosomal dominant polycystic kidney disease.

Inhibition of DNA and RNA methylation disturbs root development of moso bamboo.

DNA methylation (5mC) and N6-methyladenosine (m6A) are two important epigenetics regulation, which presents profound impact on plant growth development. Phyllostachys edulis (P. edulis) is one of the fastest spreading plants due to its well-developed root system. However, the association between 5mC and m6A were seldom reported in P. edulis. Especially, the connection between m6A and several post-transcriptional regulations remains uncharacterized in P. edulis. Here, our morphological and electron microscope observation shown phenotype of increased lateral root under RNA methylation inhibitor (DZnepA) and DNA methylation inhibitor (5-azaC) treatment. RNA epitranscriptome based on Nanopore direct RNA sequencing (DRS) revealed that DZnepA treatment exhibits significantly decreased m6A level in 3' UTR, which was accompanied by increased gene expression, full-length ratio, higher proximal poly(A) site usage, and shorter poly(A) tail length, respectively. DNA methylation levels of CG and CHG were reduced in both CDS and transposable element (TE) upon 5-azaC treatment. Cell wall synthesis was impaired under methylation inhibition. Especially, differentially expressed genes (DEGs) presented high percentage of overlap between DZnepA and 5-azaC treatment, which suggested a potential correlation between two methylations. This study provides preliminary information for a better understanding of the link between m6A and 5mC in root development of moso bamboo.

AtSAMS regulates floral organ development by DNA methylation and ethylene signaling pathway

S-adenosylmethionine synthase is the key enzyme involved in the biosynthesis of S-adenosylmethionine, which serves as the universal methyl group donor and a common precursor for the biosynthesis of ethylene and polyamines. However, little is known about how SAMS controls plant development. Here, we report that the abnormal floral organ development in the AtSAMS-overexpressing plants is caused by DNA demethylation and ethylene signaling. The whole-genome DNA methylation level decreased, and ethylene content increased in SAMOE. Wild-type plants treated with DNA methylation inhibitor mimicked the phenotypes and the ethylene levels in SAMOE, suggesting that DNA demethylation enhanced ethylene biosynthesis, which led to abnormal floral organ development. DNA demethylation and elevated ethylene resulted in changes in the expression of ABCE genes, which is essential for floral organ development. Furthermore, the transcript levels of ACE genes were highly correlated to their methylation levels, except for the down-regulation of the B gene, which might have resulted from demethylation-independent ethylene signaling. SAMS-mediated methylation and ethylene signaling might create crosstalk in the process of floral organ development. Together, we provide evidence that AtSAMS regulates floral organ development by DNA methylation and ethylene signaling pathway.

The role of DNA methylation in progression of neurological disorders and neurodegenerative diseases as well as the prospect of using DNA methylation inhibitors as therapeutic agents for such disorders.

This review aims to describe the role of DNA methylation in inhibiting and progressing neurological and neurodegenerative disorders and therapeutic approaches. A Systematic search of PubMed, Web of Science, and Cochrane Library was conducted for all qualified studies from 2000 to 2022. For the current need of time, we have focused on the DNA methylation role in neurological and neurodegenerative diseases and the expression of genes involved in neurodegeneration such as Alzheimer's, Depression, and Rett Syndrome. Finally, it appears that the various epigenetic changes do not occur separately and that DNA methylation and histone modification changes occur side by side and affect each other. We focused on the role of modification of DNA methylation in several genes associated with depression (NR3C1, NR3C2, CRHR1, SLC6A4, BDNF, and FKBP5), Rett syndrome (MECP2), Alzheimer's, depression (APP, BACE1, BIN1 or ANK1) and Parkinson's disease (SNCA), as well as the co-occurring modifications to histones and expression of non-coding RNAs. Understanding these epigenetic changes and their interactions will lead to better treatment strategies. This review captures the state of understanding of the epigenetics of neurological and neurodegenerative diseases. With new epigenetic mechanisms and targets undoubtedly on the horizon, pharmacological modulation and regulation of epigenetic processes in the brain holds great promise for therapy.

