CpG Island Hypermethylation Mediated by DNMT3A Is a Consequence of AML Progression.

DNMT3A mutations occur in ∼25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3AR882H, has dominant negative activity that reduces DNA methylation activity by ∼80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3AR882 mutations. Non-leukemic hematopoietic cells with DNMT3AR882H displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing and was essentially absent in AMLs with DNMT3AR882 mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3AR882, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.

Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma.

Liver cancer has the second highest worldwide cancer mortality rate and has limited therapeutic options. We analyzed 363 hepatocellular carcinoma (HCC) cases by whole-exome sequencing and DNA copy number analyses, and we analyzed 196 HCC cases by DNA methylation, RNA, miRNA, and proteomic expression also. DNA sequencing and mutation analysis identified significantly mutated genes, including LZTR1, EEF1A1, SF3B1, and SMARCA4. Significant alterations by mutation or downregulation by hypermethylation in genes likely to result in HCC metabolic reprogramming (ALB, APOB, and CPS1) were observed. Integrative molecular HCC subtyping incorporating unsupervised clustering of five data platforms identified three subtypes, one of which was associated with poorer prognosis in three HCC cohorts. Integrated analyses enabled development of a p53 target gene expression signature correlating with poor survival. Potential therapeutic targets for which inhibitors exist include WNT signaling, MDM4, MET, VEGFA, MCL1, IDH1, TERT, and immune checkpoint proteins CTLA-4, PD-1, and PD-L1.

Gene-specific somatic epigenetic mosaicism of FDFT1 underlies a non-hereditary localized form of porokeratosis.

Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.

Cross-Species Comparative DNA Methylation Reveals Novel Insights into Complex Trait Genetics among Cattle, Sheep, and Goats.

The cross-species characterization of evolutionary changes in the functional genome can facilitate the translation of genetic findings across species and the interpretation of the evolutionary basis underlying complex phenotypes. Yet, this has not been fully explored between cattle, sheep, goats, and other mammals. Here, we systematically characterized the evolutionary dynamics of DNA methylation and gene expression in 3 somatic tissues (i.e. brain, liver, and skeletal muscle) and sperm across 7 mammalian species, including 3 ruminant livestock species (cattle, sheep, and goats), humans, pigs, mice, and dogs, by generating and integrating 160 DNA methylation and transcriptomic data sets. We demonstrate dynamic changes of DNA hypomethylated regions and hypermethylated regions in tissue-type manner across cattle, sheep, and goats. Specifically, based on the phylo-epigenetic model of DNA methylome, we identified a total of 25,074 hypomethylated region extension events specific to cattle, which participated in rewiring tissue-specific regulatory network. Furthermore, by integrating genome-wide association studies of 50 cattle traits, we provided novel insights into the genetic and evolutionary basis of complex phenotypes in cattle. Overall, our study provides a valuable resource for exploring the evolutionary dynamics of the functional genome and highlights the importance of cross-species characterization of multiomics data sets for the evolutionary interpretation of complex phenotypes in cattle livestock.

Cellular and clinical impact of protein phosphatase enzyme epigenetic silencing in multiple cancer tissues.

BACKGROUND: Protein Phosphatase Enzymes (PPE) and protein kinases simultaneously control phosphorylation mechanisms that tightly regulate intracellular signalling pathways and stimulate cellular responses. In human malignancies, PPE and protein kinases are frequently mutated resulting in uncontrolled kinase activity and PPE suppression, leading to cell proliferation, migration and resistance to anti-cancer therapies. Cancer associated DNA hypermethylation at PPE promoters gives rise to transcriptional silencing (epimutations) and is a hallmark of cancer. Despite recent advances in sequencing technologies, data availability and computational capabilities, only a fraction of PPE have been reported as transcriptionally inactive as a consequence of epimutations. METHODS: In this study, we examined promoter-associated DNA methylation profiles in Protein Phosphatase Enzymes and their Interacting Proteins (PPEIP) in a cohort of 705 cancer patients in five tissues (Large intestine, Oesophagus, Lung, Pancreas and Stomach) in three cell models (primary tumours, cancer cell lines and 3D embedded cancer cell cultures). As a subset of PPEIP are known tumour suppressor genes, we analysed the impact of PPEIP promoter hypermethylation marks on gene expression, cellular networks and in a clinical setting. RESULTS: Here, we report epimutations in PPEIP are a frequent occurrence in the cancer genome and manifest independent of transcriptional activity. We observed that different tumours have varying susceptibility to epimutations and identify specific cellular signalling networks that are primarily affected by epimutations. Additionally, RNA-seq analysis showed the negative impact of epimutations on most (not all) Protein Tyrosine Phosphatase transcription. Finally, we detected novel clinical biomarkers that inform on patient mortality and anti-cancer treatment sensitivity. CONCLUSIONS: We propose that DNA hypermethylation marks at PPEIP frequently contribute to the pathogenesis of malignancies and within the precision medicine space, hold promise as biomarkers to inform on clinical features such as patient survival and therapeutic response.

