TET2 Expression Research Articles

Hypermethylation of CDKN2A CpG island drives resistance to PRC2 inhibitors in SWI/SNF loss-of-function tumors.

Polycomb repressive complex 2 (PRC2) catalyzes the writing of the tri-methylated histone H3 at Lys27 (H3K27me3) epigenetic marker and suppresses the expression of genes, including tumor suppressors. The function of the complex can be partially antagonized by the SWI/SNF chromatin-remodeling complex. Previous studies have suggested that PRC2 is important for the proliferation of tumors with SWI/SNF loss-of-function mutations. In the present study, we have developed an EED-directed allosteric inhibitor of PRC2 termed BR0063, which exhibits anti-proliferative properties in a subset of solid tumor cell lines harboring mutations of the SWI/SNF subunits, SMARCA4 or ARID1A. Tumor cells sensitive to BR0063 exhibited several distinct phenotypes, including cell senescence, which was mediated by the up-regulation of CDKN2A/p16. Further experiments revealed that the expression of p16 was suppressed in the BR0063-resistant cells via DNA hypermethylation in the CpG island (CGI) promoter region, rather than via PRC2 occupancy. The expression of TET1, which is required for DNA demethylation, was found to be inversely correlated with p16 CGI methylation, and this may serve as a biomarker for the prediction of resistance to PRC2 inhibitors in SWI/SNF LOF tumors.

The implication of TET1, miR-200, and miR-494 expression with tumor formation in colorectal cancer: through targeting Wnt signaling

ObjectiveColorectal cancer (CRC) is a diverse and multifaceted disease characterized by genetic and epigenetic changes that contribute to tumor initiation and progression. CRC pathophysiology has been linked to the deregulation of the Wnt signaling pathway and the ten-eleven translocation (TET) DNA demethylases. This study aimed to evaluate the expression level of selective miRNAs (miR-200 and miR-494), TET1, and Wnt1 in colorectal polyps, actual colorectal tumors, and normal adjacent tissues. We also evaluated the effect of 5-aza cytidine on the expression level of TET1 and wnt1 in the HT29 cell line.Materials and methodsIn this study, we assessed TET1 and Wnt1 expression in 5-azacytidine-treated HT29 cells, a demethylating agent commonly used in cancer therapy. Additionally, we enrolled 114 individuals who underwent radical surgical colon resection, including 47 with cancerous tissues and 67 with polyps. We utilized qRT-PCR to measure miR-200, miR-494, TET1, and Wnt1 mRNA levels in colorectal polyps, actual colorectal tumors, and normal adjacent tissues.ResultsOur study revealed that TET1 expression was notably lower in both polyps and CRC tissue compared to adjacent normal tissue, with higher TET1 expression in tumors than polyps. We also observed significant differences in miR-200 and miR-494 expression in tumor samples compared to adjacent normal tissue. Our in vitro experiments revealed that 5-azacytidine administration increased TET1 and decreased Wnt1 expression in CRC cell lines. This suggests that DNA-demethylating drugs may have a therapeutic role in modifying TET1 and Wnt signaling in the development of CRC.ConclusionsOverall, our findings shed light on the intricate interactions between TET1, Wnt1, and specific miRNAs in colorectal cancer (CRC) and their potential implications for diagnosis and treatment.

Wu Mei Wan suppresses colorectal cancer stemness by regulating Sox9 expression via JAK2/STAT3 pathway

