OBJECTIVE: To observe the clinical significance of translocator proteins (TSPO) gene in the treatment of FLT3-ITD/DNMT3A R882 double-mutated acute myeloid leukemia (AML). METHODS: Seventy-six patients with AML hospitalized in the Department of Hematology of the Affiliated People's Hospital of Ningbo University from June 2018 to June 2020 were selected, including 34 patients with FLT3-ITD mutation, 27 patients with DNMT3A R882 mutation, 15 patients with FLT3-ITD/DNMT3A R882 double mutation, as well as 19 patients with immune thrombocytopenia (ITP) hospitalized during the same period as control group. RNA was routinely extracted from 3 ml bone marrow retained during bone puncture, and TSPO gene expression was detected by transcriptome sequencing (using 2-deltadeltaCt calculation). RESULTS: The expression of TSPO gene in FLT3-ITD group and DNMT3A R882 group at first diagnosis was 2.02±1.04 and 1.85±0.76, respectively, which were both higher than 1.00±0.06 in control group, but the differences were not statistically significant (P=0.671, P=0.821). The expression of TSPO gene in the FLT3-ITD/DNMT3A R882 group was 3.98±1.07, wich was significantly higher than that in the FLT3-ITD group and DNMT3A R882 group, the differences were statistically significant (P=0.032, P=0.021). The expression of TSPO gene in patients who achieved complete response after chemotherapy in the FLT3-ITD/DNMT3A R882 group was 1.19±0.87, which was significantly lower than that at first diagnosis, and the difference was statistically significant (P=0.011). CONCLUSION TSPO gene may be used as an indicator of efficacy in FLT3-ITD /DNMT3A R882 double-mutated AML.
Humans ; DNA (Cytosine-5-)-Methyltransferases/genetics* ; DNA Methyltransferase 3A ; Mutation ; Leukemia, Myeloid, Acute/drug therapy* ; Nucleophosmin ; Prognosis ; fms-Like Tyrosine Kinase 3/genetics* ; Receptors, GABA/therapeutic use*

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.
Humans ; Apolipoprotein E4/genetics* ; Cytosine ; Mutation ; Blastocyst ; Heterozygote ; Gene Editing ; CRISPR-Cas Systems

RNA methylation is of great significance in the regulation of gene expression, among which the more important methylation modifiers are N6-methyladenosine (m6A) and 5-methylcytosine (m5C). The methylation process is mainly regulated by 3 kinds of proteins: methyltransferase, demethylase, and reader. m6A, m5C, and their related proteins have high abundance in the brain, and they have important roles in the development of the nervous system and the repair and remodeling of the vascular system. The neurovascular unit (NVU) is a unit of brain structure and function composed of neurons, capillaries, astrocytes, supporting cells, and extracellular matrix. The local microenvironment for NVU has an important role in nerve cell function repair, and the remodeling of NVU is of great significance in the prognosis of various neurological diseases.
5-Methylcytosine ; Adenosine/metabolism* ; Methylation ; Methyltransferases/metabolism* ; RNA

The CRISPR system is able to accomplish precise base editing in genomic DNA, but relies on the cellular homology-directed recombination repair pathway and is therefore extremely inefficient. Base editing is a new genome editing technique developed based on the CRISPR/Cas9 system. Two base editors (cytosine base editor and adenine base editor) were developed by fusing catalytically disabled nucleases with different necleobase deaminases. These two base editors are able to perform C>T (G>A) or A>G (T>C) transition without generating DNA double-stranded breaks. The base editing technique has been widely used in gene therapy, animal models construction, precision animal breeding and gene function analysis, providing a powerful tool for basic and applied research. This review summarized the development process, technical advantages, current applications, challenges and perspectives for base editing technique, aiming to help the readers better understand and use the base editing technique.
Adenine ; Animals ; CRISPR-Cas Systems/genetics* ; Cytosine ; DNA Breaks, Double-Stranded ; Gene Editing

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.
5-Methylcytosine/analogs & derivatives* ; Animals ; DNA Methylation ; DNA-Binding Proteins/genetics* ; Epigenesis, Genetic ; Humans ; Mixed Function Oxygenases/metabolism* ; Prospective Studies ; Proto-Oncogene Proteins/metabolism* ; Regenerative Medicine ; Stem Cells/metabolism*