Genome wide inherited modifications of the tomato epigenome by trans-activated bacterial CG methyltransferase.

BACKGROUND: Epigenetic variation is mediated by epigenetic marks such as DNA methylation occurring in all cytosine contexts in plants. CG methylation plays a critical role in silencing transposable elements and regulating gene expression. The establishment of CG methylation occurs via the RNA-directed DNA methylation pathway and CG methylation maintenance relies on METHYLTRANSFERASE1, the homologue of the mammalian DNMT1. PURPOSE: Here, we examined the capacity to stably alter the tomato genome methylome by a bacterial CG-specific M.SssI methyltransferase expressed through the LhG4/pOP transactivation system. RESULTS: Methylome analysis of M.SssI expressing plants revealed that their euchromatic genome regions are specifically hypermethylated in the CG context, and so are most of their genes. However, changes in gene expression were observed only with a set of genes exhibiting a greater susceptibility to CG hypermethylation near their transcription start site. Unlike gene rich genomic regions, our analysis revealed that heterochromatic regions are slightly hypomethylated at CGs only. Notably, some M.SssI-induced hypermethylation persisted even without the methylase or transgenes, indicating inheritable epigenetic modification. CONCLUSION: Collectively our findings suggest that heterologous expression of M.SssI can create new inherited epigenetic variations and changes in the methylation profiles on a genome wide scale. This open avenues for the conception of epigenetic recombinant inbred line populations with the potential to unveil agriculturally valuable tomato epialleles.

Downregulation of JAM3 occurs in cholangiocarcinoma by hypermethylation: A potential molecular marker for diagnosis and prognosis.

Junctional adhesion molecular 3 (JAM3) is downregulated by hypermethylation in cancers but is unclear in cholangiocarcinoma. The JAM3 expression level was checked in cholangiocarcinoma cell lines and tissues. Methylated JAM3 was detected in cell lines, tissues and plasma cell-free DNAs (cfDNA). The roles of JAM3 in cholangiocarcinoma were studied by transfection of siRNA and pCMV3-JAM3. The survival analysis was based on the Gene Set Cancer Analysis (GSCA) database. JAM3 was downregulated in HCCC-9810 and HuCCT1 cell lines and tissues by hypermethylation. Methylated JAM3 was detected in cfDNAs with 53.3% sensitivity and 96.6% specificity. Transfection of pCMV3-JAM3 into HCCC-9810 and HuCCT1 induced apoptosis and suppressed cell proliferation, migration and invasion. The depletion of JAM3 in RBE cells using siRNA decreased apoptosis and increased cell proliferation, migration and invasion. Hypermethylation of JAM3 was associated with tumour differentiation, metastasis and TNM stage. Downregulation and hypermethylation of JAM3 were related to poor progression-free survival. Junctional adhesion molecular 3 may function as a tumour suppressor in cholangiocarcinoma. Methylated JAM3 DNA may represent a non-invasive molecular marker for the early detection of cholangiocarcinoma and prognosis.

SIAH3 is frequently epigenetically silenced in cancer and regulates mitochondrial metabolism.