Ethnopharmacological relevanceWu Mei Wan (WMW) is a traditional Chinese herbal formula with a long-standing history in Chinese medicine, valued for its therapeutic properties. However, its potential anti-cancer effects, especially against colorectal cancer (CRC), have not been fully elucidated. Aim of the studyThis study aims to investigate the effects of WMW on colorectal cancer stemness and to elucidate the underlying molecular mechanisms, focusing on the modulation of Sox9 expression via the JAK2/STAT3 signaling pathway. Materials and methodsWMW was prepared and analyzed using UPLC-MS to identify their main components. To study the therapeutic effects of WMW, AOM/DSS-induced CRC mouse models were established. A comprehensive suite of experimental techniques, including in vivo imaging, cell culture, transfection, CCK-8 assays, colony formation assays, wound healing assays, cell migration assays, Western blotting, dot blot analysis, RT-qPCR, immunohistochemistry, cell transcriptome sequencing, and gene set enrichment analysis, were utilized to explore the pharmacological effects and mechanisms of WMW. ResultsWMW significantly inhibited CRC cell viability, proliferation, invasion, and migration in vitro. Mechanistically, WMW suppressed CRC stemness by downregulating Sox9 expression through the JAK2/STAT3 signaling pathway. Additionally, the regulation of methylation and demethylation mediated by TET1 and DNMT3a expression was directly associated with the JAK2/STAT3 pathway's modulation of Sox9 expression. In vivo, WMW treatment attenuated CRC progression and metastasis with minimal toxicity. ConclusionThese findings suggest that WMW exerts potent anti-CRC stemness effects by regulating Sox9 via the JAK2/STAT3 signaling pathway, underscoring its potential as a promising therapeutic agent for CRC treatment.

Injured inflammatory environment overrides the TET2 shaped epigenetic landscape of pluripotent stem cell derived human neural stem cells

Spinal cord injury creates an inflammatory microenvironment that regulates the capacity of transplanted human Neural Stem Cells (hNSC) to migrate, differentiate, and repair injury. Despite similarities in gene expression and markers detected by immunostaining, hNSC populations exhibit heterogeneous therapeutic potential. This heterogeneity derives in part from the epigenetic landscape in the hNSC genome, specifically methylation (5mC) and hydroxymethylation (5hmC) state, which may affect the response of transplanted hNSC in the injury microenvironment and thereby modulate repair capacity. We demonstrate a significant up-regulation of methylcytosine dioxygenase 2 gene (TET2) expression in undifferentiated hNSC derived from human embryonic stem cells (hES-NSC), and report that this is associated with hES-NSC competence for differentiation marker expression. TET2 protein catalyzes active demethylation and TET2 upregulation could be a signature of pluripotent exit, while shaping the epigenetic landscape in hES-NSC. We determine that the inflammatory environment overrides epigenetic programming in vitro and in vivo by directly modulating TET2 expression levels in hES-NSC to change cell fate. We also report the effect of cell fate and microenvironment on differential methylation 5mC/5hmC balance. Understanding how the activity of epigenetic modifiers changes within the transplantation niche in vivo is crucial for assessment of hES-NSC behavior for potential clinical applications.

TET2 promotes tumor antigen presentation and T cell IFN-γ which is enhanced by vitamin C.

Immune evasion by tumors is promoted by low T cell infiltration, ineffective T cell activity directed against the tumor and reduced tumor antigen presentation. The TET2 DNA dioxygenase gene is frequently mutated in hematopoietic malignancies and loss of TET enzymatic activity is found in a variety of solid tumors. We showed previously that vitamin C (VC), a co-factor of TET2, enhances tumor-associated T cell recruitment and checkpoint inhibitor therapy responses in a TET2-dependent manner. Using single-cell RNA sequencing (scRNA-seq) analysis performed on B16-OVA melanoma tumors, we have shown here that an additional function for TET2 in tumors is to promote expression of certain antigen presentation machinery genes, which is potently enhanced by VC. Consistently, VC promoted antigen presentation in cell-based and tumor assays in a TET2-dependent manner. Quantifying intercellular signaling from the scRNA-seq dataset showed that T cell-derived IFNγ-induced signaling within the tumor and tumor microenvironment requires tumor-associated TET2 expression which is enhanced by VC treatment. Analysis of patient tumor samples indicated that TET activity directly correlates with antigen-presentation gene expression and with patient outcomes. Our results demonstrate the importance of tumor-associated TET2 activity as a critical mediator of tumor immunity which is augmented by high-dose VC therapy.

