Targeting GATA1 and p2x7r Locus Binding in Spinal Astrocytes Suppresses Chronic Visceral Pain by Promoting DNA Demethylation / 神经科学通报·英文版

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1-TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.

The overall action molecular mechanism of anti-hepatitis B active extracts in Flos chrysanthemi indici based on epigenetics and metabonomics / 药学学报

Using the concepts and methods of epigenetics and metabolomics, to investigate the overall action molecular mechanism of Chrysanthemi indici C (CIC), the anti-hepatitis B virus (HBV) active extracts from Flos chrysanthemi indici. The inhibitory effects of CIC on proliferation and hepatitis B surface antigen (HBsAg), hepatitis B envelope antigen (HBeAg) and HBV-DNA of HepG2.2.15 cells were detected by CCK-8 and antigen kit. The DNA methyltransferases (DNMTs)/ten-eleven-translocation-2 (TET2) equilibrium was detected by ELISA. Illumina 850K methylation chip, pyrosequencing and qPCR were used to determine the action pathway and target of CIC by GO and KEGG analysis. Cell metabolites were extracted with 80% methanol, and the changes of differential metabolites, differential metabolic pathways and cell microenvironment were detected by LC-MS and other metabolomics methods. The results showed that CIC could inhibit the proliferation, HBsAg, HBeAg and HBV-DNA of HepG2.2.15 cells obviously, down-regulate DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a) and DNA methyltransferase 3b (DNMT3b), up-regulate TET2, and restore the balance of DNMTs/TET2. The action targets of CIC were phospholipase C gamma 2 (PLCG2), phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), 5-hydroxytryptamine receptor 2B (HTR2B), nerve growth factor (NGF), mainly involved in lipid metabolism, inflammation mediated regulation of transient receptor potential (TRP), phospholipase D signaling and advanced glycation end product-receptor for AGE (AGE-RAGE) signaling in diabetic complications pathways. CIC could significantly affect fatty acid metabolism and had great influence on phenolic acid, alkaloid and lipid metabolites in cell microenvironment. These results suggest that the action mechanism of CIC may be the synergistic action of multiple pathways and multiple targets, including related inflammatory pathways, immune pathways and lipid metabolism, through regulating epigenetic expression balance and restoring the balance of cell microenvironment.

Roles of ten eleven translocation proteins family and 5-hydroxymethylcytosine in epigenetic regulation of stem cells and regenerative medicine / 北京大学学报(医学版)

The methylation of cytosine is one of the most fundamental epigenetic modifications in mammalian genomes, and is involved in multiple crucial processes including gene expression, cell differentiation, embryo development and oncogenesis. In the past, DNA methylation was thought to be an irreversible process, which could only be diluted passively through DNA replication. It is now becoming increa-singly obvious that DNA demethylation can be an active process and plays a crucial role in biological processes. Ten eleven translocation (TET) proteins are the key factors modulating DNA demethylation. This family contains three members: TET1, TET2 and TET3. Although three TET proteins have relatively conserved catalytic domains, their roles in organisms are not repeated, and their expression has significant cell/organ specificity. TET1 is mainly expressed in embryonic stem cells, TET2 is mainly expressed in hematopoietic system, and TET3 is widely expressed in cerebellum, cortex and hippocampus. This family catalyzes 5-methylcytosine to 5-hydroxymethylcytosine and other oxidative products, reactivates silenced-gene expression, in turn maintains stem cell pluripotency and regulates lineage specification. With the development of tissue engineering, organ transplantation, autologous tissue transplantation and artificial prosthesis have been widely used in clinical treatment, but these technologies have limitations. Regenerative medicine, which uses stem cells and stem cell related factors for treatment, may provide alternative therapeutic strategies for multiple diseases. Among all kinds of human stem cells, adipose-derived stem cells (ADSCs) are the most prospective stem cell lineage since they have no ethical issues and can be easily obtained with large quantities. To date, ADSCs have been shown to have strong proli-feration capacity, secrete numerous soluble factors and have multipotent differentiation ability. However, the underlying mechanism of the proliferation, secretion, acquired pluripotency, and lineage specific differentiation of ADSCs are still largely unknown. Some studies have explored the role of epigenetic regulation and TET protein in embryonic stem cells, but little is known about its role in ADSCs. By studying the roles of TET proteins and 5-hydroxymethylcytosine in ADSCs, we could provide new theoretical foundation for the clinical application of ADSCs and the stem cell-based therapy. In the future, combined with bioprinting technology, ADSCs may be used in tissue and organ regeneration, plastic surgery reconstruction and other broader fields.

Research progress on the relationship between TET1 and tumor invasion and metastasis / 国际外科学杂志

TET1 has been discovered capable of promoting DNA demethylation by mediating 5-methylcytosine to inhibit the genesis and development of cancer.Epithelial-Mesenchymal transition is a complex cellular program in cancer progression and metastasis,whose hallmark is loss of cell polarity and cell adhesion and acquirement of migration and invasion abilities,leading tumor cells into blood vessel and lymphatic vessels to form new metastatic lesions.Epigenetics plays a vital role in epithelial-mesenchymal transition.This review focuses on the relationship of TET1,epigenetics and epithelial-mesenchymall transition,and the research progression of TET1's role in cancer invasion and metastasis.