Of the seven in absentia homologue (SIAH) family, three members have been identified in the human genome. In contrast to the E3 ubiquitin ligase encoding SIAH1 and SIAH2, little is known on the regulation and function of SIAH3 in tumorigenesis. In this study, we reveal that SIAH3 is frequently epigenetically silenced in different cancer entities, including cutaneous melanoma, lung adenocarcinoma and head and neck cancer. Low SIAH3 levels correlate with an impaired survival of cancer patients. Additionally, induced expression of SIAH3 reduces cell proliferation and induces cell death. Functionally, SIAH3 negatively affects cellular metabolism by shifting cells form aerobic oxidative phosphorylation to glycolysis. SIAH3 is localized in the mitochondrion and interacts with proteins involved in mitochondrial ribosome biogenesis and translation. We also report that SIAH3 interacts with ubiquitin ligases, including SIAH1 or SIAH2, and is degraded by them. These results suggest that SIAH3 acts as an epigenetically controlled tumor suppressor by regulating cellular metabolism through the inhibition of oxidative phosphorylation.

DNMT1/DNMT3a-mediated promoter hypermethylation and transcription activation of ICAM5 augments thyroid carcinoma progression.

Promoter methylation is one of the most studied epigenetic modifications and it is highly relevant to the onset and progression of thyroid carcinoma (THCA). This study investigates the promoter methylation and expression pattern of intercellular adhesion molecule 5 (ICAM5) in THCA. CpG islands with aberrant methylation pattern in THCA, and the expression profiles of the corresponding genes in THCA, were analyzed using bioinformatics. ICAM5 was suggested to have a hypermethylation status, and it was highly expressed in THCA tissues and cells. Its overexpression promoted proliferation, mobility, and tumorigenic activity of THCA cells. As for the downstream signaling, ICAM5 was found to activate the MAPK/ERK and MAPK/JNK signaling pathways. Either inhibition of ERK or JNK blocked the oncogenic effects of ICAM5. DNA methyltransferases 1 (DNMT1) and DNMT3a were found to induce promoter hypermethylation of ICAM5 in THCA cells. Knockdown of DNMT1 or DNMT3a decreased the ICAM5 expression and suppressed malignant properties of THCA cells in vitro and in vivo, which were, however, restored by further artificial ICAM5 overexpression. Collectively, this study reveals that DNMT1 and DNMT3a mediates promoter hypermethylation and transcription activation of ICAM5 in THCA, which promotes malignant progression of THCA through the MAPK signaling pathway.

BRD7 facilitates ferroptosis via modulating clusterin promoter hypermethylation and suppressing AMPK signaling in diabetes-induced testicular damage.

BACKGROUND: Diabetes mellitus (DM)-induced testicular damage is associated with sexual dysfunction and male infertility in DM patients. However, the pathogenesis of DM-induced testicular damage remains largely undefined. METHODS: A streptozotocin (STZ)-induced diabetic model and high glucose (HG)-treated in vitro diabetic model were established. The histological changes of testes were assessed by H&E staining. Serum testosterone, iron, MDA and GSH levels were detected using commercial kits. Cell viability and lipid peroxidation was monitored by MTT assay and BODIPY 581/591 C11 staining, respectively. qRT-PCR, immunohistochemistry (IHC) or Western blotting were employed to detect the levels of BRD7, Clusterin, EZH2 and AMPK signaling molecules. The associations among BRD7, EZH2 and DNMT3a were detected by co-IP, and the transcriptional regulation of Clusterin was monitored by methylation-specific PCR (MSP) and ChIP assay. RESULTS: Ferroptosis was associated with DM-induced testicular damage in STZ mice and HG-treated GC-1spg cells, and this was accompanied with the upregulation of BRD7. Knockdown of BRD7 suppressed HG-induced ferroptosis, as well as HG-induced Clusterin promoter methylation and HG-inactivated AMPK signaling in GC-1spg cells. Mechanistical studies revealed that BRD7 directly bound to EZH2 and regulated Clusterin promoter methylation via recruiting DNMT3a. Knockdown of Clusterin or inactivation of AMPK signaling reverses BRD7 silencing-suppressed ferroptosis in GC-1spg cells. In vivo findings showed that lack of BRD7 protected against diabetes-induced testicular damage and ferroptosis via increasing Clusterin expression and activating AMPK signaling. CONCLUSION: BRD7 suppressed Clusterin expression via modulating Clusterin promoter hypermethylation in an EZH2 dependent manner, thereby suppressing AMPK signaling to facilitate ferroptosis and induce diabetes-associated testicular damage.