EPO modified MSCs protects SH-SY5Y cells against ischemia/hypoxia-induced apoptosis via REST-dependent epigenetic remodeling

Hypoxic-ischemic encephalopathy (HIE) is a diffuse brain tissue injury caused by acute ischemia and hypoxia, and it is most commonly found in newborn infants but can also occur in adults. Mesenchymal stem cell (MSC) therapies have showed improved outcomes for treating HIE-induced neuronal defects. However, many key issues associated with poor cell viability and tolerance of grafted MSCs after HIE remain to be resolved. Genetic engineering could endow MSCs with more robust regenerative capacities. Our research, along with that of other scientists, has found that the expression of intracellular erythropoietin (EPO) in human umbilical cord MSCs (hUC-MSCs) increases proportionally with the duration of hypoxia exposure. Furthermore, we observed that EPO, when introduced into the EPO gene-modified hUC-MSCs, can be secreted into the extracellular space. However, the underlying mechanisms that support the neuroprotective effects of EPO-MSCs remain unclear. EPO-MSCs, hUC-MSCs, and NC-MSCs were identified by flow cytometry, osteogenic, and adipogenic differentiation assays. The oxygen-glucose deprivation (OGD)-induced SH-SY5Y cell-line was established, and five groups were set up: control, 24-h ischemia-hypoxia, co-cultured with hUC-MSCs, NC-MSCs, and EPO-MSCs after hypoxia. LEGENDplex™ multi-factor flow cytometry was used to detect the secretion of inflammatory factors in cell supernatants and cerebrospinal fluid. Chromosome-targeted excision and tagging (CUT&Tag) sequencing was applied to detect genomic H3K4me2 modifications, and conjoint analysis with transcriptome sequencing (RNA-seq) was performed. Lentiviral vector infection was used to construct SH-SY5Y cells with stable knockdown of RE1-silencing transcription factor (REST), and flow cytometry was used to detect alterations in apoptosis. Finally, the molecular mechanism underlying the neuroprotective and anti-apoptotic effects of EPO-MSCs was investigated using RNA sequencing, qRT-PCR, and western blot assays. Our results suggest that EPO-MSCs are genetically engineered to secrete significantly more EPO. EPO-MSCs treatment has anti-apoptotic properties and offers neuronal protection during ischemic-hypoxic injury. Furthermore, RNA-seq results suggest that multiple inflammation-related genes were down-regulated after EPO-MSCs treatment. Application of RNA-seq and CUT&Tag combined analysis found that the expressions of REST were significantly up-regulated. Lentiviral vector infection to construct REST knockdown SH-SY5Y failed to rescue apoptosis after hypoxia and co-culture with EPO-MSCs, and SETD2-mediated H3K36me3 protein level expression was reduced. EPO-MSCs may promote neuronal survival by affecting H3K4me2 and thus activating the expression of REST and TET3. EPO-MSCs also upregulated the modification level of SETD2-mediated H3K36me3 and regulated the expression of inflammation-related genes such as PLCG2, as well as apoptosis genes BCL2A1. To investigate the neuroprotective effects of EPO-modified hUC-MSCs and the underlying epigenetic regulatory mechanisms, this study aims to provide a theoretical foundation for the potential application of EPO gene-modified hUC-MSCs in the treatment of HIE.

TET1 overexpression affects cell proliferation and apoptosis in aging ovaries.

Along with the progress of society, human life expectancy has been increasing, and late marriage and late childbearing are the current trend. Since reproductive aging affects fertility, ovarian aging in women has become a major reproductive health issue in the current society. During ovarian aging, DNA methylation levels may change. The ten-eleven translocation (TET) protein family proteins TET1, TET2, and TET3 are important DNA demethylation enzymes, and differential expression of TET1, TET2, and TET3 may affect the proliferation and apoptosis of aging ovarian cells. The aim of this study was to investigate the role of TET1 in the regulation of ovarian aging. The expression of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) was analyzed by immunofluorescence (IF) in young and aging ovaries of six 6-8-week-old female mice and six 6-8-month-old female mice. Then, the expression pattern of the TET protein family in young and aging ovaries of mice was investigated. To determine the impact of TET1 on ovarian development, the aging of IOSE-80, KGN, and SKOV-3 cells was induced with D-galactosidase (D-gal). Cells were then transfected using the TET1 overexpression vector or si-TET1. We assessed the proliferation and apoptosis of aging cells after transfection and analyzed the regulatory effect of TET1 expression on aging cells. Additionally, we verified the Tet1 expression in Tet1-KO mice. The 5mC to 5hmC transition, oocyte maturation, and blastocyst rate were reduced in aging mice compared to young mice. In aging mice ovaries, the expression levels of Tet1, Tet2, and Tet3 were reduced significantly, with Tet1 being particularly pronounced. The overexpression of TET1 promoted proliferation and inhibited apoptosis in aging human ovarian cells. Furthermore, Tet1 expression was very low in Tet1-KO C57BL/6J mice ovaries. This study demonstrates that the expression levels of TET family proteins are low in aging ovaries, and the overexpression of TET1 can promote proliferation and inhibit apoptosis in aging ovarian cells.