DNA methylation is a significant epigenetic modification mode, which plays an important role in embryo reprogramming, stem cell differentiation and tumor occurrence. The ten-eleven translocation (TET) enzyme is a crucial demethylation enzyme, which can catalyze 5-methylcytosine(5mC) to 5-hydroxymethylcytosine(5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine(5caC). These bases represent the epigenetic modifications of DNA and regulate the process of DNA methylation. Understanding the role of TET enzyme in regulating the DNA methylation modification and gene expression can help us to gain the knowledge for the normal growth development and epigenetic regulation in human diseases.
5-Methylcytosine ; metabolism ; Cell Differentiation ; DNA ; DNA Methylation ; DNA-Binding Proteins ; Epigenesis, Genetic ; Humans

OBJECTIVE: To explore the efficacy and safety of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of relapsed or refractory peripheral T-cell lymphoma(PTCL). METHODS: The clinical data of 6 patients with relapsed or refractory PTCL undergoing allo-HSCT from Sep. 2014 to Sep. 2018 in the department of hematology, aerospace center hospital were retrospectively analyzed. Complications and disease-free survival after HSCT were observed. RESULTS: All the patients could well tolerate the conditioning regimen and acquired hematopoietic recon-struction. Following up till December 2018, with a median time of 11.5 months (1-51); acute GVHD developed in 2 cases and chronic GVHD developed in 5 cases, Among 6 cases one case died of viral pheumonia and the other 5 patients remained disease-free survival. The longest disease-free survival time has reached 51 months. CONCLUSION allo-HSCT is a safe and effective method for relapsed or refractory peripheral T-cell lymphoma, which can be chosen as salvage treatment method for patients with primary resistance. Optimization of the conditioning regimen may result in better efficacy of allo-HSCT.
DNA (Cytosine-5-)-Methyltransferases ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myeloid, Acute ; Mutation ; Retrospective Studies ; Transplantation Conditioning ; fms-Like Tyrosine Kinase 3

Tripterygium Glycosides Tablets has good anti-inflammatory and immunomodulatory activities,but its reproductive damage is significant. Previous studies of the research group have found that Cuscutae Semen flavonoids can improve spermatogenic cell damage caused by Tripterygium Glycosides Tablets by regulating spermatogenic cell cycle,apoptosis and related protein expression,but the mechanism of action at the gene level is still unclear. In this study,Illumina high-throughput sequencing platform was applied in transcriptional sequencing of spermatogenic cells of rats after the intervention of Cuscutae Semen flavonoids and Tripterygium Glycosides Tablets. Differentially expressed genes were screened out and the GO enrichment and KEGG pathway analysis of differentially expressed genes were conducted to explore the mechanism of Cuscutae Semen flavonoids in improving reproductive injury caused by Tripterygium Glycosides Tablets. The results showed that 794 up-regulated genes and 491 down-regulated genes were screened in Tripterygium Glycosides Tablets group compared with the blank group. Compared with Tripterygium Glycosides Tablets,440 up-regulated genes and 784 down-regulated genes were screened in the Cuscutae Semen flavonoids+Tripterygium Glycosides Tablets group. Among them,the gene closely related to reproductive function is DNMT3 L. Analysis of GO function and KEGG signaling pathway enrichment showed that the above differentially expressed genes were mainly enriched in cell,cell process,catalytic activity,binding,ovarian steroid synthesis,thyroid hormone and other functions and pathways. The thyroid hormone signaling pathway was the common enrichment pathway of the two control groups. In a word,Cuscutae Semen flavonoids has a good treatment effect on male reproductive damage caused by Tripterygium Glycosides Tablets. The mechanism may be closely related to up-regulation of DNMT3 L genes and intervention of thyroid hormone signaling pathway. At the same time,the discovery of many different genes provides valuable information for study on the mechanism of Cuscutae Semen flavonoids and Tripterygium Glycosides Tablets compatibility decreasing toxicity and increasing efficiency.
Animals ; Cuscuta ; chemistry ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Female ; Flavonoids ; pharmacology ; Genitalia ; drug effects ; pathology ; Glycosides ; toxicity ; High-Throughput Nucleotide Sequencing ; Male ; Rats ; Seeds ; chemistry ; Signal Transduction ; Tablets ; Thyroid Hormones ; genetics ; Transcriptome ; Tripterygium ; toxicity