Recurrent carotid paragangliomas in a syndromic patient with a heterozygous missense variant in DNA Methyltransferase 3 Alpha.

We report a 40-year-old African American female with a novel variant in exon 8 of DNA methyltransferase 3 alpha (DNMT3A), (NM_022552.4: c.905G>C, p.G302A) who presented with a history of recurrent carotid paragangliomas, mediastinal mass, intellectual disability, dysarthria, cholelithiasis, diabetes mellitus, hypertension, and dysmorphic features. We interpret this novel variant as likely pathogenic and causative for the patient's syndromic features of Heyn-Sproul-Jackson syndrome. Heyn-Sproul-Jackson syndrome is a condition caused by gain-of-function genetic changes in DNMT3A. Paragangliomas have also been observed in non-syndromic patients with genetic alterations in DNMT3A. We describe a patient with clinical features of Heyn-Sproul-Jackson syndrome such as intellectual disability, dysarthria, brachydactyly, and lack of brain MRI findings to add evidence to associate paragangliomas with DNMT3A and draw particular attention to the potential involvement of the proline-tryptophan-tryptophan-proline domain of DNMT3A.

Copper nanoparticles and silver nanoparticles impair lymphangiogenesis in zebrafish.

Lymphatic system distributes in almost all vertebrate tissues and organs, and plays important roles in the regulation of body fluid balance, lipid absorption and immune monitoring. Although CuNPs or AgNPs accumulation has been reported to be closely associated with delayed hatching and motor dysfunction in zebrafish embryos, their biological effects on lymphangiogenesis remain unknown. In this study, thoracic duct was observed to be partially absent in both CuNPs and AgNPs stressed zebrafish larvae. Specifically, CuNPs stress induced hypermethylation of E2F7/8 binding sites on CCBE1 promoters via their producing ROS, thereby leading to the reduction of binding enrichment of E2F7/8 on CCBE1 promoter and its subsequently reduced expression, then resulting in defective lymphatic vessel formation. Differently, AgNPs stress induced down-regulated CCBE1 expression via down-regulating mRNA and protein levels of E2F7/8 transcription factors, thereby resulting in defective lymphatic vessel formation. This study may be the first to demonstrate that CuNPs and AgNPs damaged lymphangiogenesis during zebrafish embryogenesis, mechanistically, CuNPs epigenetically regulated the expression of lymphangiogenesis regulator CCBE1 via hypermethylating its promoter binding sites of E2F7/8, while AgNPs via regulating E2F7/8 expression. Meanwhile, overexpression of ccbe1 mRNA effectively rescued the lymphangiogenesis defects in both AgNPs and CuNPs stressed larvae, while overexpression of e2f7/8 mRNA effectively rescued the lymphangiogenesis defects in AgNPs rather than CuNPs stressed larvae. The results in this study will shed some light on the safety assessment of nanomaterials applied in medicine and on the ecological security assessments of nanomaterials. Video Abstract.

SETDB1 deletion causes DNA demethylation and upregulation of multiple zinc-finger genes.