TET3-overexpressing macrophages promote endometriosis.

Endometriosis is a debilitating, chronic inflammatory disease affecting ~10% of reproductive age women worldwide with no cure. While macrophages have been intrinsically linked to the pathophysiology of endometriosis, targeting them therapeutically has been extremely challenging due to their high heterogeneity and because these disease-associated macrophages (DAMs) can be either pathogenic or protective. Here, we reported identification of pathogenic macrophages characterized by TET3 overexpression in human endometriosis lesions. We showed that factors from the disease microenvironment upregulated TET3 expression transforming macrophages into pathogenic DAMs. TET3 overexpression stimulated pro-inflammatory cytokine production via a feedback mechanism involving inhibition of let-7 miRNA expression. Remarkably, these cells relied on TET3 overexpression for survival, hence vulnerable to TET3 knockdown. We demonstrated that Bobcat339, a synthetic cytosine derivative, triggered TET3 degradation both in human and mouse macrophages. This degradation was dependent on a VHL E3 ubiquitin ligase whose expression was also upregulated in TET3-overexpressing macrophages. Furthermore, depleting TET3-overexpressing macrophages either through myeloid-specific Tet3 ablation or using Bobcat339 strongly inhibited endometriosis progression in mice. Our results defined TET3-overexpressing macrophages as key pathogenic contributors to and attractive therapeutic targets for endometriosis. Our findings may also be applicable to other chronic inflammatory diseases where DAMs have important roles.

AMPK Activation in TET2 Downregulated Leukemia Cells Upon Glutamine Limitation.

Metabolic rewiring is a characteristic of cancer cells. Cancer cells require more nutrients for survival and proliferation. Although glutamine can be produced in cells via a series of enzymatic reactions, a group of cancer cells are dependent on extracellular glutamine for survival. TET2 plays a role in DNA demethylation and is a tumor suppressor gene. The TET2 gene is frequently mutated in various cancers, including acute myeloid leukemia (AML). Our study aimed to investigate the association between TET2-knockdown AML cell line HL-60 cells and glutamine metabolism. To evaluate the association between TET2 expression and glutamine limitation, TET2 was downregulated in HL-60 cells using shRNA plasmids. The proliferation of TET2-knockdown HL-60 cells was calculated in normal and glutamine-deficient medium. GLUL mRNA expression was investigated using quantitative reverse transcription polymerase chain reaction and protein levels were evaluated using immunoblotting. The numbers and viability of TET2-knockdown HL-60 cells were decreased in low glutamine-containing medium, but the viability of TET2-knockdown HL-60 cells was higher than that of control cells. GLUL mRNA expressions were increased in TET2-knockdown cells in low glutamine. In addition, P-AMPKα protein expression was increased in TET2-knockdown HL-60 cells in low glutamine-containing medium. Our findings indicate that TET2-knockdown HL-60 cells may be more resistant to glutamine deprivation. In glutamine-deficient medium, the mRNA expression of glutamine synthetase is increased, which could be related to glutamine addiction in cells. In addition, low-glutamyl medium increased the P-AMPKα protein level in TET2-knockdown HL-60 cells.

Experimental Varicocele Impairs DNA Methylation and Demethylation in Germ Cells, TESE, and Epididymal Spermatozoa, Impacting Active DNA Demethylation in Zygotes.