BACKGROUND: DNA methylation is involved in numerous biologic events and associates with transcriptional gene silencing, playing an important role in the pathogenesis of endometrial cancer. ESR1/PGR frequently undergoes de novo methylation and loss expression in a wide variety of tumors, including breast, colon, lung, and brain tumors. However, the mechanisms underlying estrogen and progesterone receptors (ER/PR) loss in endometrial cancer have not been studied extensively. The aims of this study were to determine the expression of DNA (cytosine-5)-methyltransferase 3A/3B (DNMT3A/3B) in endometrial cancer to investigate whether the methylation catalyzed by DNMT3A/3B contributes to low ER/PR expression. METHODS: The clinicopathologic information and RNA-Seq expression data of DNMT3A/3B of 544 endometrial cancers were derived from The Cancer Genome Atlas (TCGA) uterine cancer cohort in May 2018. RNA-Seq level of DNMT3A/3B was compared between these clinicopathologic factors with t-test or one-way analysis of variance. RESULTS: DNMT3A/3B was overexpressed in endometrioid carcinoma (EEC) and was even higher in non-endometrioid carcinoma (NEEC) (DNMT3A, EEC vs. NEEC: 37.6% vs. 69.9%, t = -7.440, P < 0.001; DNMT3B, EEC vs. NEEC: 42.4% vs. 72.8%, t = -6.897, P < 0.001). In EEC, DNMT3A overexpression was significantly correlated with the hypermethylation and low expression of the ESR1 and PGR (P < 0.05). The same trend was observed in the DNMT3B overexpression subgroup. In the ESR1/PGR low-expression subgroups, as much as 83.1% of ESR1 and 59.5% of PGR were hypermethylated, which was significantly greater than the ESR1/PGR high-expression subgroups (31.3% and 11.9%, respectively). However, the above phenomena were absent in NEEC, while DNMT3A/3B overexpression, ESR1/PGR hypermethylation, and low ER/PR expression occurred much more often. In univariate analysis, DNMT3A/3B overexpressions were significantly correlated with worse prognosis. In multivariate analysis, only DNMT3A was an independent predictor of disease-free survival (P < 0.05). CONCLUSIONS DNMT3A/3B expression increases progressively from EEC to NEEC and is correlated with poor survival. The mechanisms underlying low ER/PR expression might be distinct in EEC vs. NEEC. In EEC, methylation related to DNMT3A/3B overexpression might play a major role in ER/PR downregulation.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Endometrioid ; genetics ; metabolism ; pathology ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Methylation ; genetics ; Endometrial Neoplasms ; genetics ; metabolism ; pathology ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Prognosis

PURPOSE: In the present study, human neural stem cells (hNSCs) with tumor-tropic behavior were used as drug delivery vehicle to selectively target melanoma. A hNSC line (HB1.F3) was transduced into two types: one expressed only the cytosine deaminase (CD) gene (HB1.F3. CD) and the other expressed both CD and human interferon-β (IFN-β) genes (HB1.F3.CD. IFN-β). MATERIALS AND METHODS: This study verified the tumor-tropic migratory competence of engineered hNSCs on melanoma (A375SM) using a modified Boyden chamber assay in vitro and CM-DiI staining in vivo. The antitumor effect of HB1.F3.CD and HB1.F3.CD.IFN-β on melanoma was also confirmed using an MTT assay in vitro and xenograft mouse models. RESULTS: A secreted form of IFN-β from the HB1.F3.CD.IFN-β cells modified the epithelial-mesenchymal transition (EMT) process and metastasis of melanoma. 5-Fluorouracil treatment also accelerated the expression of the pro-apoptotic protein BAX and decelerated the expression of the anti-apoptotic protein Bcl-xL on melanoma cell line. CONCLUSION: Our results illustrate that engineered hNSCs prevented malignant melanoma cells from proliferating in the presence of the prodrug, and the form that secreted IFN-β intervened in the EMT process and melanoma metastasis. Hence, neural stem cell-directed enzyme/prodrug therapy is a plausible treatment for malignant melanoma.
Animals ; Cell Line ; Cytosine Deaminase ; Epithelial-Mesenchymal Transition ; Flucytosine ; Fluorouracil ; Heterografts ; Humans ; In Vitro Techniques ; Melanoma ; Mental Competency ; Mice ; Neoplasm Metastasis ; Neural Stem Cells ; Stem Cells