Targeting the ADPKD methylome using nanoparticle-mediated combination therapy.

DNA methylation aberrancies are found in autosomal dominant polycystic kidney disease (ADPKD), which suggests the methylome to be a promising therapeutic target. However, the impact of combining DNA methylation inhibitors (DNMTi) and ADPKD drugs in treating ADPKD and on disease-associated methylation patterns has not been fully explored. To test this, ADPKD drugs, metformin and tolvaptan (MT), were delivered in combination with DNMTi 5-aza-2'-deoxycytidine (Aza) to 2D or 3D cystic Pkd1 heterozygous renal epithelial cells (PKD1-Het cells) as free drugs or within nanoparticles to enable direct delivery for future in vivo applications. We found Aza synergizes with MT to reduce cell viability and cystic growth. Reduced representation bisulfite sequencing (RRBS) was performed across four groups: PBS, Free-Aza (Aza), Free-Aza+MT (F-MTAza), and Nanoparticle-Aza+MT (NP-MTAza). Global methylation patterns showed that while Aza alone induces a unimodal intermediate methylation landscape, Aza+MT recovers the bimodality reminiscent of somatic methylomes. Importantly, site-specific methylation changes associated with F-MTAza and NP-MTAza were largely conserved including hypomethylation at ADPKD-associated genes. Notably, we report hypomethylation of cancer-associated genes implicated in ADPKD pathogenesis as well as new target genes that may provide additional therapeutic effects. Overall, this study motivates future work to further elucidate the regulatory mechanisms of observed drug synergy and apply these combination therapies in vivo.

5-Azacytidine- and retinoic-acid-induced reprogramming of DCCs into dormancy suppresses metastasis via restored TGF-β-SMAD4 signaling

Disseminated cancer cells (DCCs) in secondary organs can remain dormant for years to decades before reactivating into overt metastasis. Microenvironmental signals leading to cancer cell chromatin remodeling and transcriptional reprogramming appear to control onset and escape from dormancy. Here, we reveal that the therapeutic combination of the DNA methylation inhibitor 5-azacytidine (AZA) and the retinoic acid receptor ligands all-trans retinoic acid (atRA) or AM80, an RARα-specific agonist, promotes stable dormancy in cancer cells. Treatment of head and neck squamous cell carcinoma (HNSCC) or breast cancer cells with AZA+atRA induces a SMAD2/3/4-dependent transcriptional program that restores transforming growth factor β (TGF-β)-signaling and anti-proliferative function. Significantly, either combination, AZA+atRA or AZA+AM80, strongly suppresses HNSCC lung metastasis formation by inducing and maintaining solitary DCCs in a SMAD4+/NR2F1+ non-proliferative state. Notably, SMAD4 knockdown is sufficient to drive resistance to AZA+atRA-induced dormancy. We conclude that therapeutic doses of AZA and RAR agonists may induce and/or maintain dormancy and significantly limit metastasis development.

Characteristics of Salvia miltiorrhiza methylome and the regulatory mechanism of DNA methylation in tanshinone biosynthesis.