Varicocele causes infertility. The current study has investigated the impact of experimental varicocele on DNA methylation, demethylation, and damage in the germ cells, TESE-derived and epididymal spermatozoa. Moreover, the results were compared between epidydimal and TESE-derived spermatozoa. Finally, the varicocele-induced effect on active DNA demethylation (ADD) of male pronucleus and pre-implantation embryo development was assessed. The mature male rats were divided into control, control-sham (undergone simple laparotomy), and experimental varicocele-induced groups (n = 6/each group). The left renal vein semi-ligation was considered to induce varicocele. The expression levels of DNA methyltransferase 1 (DNMT1) and ten-eleven-translocation proteins (TET1, 2, 3), and global DNA methylation in testicular tissue, TESE, and epididymis-derived spermatozoa, and the ADD in zygotes male pronucleus as well as pre-implantation embryo development were assessed. The expression levels of DNMT1 and TET1, 2, 3 in testicles, TESE, and epididymis-derived spermatozoa were decreased in the varicocele group compared to the control and control-sham groups. The TESE-derived spermatozoa exhibited higher DNMT1, higher DNMT1, and TET 1, 2, and no change in TET3 expression compared to epididymis-derived spermatozoa. The varicocele group represented lower DNA methylation in the testicles, TESE-derived and epididymal spermatozoa, higher 5mC+ signal in male pronucleus, and a lower pre-implantation embryo development compared to control and control-sham rats. The TESE-derived spermatozoa exhibited higher 5mC protein expression compared to epididymal spermatozoa. In conclusion, varicocele can negatively impact the DNA methylation/demethylation processes impairing spermatogenesis and leading to fertilization failure, which may ultimately result in a decrease in embryo development by increasing susceptibility to DNA damage.

Selective Estrogen Receptor Modulators' (SERMs) Influence on TET3 Expression in Breast Cancer Cell Lines with Distinct Biological Subtypes.

Tamoxifen, a selective estrogen receptor modulator (SERM), exhibits dual agonist or antagonist effects contingent upon its binding to either G-protein-coupled estrogen receptor (GPER) or estrogen nuclear receptor (ESR). Estrogen signaling plays a pivotal role in initiating epigenetic alterations and regulating estrogen-responsive genes in breast cancer. Employing three distinct breast cancer cell lines-MCF-7 (ESR+; GPER+), MDA-MB-231 (ESR-; GPER-), and SkBr3 (ESR-; GPER+)-this study subjected them to treatment with two tamoxifen derivatives: 4-hydroxytamoxifen (4-HT) and endoxifen (Endox). Through 2D high-performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS), varying levels of 5-methylcytosine (5-mC) were found, with MCF-7 displaying the highest levels. Furthermore, TET3 mRNA expression levels varied among the cell lines, with MCF-7 exhibiting the lowest expression. Notably, treatment with 4-HT induced significant changes in TET3 expression across all cell lines, with the most pronounced increase seen in MCF-7 and the least in MDA-MB-231. These findings underscore the influence of tamoxifen derivatives on DNA methylation patterns, particularly through modulating TET3 expression, which appears to be contingent on the presence of estrogen receptors. This study highlights the potential of targeting epigenetic modifications for personalized anti-cancer therapy, offering a novel avenue to improve treatment outcomes.

Upregulation of TET2 and Resistance to DNA Methyltransferase (DNMT) Inhibitors in DNMT1-Deleted Cancer Cells.

Ten-eleven-translocation (TET) 2 is a member of the TET family of proteins (TET1-3). DNMT1 gene deletion confers resistance to DNA methyltransferase (DNMT) inhibitors in colorectal, breast, and ovarian cancer cells. Currently, the effect of DNMT1 gene status on TET2 phenotype following DNMT inhibitor treatment is unclear in human malignancies. Human colorectal carcinoma HCT116 cells (DNMT+/+) and their isogenic DNMT1 knockout (DNMT1-/-) counterpart were treated with DNMT inhibitors. Expression of TET2 and tumor suppressor (p16ink4A and p15ink4B) proteins were examined by Western blot. Apoptosis and CDKN2A promoter demethylation following drug treatment were detected by Annexin-V apoptosis assay and methylation-specific PCR. TET2 expression was robustly increased in DNMT1-/- cells by 0.5 µM and 5 µM decitabine and azacitidine treatment. Augmentation of TET2 expression was accompanied by re-expression of p16ink4A and p15ink4B proteins and CDKN2A promoter demethylation. TET2 upregulation and tumor suppressor re-expression were associated with resistance conferred by DNMT1 deletion. Treatment with 5-aza-4'-thio-2'-deoxycytidine at a low 0.5 µM dose only upregulated TET2 and reduced CDKN2A promoter methylation, and re-expression of p16ink4A in DNMT1-/- cells. DNMT inhibitors showed minimal effects on TET2 upregulation and re-expression of tumor suppressor proteins in cells with intact DNMT1. DNMT1 gene deletion made cancer cells prone to TET2 upregulation and activation of tumor suppressor expression upon DNMT inhibitor challenge. TET2 augmentation is concomitant with resistance to DNMT inhibitors in a DNMT1-deleted state.