Expression of TET and 5-HmC in Trophoblast Villi of Women with Normal Pregnancy and with Early Pregnancy Loss / 华中科技大学学报（医学）（英德文版）

Increasing evidence suggests that epigenetic dysfunction may influence the stability of normal pregnancy.The ten-eleven translocation (TET) family and 5-hydroxymethylcytosine (5-hmC) were found to be linked with epigenetic reprogramming.The present study aimed to examine the expression of the TET family and 5-hmC in the villi of human embryos and compared their expression between normal pregnancy and early pregnancy loss (EPL).Embryonic villi were collected from normal pregnant women (control) experiencing medical abortion and from EPL patients at gestation ages of 6,7 and 8 weeks.The mRNAs of TET family were analysed using quantitative polymerase chain reaction (qPCR),and TET proteins using Western blotting and immunohistochemical analysis.The MethylFlashTM Kit was used to quantify the absolute amount of 5-methylcytosine (5-mC) and 5-hmC.Our results showed that the expression of the TETs and 5-hmC in the normal villus decreased with increasing gestational age.Immunohistochemistry revealed that the TET proteins were expressed in the cytoplasm of trophoblasts and their expression was the highest in the 6-week tissue samples,which was consistent with the qPCR and Western blot results.The expression of TET1,TET2,and TET3 was lower in the villi in EPL group than in normal pregnancy group (P＜0.05 for all).It was concluded that the TET family and 5-hmC are critical in epigenetic reprogramming of human embryo.The findings also suggest that a deficiency of TETs in the villus might be associated with human EPL.

Expression of TET and 5-HmC in Trophoblast Villi of Women with Normal Pregnancy and with Early Pregnancy Loss / 华中科技大学学报（医学）（英德文版）

Increasing evidence suggests that epigenetic dysfunction may influence the stability of normal pregnancy.The ten-eleven translocation (TET) family and 5-hydroxymethylcytosine (5-hmC) were found to be linked with epigenetic reprogramming.The present study aimed to examine the expression of the TET family and 5-hmC in the villi of human embryos and compared their expression between normal pregnancy and early pregnancy loss (EPL).Embryonic villi were collected from normal pregnant women (control) experiencing medical abortion and from EPL patients at gestation ages of 6,7 and 8 weeks.The mRNAs of TET family were analysed using quantitative polymerase chain reaction (qPCR),and TET proteins using Western blotting and immunohistochemical analysis.The MethylFlashTM Kit was used to quantify the absolute amount of 5-methylcytosine (5-mC) and 5-hmC.Our results showed that the expression of the TETs and 5-hmC in the normal villus decreased with increasing gestational age.Immunohistochemistry revealed that the TET proteins were expressed in the cytoplasm of trophoblasts and their expression was the highest in the 6-week tissue samples,which was consistent with the qPCR and Western blot results.The expression of TET1,TET2,and TET3 was lower in the villi in EPL group than in normal pregnancy group (P＜0.05 for all).It was concluded that the TET family and 5-hmC are critical in epigenetic reprogramming of human embryo.The findings also suggest that a deficiency of TETs in the villus might be associated with human EPL.

Tet: novel anti-tumor drug target based on DNA demethylation / 上海交通大学学报（医学版）

Tet (ten-eleven translocation) proteins belong to α-ketoglutaric acid (α-KG or 2-OG) and Fe2+ dependent dioxygenases. Tets are found to be involved in the unique mammalian DNA active demethylation process by specifically oxidizing the methyl group of 5-methylcytosine (5mC) in mammalian genome, and play critical roles in gene regulation in early embryonic development and stem cell differentiation via regulating the dynamic balance distribution of 5mC. Abnormal expression and function of Tets are closely associated with various hematological malignances, including myelodysplastic syndrome, chronic myelomonocytic leukemia, and acute lymphoblastic leukemia, as well as solid tumors. Hence, Tets and Tets-mediated DNA demethylation are novel anti-tumor drug targets. Investigation of biological function and catalytic mechanism of Tets is helpful for further understanding mechanisms of tumor incidence and development relevant to DNA demethylation pathway and can provide reference for developing new anti-tumor targeted drugs.

The effect of TET1 on the proliferation of renal cancer 786-O cells and its related mechanism / 肿瘤

Objective: To silence the expression of TET 1 (ten-eleven translocation 1) gene by siRNA (small interfering RNA) and to investigate its impact on the proliferation of human renal cancer 786-O cells and explore its possible mechanism. Methods: The TET1-siRNA targeting human TET 1 gene was transfected into 786-O cells by LipofectAMINE 2000.Three groups were designed as blank control, negative control and siRNA interference (TET1-siRNA) in this study. The expression levels of TET1, SUZ12 (suppressor of zeste 12), EZH2 (enhancer of zeste homolog 2) and EED (embryonic ectoderm development) mRNAs and proteins after transfection with TET1-siRNA were determined by real-time fluorescence quantitative-PCR and Western blotting, respectively. The MTT method, colony formation assay and flow cytometry were performed to detect the proliferation ability, cell colony formation rate and cell cycle distribution of 786-O cells, respectively. Results: As compared with the blank control, the expression levels of TET1 mRNA and protein in 786-O cells were reduced by (85.13±0.03)% and (56.41±0.09)% respectively after transfection with TET1-siRNA (P 0.05). Conclusion: TET1-siRNA can down-regulate the expression levels of TET1 mRNA and protein in human renal cancer 786-O cells, suppress the proliferation of 786-O cells, and block the cells at G1 phase. This mechanism may be associated with the recruitment of PRC2 (polycomb repressive complex 2) influenced by TET1 which regulates the expression of SUZ12. Copyright © 2012 by TUMOR.