Salvia miltiorrhiza is a model medicinal plant with significant economic and medicinal value. Its roots produce a group of diterpenoid lipophilic bioactive components, termed tanshinones. Biosynthesis and regulation of tanshinones has attracted widespread interest. However, the methylome of S. miltiorrhiza has not been analysed and the regulatory mechanism of DNA methylation in tanshinone production is largely unknown. Here we report single-base resolution DNA methylomes from roots and leaves. Comparative analysis revealed differential methylation patterns for CG, CHG, and CHH contexts and the association between DNA methylation and the expression of genes and small RNAs. Lowly methylated genes always had higher expression levels and 24-nucleotide sRNAs could be key players in the RdDM pathway in S. miltiorrhiza. DNA methylation variation analysis showed that CHH methylation contributed mostly to the difference. Go enrichment analysis showed that diterpenoid biosynthetic process was significantly enriched for genes with downstream overlapping with hypoCHHDMR in July_root when comparing with those in March_root. Tanshinone biosynthesis-related enzyme genes, such as DXS2, CMK, IDI1, HMGR2, DXR, MDS, CYP76AH1, 2OGD25, and CYP71D373, were less CHH methylated in gene promoters or downstream regions in roots collected in July than those collected in March. Consistently, gene expression was up-regulated in S. miltiorrhiza roots collected in July compared with March and the treatment of DNA methylation inhibitor 5-azacytidine significantly promoted tanshinone production. It suggests that DNA methylation plays a significant regulatory role in tanshinone biosynthesis in S. miltiorrhiza through changing the levels of CHH methylation in promoters or downstreams of key enzyme genes.

Role of histone H3K4 methyltransferase in regulating Monascus pigments production by red light-coupled magnetic field.

Light, magnetic field, and methylation affected the growth and secondary metabolism of fungi. The regulation effect of the three factors on the growth and Monascus pigments (MPs) synthesis of Monascus purpureus was investigated in this study. 5-azacytidine (5-AzaC), DNA methylation inhibitor, was used to treat M. purpureus (wild-type, WT). Twenty micromolar 5-AzaC significantly promoted the growth, development, and MPs yield. Moreover, 250 lux red light and red light coupled magnetic field (RLCMF) significantly promoted the biomass. For WT, red light, and RLCMF significantly promoted MPs yield. But compared with red light treatment, only 0.2 mT RLCMF promoted the alcohol-soluble MPs yield. For histone H3K4 methyltransferase complex subunit Ash2 gene knockout strain (ΔAsh2), only 0.2 mT RLCMF significantly promoted water-soluble MPs yield. Yet red light, 1.0 and 0.2 mT RLCMF significantly promoted alcohol-soluble MPs yield. This indicated that methylation affected the MPs biosynthesis. Red light and weaker MF had a synergistic effect on the growth and MPs synthesis of ΔAsh2. This result was further confirmed by the expression of related genes. Therefore, histone H3K4 methyltransferase was involved in the regulation of the growth, development, and MPs synthesis of M. purpureus by the RLCMF.

Combined inhibition of histone deacetylase and cytidine deaminase improves epigenetic potency of decitabine in colorectal adenocarcinomas

BackgroundTargeting the epigenome of cancerous diseases represents an innovative approach, and the DNA methylation inhibitor decitabine is recommended for the treatment of hematological malignancies. Although epigenetic alterations are also common to solid tumors, the therapeutic efficacy of decitabine in colorectal adenocarcinomas (COAD) is unfavorable. Current research focuses on an identification of combination therapies either with chemotherapeutics or checkpoint inhibitors in modulating the tumor microenvironment. Here we report a series of molecular investigations to evaluate potency of decitabine, the histone deacetylase inhibitor PBA and the cytidine deaminase (CDA) inhibitor tetrahydrouridine (THU) in patient derived functional and p53 null colon cancer cell lines (CCCL). We focused on the inhibition of cell proliferation, the recovery of tumor suppressors and programmed cell death, and established clinical relevance by evaluating drug responsive genes among 270 COAD patients. Furthermore, we evaluated treatment responses based on CpG island density.ResultsDecitabine caused marked repression of the DNMT1 protein. Conversely, PBA treatment of CCCL recovered acetylation of histone 3 lysine residues, and this enabled an open chromatin state. Unlike single decitabine treatment, the combined decitabine/PBA treatment caused > 95% inhibition of cell proliferation, prevented cell cycle progression especially in the S and G2-phase and induced programmed cell death. Decitabine and PBA differed in their ability to facilitate re-expression of genes localized on different chromosomes, and the combined decitabine/PBA treatment was most effective in the re-expression of 40 tumor suppressors and 13 genes typically silenced in cancer-associated genomic regions of COAD patients. Furthermore, this treatment repressed expression of 11 survival (anti-apoptotic) genes and augmented expression of X-chromosome inactivated genes, especially the lncRNA Xist to facilitate p53-mediated apoptosis. Pharmacological inhibition of CDA by THU or its gene knockdown prevented decitabine inactivation. Strikingly, PBA treatment recovered the expression of the decitabine drug-uptake transporter SLC15A1, thus enabling high tumor drug-loads. Finally, for 26 drug responsive genes we demonstrated improved survival in COAD patients.ConclusionThe combined decitabine/PBA/THU drug treatment improved drug potency considerably, and given their existing regulatory approval, our findings merit prospective clinical trials for the triple combination in COAD patients.

Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer

BackgroundMethylation of the p16 promoter resulting in epigenetic gene silencing—known as p16 epimutation—is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis.MethodsWe established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious.ResultsMice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone.ConclusionsOur study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

ASXL1 disruption preserves CD8 T cell exhaustion-progenitor epigenetic programming during chronic stimulation

Abstract Differentiation of CD8 T cells into a terminally exhausted state is a major hurdle limiting efficacy of both immune checkpoint blockade and adoptive cellular immunotherapy approaches. Prior studies describing disruption of the epigenetic regulators DNMT3A and TET2 in CD8 T cells have revealed a critical role of DNA methylation in repressing the proliferative capacity and effector potential of CD8 T cells during exhaustion. Disruption of these genes has also been noted to preserve the proliferative capacity of hematopoietic stem cells (HSCs), resulting in clonal hematopoiesis (CH) and establishing the broader concept that these epigenetic regulators control cellular developmental potential. In addition to DNMT3A and TET2, the histone modifier ASXL1 is a critical regulator controlling CH. Here we report that disruption of ASXL1 in CD8 T cells preserved a stem-like phenotype denoted by increased TCF1 expression, IFN-γ production during chronic infection. At the chronic stage of infection, ASXL1 KO CD8 T cells retained a heightened ability to undergo a proliferative burst during PD-L1 blockade. Genome-wide epigenetic profiling demonstrated that disruption of ASXL1 resulted in inhibition of DNA methylation accompanied by changes in chromatin accessibility. These data document crosstalk between histone modifications and DNA methylation in regulating the transition of stem-like progenitor T cells into terminally exhausted T cells. Collectively, these data demonstrate that ASXL1 is a novel epigenetic regulator controlling development of exhausted CD8 T cells and represents a novel immunotherapeutic target for improving responses to T cell-based cancer immunotherapy.

DNA methylation inhibitor regulates guanylyl cyclase/natriuretic peptide receptor-A gene expression via 5'-untranslated region in mesangial and smooth muscle cells

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is activated by cardiac hormones atrial and brain natriuretic peptides (ANP, BNP) and produces intracellular second messenger cGMP. The physiological actions of ANP/NPRA/cGMP signaling cascade largely include diuresis, natriuresis, vasorelaxation, and organ protection, which maintains blood pressure and opposed renal, cardiac, and vascular injury and remodeling. However, the mechanisms that contribute to the activation of Npr1 (coding for GC-A/NPRA) expression is not well understood. The present study was aimed at understanding the epigenetic mechanisms of DNA methyltransferase (DNMT) inhibitor, 5-Azacytidine (5-Aza)-mediated Npr1 gene transcription. The studies were carried out in mouse mesangial cells (MMCs) and rat thoracic aortic smooth muscle (RTASM) cells, cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin, transferrin, and sodium selenite) and treated with 5-Aza for 24 h. Western blot analysis showed that 5-Aza enhanced NPRA protein levels by 5-fold and 7-fold in a dose-dependent manner in MMCs and RTASM cells, respectively, and greatly stimulated intracellular accumulation of cGMP in ANP-treated cells. Treatment with 5-Aza attenuated DNMT activity and repressed DNMT1 protein levels in cells. The MethPrimer search result showed the presence of three cytosine-phosphate-guanine (CpG) islands in the Npr1 full length promoter and 5'-untranslated region (UTR), namely island 1 (-886 to -752, 134 bp), island 2 (-310 to -158, 152 bp), and island 3 (-154 to +310, 464 bp) from the transcription start site. Addition of 5'-UTR encompassing +56 to +359 bp region to the Npr1 promoter constructs, (-1982/+55) and proximal promoter (-356/+55), was inhibitory to Npr1 transcriptional activity, independent of the cell type. Furthermore, treatment with 5-Aza distinctly induced Npr1 promoter activity of the construct -356/5'-UTR by 10-fold in MMCs suggesting that the repressive effect of DNA methylation on Npr1 gene transcription might be via 5'-UTR. The results demonstrate that 5-Aza acts as a potent inducer of Npr1 expression and signaling and provides new insights in the epigenetic control of Npr1, an important player in the control of hypertension and cardiorenal axis. Supported by the NIH grants (HL062147 and DK133833). This is the full abstract presented at the American Physiology Summit 2023 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.