Targeting Nr2e3 to Modulate Tet2 Expression: Therapeutic Potential for Depression Treatment.

Epigenetic mechanisms such as DNA methylation and hydroxymethylation play a significant role in depression. This research has shown that Ten-eleven translocation 2 (Tet2) deficiency prompts depression-like behaviors, but Tet2's transcriptional regulation remains unclear. In the study, bioinformatics is used to identify nuclear receptor subfamily 2 group E member 3 (Nr2e3) as a potential Tet2 regulator. Nr2e3 is found to enhance Tet2's transcriptional activity by binding to its promoter region. Nr2e3 knockdown in mouse hippocampus leads to reduced Tet2 expression, depression-like behaviors, decreased hydroxymethylation of synaptic genes, and downregulation of synaptic proteins like postsynaptic density 95 KDa (PSD95) and N-methy-d-aspartate receptor 1 (NMDAR1). Fewer dendritic spines are also observed. Nr2e3 thus appears to play an antidepressant role under stress. In search of potential treatments, small molecule compounds to increase Nr2e3 expression are screened. Azacyclonal (AZA) is found to enhance the Nr2e3/Tet2 pathway and exhibited antidepressant effects in stressed mice, increasing PSD95 and NMDAR1 expression and dendritic spine density. This study illuminates Tet2's upstream regulatory mechanism, providing a new target for identifying early depression biomarkers and developing treatments.

Microbiota regulates the TET1-mediated DNA hydroxymethylation program in innate lymphoid cell differentiation

Innate lymphoid cell precursors (ILCPs) develop into distinct subsets of innate lymphoid cells (ILCs) with specific functions. The epigenetic program underlying the differentiation of ILCPs into ILC subsets remains poorly understood. Here, we reveal the genome-wide distribution and dynamics of the DNA methylation and hydroxymethylation in ILC subsets and their respective precursors. Additionally, we find that the DNA hydroxymethyltransferase TET1 suppresses ILC1 but not ILC2 or ILC3 differentiation. TET1 deficiency promotes ILC1 differentiation by inhibiting TGF-β signaling. Throughout ILCP differentiation at postnatal stage, gut microbiota contributes to the downregulation of TET1 level. Microbiota decreases the level of cholic acid in the gut, impairs TET1 expression and suppresses DNA hydroxymethylation, ultimately resulting in an expansion of ILC1s. In adult mice, TET1 suppresses the hyperactivation of ILC1s to maintain intestinal homeostasis. Our findings provide insights into the microbiota-mediated epigenetic programming of ILCs, which links microbiota-DNA methylation crosstalk to ILC differentiation.

Expression of genes related to gonadal development and construction of gonadal DNA methylation maps of Trachinotus blochii under hypoxia

Chronic hypoxia can affect the growth and metabolism of fish and potentially impact gonadal development through epigenetic regulation. Trachinotus blochii (Golden pompano) is widely cultured near the coast and is sensitive to low oxygen conditions. We found that hypoxia and reoxygenation processes acted on multiple targets on the HPG axis, leading to endocrine disorders. Changes in the expression of key genes in the brain (gnrh), pituitary (fsh and lh), ovaries (cyp19a1a, foxl2, and er), and testes (dmrt1, ar, sox9, and gsdf) were associated with significant decreases in estrogen and testosterone levels. Hypoxia and reoxygenation lead to changes in DNA methylation levels in the gonads. Hypoxia upregulated the expression of dnmt1, dnmt3a, dnmt3b, tet1, and tet2 in females and dnmt3a and dnmt3b in males, while reoxygenation down-regulated the expression of dnmt1, dnmt3a, dnmt3b, tet1, and tet2 in males. Whole genome methylation sequencing showed that the number of differentially methylated regions was highest on chromosome 10 (5192) and lowest on chromosome 24 (275). Differentially methylated genes in females and males, as well as between males and females, were enriched in the oxytocin signaling pathway, fatty acid metabolism pathway, and HIF-1a pathway. In summary, hypoxia and reoxygenation can induce endocrine disorders, affect the expression of HPG axis genes, change the methylation pattern and modification pattern of gonad DNA, and then have potential effects on gonad development.