BACKGROUND: SETDB1 (SET domain bifurcated-1) is a histone H3-lysine 9 (H3K9)-specific methyltransferase that mediates heterochromatin formation and repression of target genes. Despite the assumed functional link between DNA methylation and SETDB1-mediated H3K9 trimethylations, several studies have shown that SETDB1 operates autonomously of DNA methylation in a region- and cell-specific manner. This study analyzes SETDB1-null HAP1 cells through a linked methylome and transcriptome analysis, intending to explore genes controlled by SETDB1-involved DNA methylation. METHODS AND RESULTS: We investigated SETDB1-mediated regulation of DNA methylation and gene transcription in human HAP1 cells using reduced-representation bisulfite sequencing (RRBS) and RNA sequencing. While two-thirds of differentially methylated CpGs (DMCs) in genic regions were hypomethylated in SETDB1-null cells, we detected a plethora of C2H2-type zinc-finger protein genes (C2H2-ZFP, 223 of 749) among the DMC-associated genes. Most C2H2-ZFPs with DMCs in their promoters were found hypomethylated in SETDB1-KO cells, while other non-ZFP genes with promoter DMCs were not. These C2H2-ZFPs with DMCs in their promoters were significantly upregulated in SETDB1-KO cells. Similarly, C2H2-ZFP genes were upregulated in SETDB1-null 293T cells, suggesting that SETDB1's function in ZFP gene repression is widespread. There are several C2H2-ZFP gene clusters on chromosome 19, which were selectively hypomethylated in SETDB1-KO cells. CONCLUSIONS: SETDB1 collectively and specifically represses a substantial fraction of the C2H2-ZFP gene family. Through the en-bloc silencing of a set of ZFP genes, SETDB1 may help establish a panel of ZFP proteins that are expressed cell-type specifically and thereby can serve as signature proteins for cellular identity.

Clinical Significance of Frequently Down-Regulated Phosphatidylethanolamine-Binding Protein-1 in Gallbladder Cancer.

BACKGROUND: Promoter hypermethylation of tumor suppressor genes has been demonstrated to be one of the major mechanisms of their epigenetic regulation in various reports. We have studied the promoter methylation status of PEBP1 and evaluated its correlation with gallbladder carcinogenesis. AIMS: PEBP1, an endogenous inhibitor of Raf/MEK/ERK signaling pathway, is a tumor suppressor gene. We aimed to study the expression profile of PEBP1 and understand the mechanism and significance of its deregulation in gallbladder cancer. METHODS: PEBP1 expression analysis and its promoter methylation status were investigated in 77 gallbladder carcinoma (GBC) and tissue biopsies from 28 patients of gallstone disease by RT-PCR and MS-PCR, respectively. RESULTS: Our results of the mRNA expression profiling demonstrate that PEBP1 is down-regulated in 62.3% (48/77), while 31.2% (24/77) of the gallbladder cancer biopsies show no significant change and 6.5% (5/77) show up-regulated expression compared to tissue samples of gallstone diseases. In GBC, 48.1% (N = 37) GBC biopsy samples exhibited significantly heterozygous promoter hypermethylation compared to tissue samples from gallstone diseases which show promoter hypermethylation in 3 (10.7%) samples only. In gallbladder cancer, the PEBP1 methylation is significantly associated with lymph node metastasis and shorter period of survival. CONCLUSION: PEBP1 is frequently down-regulated and hypermethylated in gallbladder cancer and its promoter hypermethylation is a frequent and early inactivating mechanism in GBC.

Loss of fragile WWOX gene leads to senescence escape and genome instability.

Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular senescence is a DDR mechanism that prevents the proliferation of cells with damaged DNA to avoid mitotic anomalies and inheritance of the damage over cell generations. Human WWOX gene resides within a common fragile site FRA16D that is preferentially prone to form breaks on metaphase chromosome upon replication stress. We report here that primary Wwox knockout (Wwox-/-) mouse embryonic fibroblasts (MEFs) and WWOX-knockdown human dermal fibroblasts failed to undergo replication-induced cellular senescence after multiple passages in vitro. Strikingly, by greater than 20 passages, accelerated cell cycle progression and increased apoptosis occurred in these late-passage Wwox-/- MEFs. These cells exhibited γH2AX upregulation and microsatellite instability, indicating massive accumulation of nuclear DNA lesions. Ultraviolet radiation-induced premature senescence was also blocked by WWOX knockdown in human HEK293T cells. Mechanistically, overproduction of cytosolic reactive oxygen species caused p16Ink4a promoter hypermethylation, aberrant p53/p21Cip1/Waf1 signaling axis and accelerated p27Kip1 protein degradation, thereby leading to the failure of senescence induction in Wwox-deficient cells after serial passage in culture. We determined that significantly reduced protein stability or loss-of-function A135P/V213G mutations in the DNA-binding domain of p53 caused defective induction of p21Cip1/Waf1 in late-passage Wwox-/- MEFs. Treatment of N-acetyl-L-cysteine prevented downregulation of cyclin-dependent kinase inhibitors and induced senescence in Wwox-/- MEFs. Our findings support an important role for fragile WWOX gene in inducing cellular senescence for maintaining genome integrity during DDR through alleviating oxidative stress.