Hypermethylated ITGA8 Facilitate Bladder Cancer Cell Proliferation and Metastasis.

DNA methylation plays a vital role during the development of tumorigenesis. The purpose of this study is to identify candidate DNA methylation drivers during progression of bladder cancer (BLCA). The methylation spectrum in bladder cancer tissues was detected by CHARM analysis, and methylated ITGA8 was selected for further study due to its low expression. Methylation levels in BLCA tissues and cells were detected with methylated-specific PCR (MSP), while mRNA expression and methylation of ITGA8 were detected by qRT-PCR and MSP. After treatment with 5-Aza-dC (DNA methylation inhibitor), the proliferation, migration, and invasion abilities of BLCA cells were determined by MTT, wound healing, and transwell assays, respectively. Flow cytometric analysis was performed to evaluate any variance in the cell cycle. In addition, the effect of demethylated ITGA8 on BLCA tumor growth was verified with an in vivo xenograft tumor model. Based on the methylation profiling of BLCA, ITGA8 was identified to be hypermethylated. ITGA8 methylation levels in BLCA tissues and cells were upregulated, and 5-Aza-dC significantly suppressed ITGA8 methylation levels and increased ITGA8 mRNA expression. Furthermore, after treatment with 5-Aza-dC, the propagation, migration, and invasiveness of the cancer cells were inhibited, and more cancer cells were arrested at the G0/G1 phase. In vivo assays further demonstrated that 5-Aza-dC could impede BLCA tumor growth by repressing methylation levels of ITGA8 and increasing ITGA8 mRNA expression. Hypermethylated ITGA8 facilitated BLCA progression, and 5-Aza-dC treatment inhibited BLCA cell propagation and metastasis by decreasing methylation levels of ITGA8 and inducing cell cycle arrest.

NXNL2 Promotes Colon Cancer Proliferation and Metastasis by Regulating AKT Pathway.

This study aimed to explore the role of nucleoredoxin-like 2 (NXNL2) in colon cancer (CC). The GEPIA and UALCAN databases were analyzed to explore genes involved in the prognosis of CC patients. DLD1 cells were treated with the DNA methylation inhibitor 5-azacitidine to validate the above findings. The methyltransferase DNMT (DNA methylation) was further knocked down by shRNA, then the expression of NXNL2 was assessed by qPCR. The role of NXNL2 on cell proliferation and metastasis was examined using corresponding assays. NXNL2 was found to exhibit the greatest impact on the prognosis of CC patients. High NXNL2 correlated with poor survival outcomes of CC. The expression of NXNL2 was regulated by DNA methylation. NXNL2 promoted CC cell proliferation and metastasis. Also, NXNL2 promoted the AKT pathway activity. In conclusion, NXNL2 could affect the cancer cell proliferation and metastasis, and has a poor survival prognosis in CC.