TET1 mediated m5C modification of RelB aggravates cerebral ischemia/reperfusion-induced neuroinflammation through regulating microglia polarization

Microglia mediated neuroinflammation is one of the major contributors to brain damage in cerebral ischemia reperfusion injury (CI/RI). Recently, RNA modification was found to contribute to the regulation of microglia polarization and the subsequent development of cerebral I/R neuroinflammation. Herein, we investigated the effect and mechanism of m5C RNA modification in the microglia induced CI/RI neuroinflammation. We found that the m5C RNA modification levels decreased in the primary microglia isolated from a mouse model of intraluminal middle cerebral artery occlusion/reperfusion (MCAO/R) and the BV2 microglial cells subjected to oxygen-glucose deprivation and reoxygenation (OGD/R), and this change was accompanied by an increase in the M1/M2 polarization ratio. Furthermore, the expression of m5C demethylase TET1 in microglia increased, which promoted M1 polarization but impeded M2 polarization. Mechanistically, the higher TET1 expression decreased the m5C modification level of RelB and enhanced its mRNA stability, which subsequently increased the M1/M2 polarization ratio. In conclusion, this study provides insight into the role of m5C RNA modification in the pathogenesis of cerebral I/R neuroinflammation and may deepen our understanding on clinical therapy targeting the TET1-RelB axis.

TET2 Is Downregulated in Early Esophageal Squamous Cell Carcinoma and Promotes Esophageal Squamous Cell Malignant Behaviors.

To explore the expression of the ten eleven translocation (TET) 2 protein in early esophageal squamous cell carcinoma (EESCC), precancerous lesions, and cell lines and to evaluate the effect of TET2 on the functional behavior of EC109 esophageal cancer cells. Thirty-one samples of EESCC and precancerous lesions collected via endoscopic submucosal dissection at Taihe Hospital, Shiyan, from February 1, 2017, to February 1, 2019, were analyzed. The study involved evaluating TET2 expression levels in lesion tissue and adjacent normal epithelium, correlating these with clinical pathological features. Techniques including 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, cell scratch assays, flow cytometry for propidium iodide (PI) staining, Hoechst 333258/PI double staining, and nude mouse tumorigenesis experiments were employed to assess the effect of TET2 on the proliferation, migration, cell cycle, apoptosis, and tumorigenic ability of esophageal cancer cells. TET2 expression was notably reduced in early esophageal cancer tissue and correlated with tumor invasion depth (P < 0.05). Overexpression of TET2 enhanced the proliferation and migration of esophageal cancer cells, increased the cell population in the G0 phase, decreased it in the S phase, and intensified cell necrosis (P < 0.05). There was a partial increase in tumorigenic ability (P = 0.087). TET2 downregulation in ESCC potentially influences the necrosis, cell cycle, and tumorigenic ability of esophageal cancer cells, suggesting a role in the onset and progression of esophageal cancer.

Inter- and trans-generational impacts of real-world PM2.5 exposure on male-specific primary hypogonadism

Exposure to PM2.5, a harmful type of air pollution, has been associated with compromised male reproductive health; however, it remains unclear whether such exposure can elicit transgenerational effects on male fertility. Here, we aim to examine the effect of paternal exposure to real-world PM2.5 on the reproductive health of male offspring. We have observed that paternal exposure to real-world PM2.5 can lead to transgenerational primary hypogonadism in a sex-selective manner, and we have also confirmed this phenotype by using an external model. Mechanically, we have identified small RNAs (sRNAs) that play a critical role in mediating these transgenerational effects. Specifically, miR6240 and piR016061, which are present in F0 PM sperm, regulate intergenerational transmission by targeting Lhcgr and Nsd1, respectively. We have also uncovered that piR033435 and piR006695 indirectly regulate F1 PM sperm methylation by binding to the 3′-untranslated region of Tet1 mRNA. The reduced expression of Tet1 resulted in hypermethylation of several testosterone synthesis genes, including Lhcgr and Gnas, impaired Leydig cell function and ultimately led to transgenerational primary hypogonadism. Our findings provide insights into the mechanisms underlying the transgenerational effects of paternal PM2.5 exposure on reproductive health, highlighting the crucial role played by sRNAs in mediating these effects. The findings underscore the significance of paternal pre-conception interventions in alleviating the adverse effects of environmental pollutants on reproductive health.