Genomic analysis links the American mink Royal pastel coat phenotype to retroviral element type 1 insertion in the HPS3 gene.

To date, only 10 of the more than 30 fur colours that had been observed in American mink (Neogale vison) have been linked to specific genes. The Royal pastel fur colour is part of a large family of brownish colours that are quite similar to one another, making breeding and selecting processes more difficult. Here we carried out whole-genome sequencing of five American minks with Royal pastel (b/b) phenotypes originating from two distinct mink populations. We identified an insertion of endogenous retroviral element type 1 (ERV1) into the first intron of the gene encoding the HPS3 protein, which regulates the trafficking of tyrosinase-containing vesicles to maturing melanosomes. With Cas9-targeted nanopore sequencing, we reconstructed the full-length sequence of the 11.7 Kb ERV1 insertion and observed hypermethylation that spread to the HPS3 gene promoter region. These findings highlight the role of HPS3 in the formation of melanosomes and melanin, as well as the genetic process regulating the intensity and spectrum of hair colour. Moreover, in mink breeding projects, these data are also useful for tracking economically important fur qualities.

Chronic low-dose chromium VI exposure induces oxidative stress and apoptosis with altered expressions of DNA repair genes and promoter hypermethylation in the liver of Swiss albino mice.

The present investigation dealt with harmful effects of hexavalent chromium (Cr [VI]) on liver of Swiss albino mice. This variant exhibited cytotoxicity, mutagenicity, and carcinogenicity. Our study focused on elucidating the hepatotoxic effects of chronic low-dose exposure to Cr (VI) (2, 5, and 10 ppm) administered via drinking water for 4 and 8 months. The observed elevation in SGPT, ALP, and SGOT and increased oxidative stress markers unequivocally confirmed the severe disruption of liver homeostasis at these low treatment doses. Noteworthy alterations in histoarchitecture, body weight, and water intake provided further evidences of the harmful effects of Cr (VI). Production of reactive oxygen species (ROS) during metabolism led to DNA damages. Immunohistochemistry and qRT-PCR analyses revealed that chronic low-dose exposure of Cr (VI) induced apoptosis in liver tissue. Our study exhibited alterations in the expression pattern of DNA repair genes (Rad51, Mutyh, Mlh1, and Ogg1), coupled with promoter hypermethylation of Mutyh and Rad51, leading to transcriptional inhibition. Our findings underscored the potential of low-dose Cr (VI) exposure on hepatotoxicity by the intricate interplay between apoptosis induction and epigenetic alterations of DNA repair genes.

LncRNA LINC00261 associates with chemoresistance and clinical prognosis in patients with epithelial ovarian cancer.

OBJECTIVE: The purpose of this experiment is to explore the role of long intergenic noncoding RNA 261 (LINC00261) gene in the chemoresistance and clinical prognosis of epithelial ovarian cancer (EOC). METHODS: We used matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to detect the methylation levels of the LINC00261 promoter region in EOC patient specimens. The expression levels of LINC00261, miR-545-3p, and MT1M in EOC patients were evaluated by quantitative real-time reverse transcriptase PCR (RT-qPCR). Spearman's correlation analysis was used for relevance analyses and clinical prognosis was counted by Kaplan-Meier analysis. Stable overexpressed LINC00261 SKOV3 cells were established to test the influence of LINC00261 on proliferation, platinum sensitivity, migration, and invasion. RESULTS: The promoter region methylation level of the LINC00261 was hypermethylated and LINC00261 was significantly downregulated in platinum-resistant EOC tissues. The methylation level of LINC00261was significantly negative correlated with its RNA expression in EOC. Moreover, hypermethylation and lower expression of LINC00261 in EOC patients were related to shorter progression-free survival (PFS) and overall survival (OS). Furthermore, Spearman's correlation analysis showed that the expression of miR-545-3p had a negative relevance with LINC00261. According to the website prediction, MT1M might be the downstream target gene of LINC00261. Expression of MT1M was negatively correlated with miR-545-3p and positively with LINC00261 in EOC tissues. And lower MT1M mRNA expression was correlated with chemotherapy resistance and worse prognosis. In vitro, overexpression of LINC00261 could inhibit cisplatin resistance, proliferation, and suppression of migration and invasion in SKOV3 cells. CONCLUSIONS: This research indicates that the aberrant hypermethylation and low expression of LINC00261 were associated with platinum resistance and adverse outcomes in EOC patients.