Mechanisms underlying LncRNA SNHG1 regulation of Alzheimer’s disease involve DNA methylation

ABSTRACT Alzheimer’s disease (AD) is a neurodegenerative disease associated with long non-coding RNAs and DNA methylation; however, the mechanisms underlying the role of lncRNA small nucleolar RNA host gene 1 (lncRNA SNHG1) and subsequent involvement of DNA methylation in AD development are not known. The aim of this study was to examine the regulatory mechanisms attributed to lncRNA SNHG1 gene utilizing 2 strains of senescence-accelerated mouse prone 8 (SAMP8) model of AD and compared to senescence-accelerated mouse resistant (SAMR) considered a control. Both strains of the mouse were transfected with either blank virus, psLenti-U6-SNHG1(low gene expression) virus, and psLenti-pA-SNHG1(gene overexpression) virus via a single injection into the brains for 2 weeks. At 2 weeks mice were subjected to a Morris water maze to determine any behavioral effects followed by sacrifice to extract hippocampal tissue for Western blotting to measure protein expression of p-tau, DNMT1, DNMT3A, DNMT3B, TET1, and p-Akt. No marked alterations were noted in any parameters following blank virus transfection. In SAMP8 mice, a significant decrease was noted in protein expression of DNMT1, DNMT3A, DNMT3B, and p-Akt associated with rise in p-tau and TET1. Transfection with ps-Lenti-U6-SNHG1 alone in SAMR1 mice resulted in a significant rise in DNMTs and p-Akt and a fall in p-tau and TET1. Transfection of SAMP8 with ps-Lenti-U6-SNHG1 blocked effects on overexpression noted in this mouse strain. However, knockdown of lncRNA SNHG1 yielded the opposite results as found in SAMR1 mice. In conclusion, the knockdown of lncRNA SNHG1 enhanced DNA methylation through the PI3K/Akt signaling pathway, thereby reducing the phosphorylation levels of tau in SAMP8 AD model mice with ameliorating brain damage attributed to p-tau accumulation with consequent neuroprotection.

Abstract 5881: Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells

Abstract Ten-eleven-translocation 2 (TET2) is a member of the TET family proteins (TET1-3) that convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and causes site-specific DNA demethylation. The effects of DNMT1 on TET2 phenotype following DNMT inhibitor treatment is unclear in human cancer. In colorectal cancer HCT116 cells with DNMT1 gene intact (DNMT1+/+) and deletion (DNMT1−/−) status, we found that TET2 expression was robustly increased in DNMT1−/− cells after treatment with 0.5 µM and 5 µM decitabine for 72 h, 96 h, and 120 h. The augmentation in TET2 expression was accompanied by a dose-dependent re-expression of p16ink4A in DNMT1−/− cells. Particularly, TET2 upregulation was associated with resistance to DNMT inhibitors, showing a lack of induction of DNA damage, cell-cycle arrest and apoptosis after drug exposure in DNMT1−/− compared to DNMT1+/+ cells. The treatment with 4’-thio-5-aza-2’-deoxycytidine (aza-T-dCyd) only up-regulated TET2 and re-expressed p16ink4A at the lower dose of 0.5 µM, which was consistent with our previous findings that high dose of aza-T-dCyd at 5 µM predominantly eliminates the cancer cells with severe DNA damage by undergoing apoptosis and cell death (Scientific Reports, 13:5964, 2023). Moreover, both decitabine and aza-T-dCyd had minimal effects on TET2 upregulation and p16ink4A re-expression in cells with intact DNMT1 gene. The data suggest that the absence of DNMT1 gene made the cells prone to TET2 upregulation upon DNMT inhibitor challenges. Future study is needed to investigate the interactive relationship between DNMT1 and TET2 as well as demethylation, and its impact on cancer treatment with DNMT inhibitors. Citation Format: Angelo B. Laranjeira, Dat Nguyen, Lorraine C. Pelosof, James H. Doroshow, Sherry X. Yang. Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5881.