Hypermethylation of the FOXP3 gene regulates Tregs immunodysregulation in chronic idiopathic thrombocytopenic purpura.

BACKGROUND: Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by a breakdown of immune tolerance; in ITP, the body's immune system mistakenly attacks and destroys platelets. This study aims to investigate the role and underlying mechanisms of FOXP3 in chronic ITP. METHODS: Flow cytometry was used to detect the proportion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) in CD4+CD25+ T lymphocytes from 20 patients with chronic ITP (CITP), 20 acute ITP (AITP) controls, and 20 healthy individuals.CD4+CD25+ Treg cells were isolated from peripheral blood of patients with CITP using magnetic beads and then treated with phosphate-buffered saline solution or decitabine (a methylation inhibitor) for 48 h. The levels of interleukin-2 (IL-2), IL-10, and transforming growth factor-beta1 (TGF-ß1) in the plasma and CD4+CD25+ Treg cells were assessed by Enzyme-linked-immunosorbent serologic assay and quantitative real-time polymerase chain reaction (qRT-PCR). FOXP3 level was measured by qRT-PCR and Western blot analysis. Methylation-specific PCR (MS-PCR) was adopted to detect the status of FOXP3 methylation. RESULTS: The number of Treg cells and the contents of IL-2, IL-10, and TGF-ß1 decreased in patients with CITP, compared to the AITP control group and normal group. FOXP3 expression was reduced and FOXP3 methylation increased in patients with CITP, compared to the AITP control group and normal group. Hypermethylation of FOXP3 promoter led to decrease in FOXP3 level in Treg cells. Inhibition of FOXP3 promoter hypermethylation promoted the secretion of IL-2, IL-10, and TGF-ß1 in Treg cells. CONCLUSION: The number of Treg cells in CITP patients decreased, and the hypermethylation of FOXP3 promoter led to reduction of its expression in Treg cells, thus affecting the immune functioning of Treg cells.

Efficacy of cell-free DNA methylation-based blood test for colorectal cancer screening in high-risk population: a prospective cohort study.

BACKGROUND: Although colonoscopy is the standard screening test for colorectal cancer (CRC), its use is limited by a poor compliance rate, the need for extensive bowel preparation, and the risk of complications. As an alternative, an FDA-approved stool-based DNA test, Cologuard, has demonstrated satisfactory detection performance for CRC, but its compliance rate remains suboptimal, primarily attributable to individuals' reluctance to provide stool samples. METHODS: We developed a noninvasive blood-based CRC test, ColonSecure, based on cell-free DNA containing cancer-specific CpG island methylation patterns. We initially screened publicly available datasets for differentially methylated CpG sites in CRC with prediction potential. Subsequently, we performed two sequential bisulfite-free methylation sequencing on blood samples obtained from CRC patients and non-cancer controls. Through rigorous evaluation of each marker and machine learning-assisted feature selection, we identified 149 hypermethylated markers from over 193,000 CpG sites. These markers were then utilized to construct the ColonSecure model, enabling accurate CRC detection. RESULTS: We validated the efficacy of our cell-free DNA methylation-based blood test for CRC screening with 3493 high-risk individuals identified from 114,136 urban residents. The ColonSecure test identified 89 out of 103 CRC patients diagnosed by the follow-up colonoscopy, outperforming CEA, CRP, and CA19-9 (with a sensitivity of 86.4% compared to 45.6%, 39.8%, and 25.2% for CEA, CRP, and CA19-9 respectively; an AUROC of 0.956 compared to an AUROC of < 0.77 for other methods). CONCLUSION: Our observations emphasize the potential of our multiple cfDNA methylation marker-based test for CRC screening in high-risk populations.