Science China-Chemistry
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2024
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IF 10.4
Xu Xing,Zeng Xi,Lin Xin,Lin Shiyan,Liang Shanshan,Tian Tian,Su Rui,Song Jia,Yang Chaoyong
Single-cell joint analysis of methylome and transcriptome reveals how the methylation regulates the transcriptional activity. However, traditional bench-top protocols for single-cell DNA methylation and RNA transcription co-detection are labor-intensive, cost-ineffective and contaminant-prone. Herein, we establish the DMF-scMT-seq, a highly-efficient and cost-effective method to simultaneously analyze single-cell DNA methylation and transcriptional activity based on digital microfluidics. DMF-scMT-seq automates the workflow of single-cell isolation, cellular hypotonic lysis, nucleic acid separation and methylome/transcriptome library construction in a contactless and addressable way. The system ensures high accuracy ( R >0.85), high gene detection ability (14,697 genes per cell at 4 million sequencing depth), and high CpG coverage (677,198 CpG sites per cell at 1 million sequencing depth). By using DMF-scMT-seq, the relationship of DNA methylation and RNA transcription under different genomic contexts is resolved. We further apply DMF-scMT-seq to study the dynamics of transcription regulation with methylation-inhibiting anti-tumor Decitabine, and identify the methylated promoter/gene body driven genes in response to Decitabine treatment. DMF-scMT-seq facilitates the construction of the correlation of DNA methylation and transcriptional activity at the single-cell level in a flexible, sensitive and accurate way, which is anticipated to be a powerful tool in studying single-cell biological systems.
研究领域:
生物信息学
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表观遗传学
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分子生物学
✓ 已验证
DOI
高影响因子
Frontiers in Medicine
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2025
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IF 3
Xiaohui Gao,Hui Zeng,Fei Sun,Xiaoyan Zhao,Haibing Wu,Minchao Yan,Yuan Li,Qinyan Fu,Gang Zhang
Purpose: This study aimed to evaluate the underlying mechanisms of decitabine (DAC) in inhibiting acute T-acute lymphoblastic leukemia (T-ALL) cell proliferation and promoting apoptosis.Methods: Human T-ALL cells (CCRF-CEM) were treated with varying concentrations of DAC, and cell proliferation was assessed using a CCK-8 assay. Flow cytometry was used to detect apoptosis and cell cycle alterations. The expression levels of apoptosis-related genes, including PI3K and miR-92b-3p, were quantified using real-time PCR (RT-PCR). Western blotting was used to analyze the expression of apoptotic proteins. Furthermore, we evaluated the in vivo antileukemic activity of DAC using a nude mouse xenograft model, monitored the body weight and tumor volume of mice to calculate inhibition rates, and examined tumor morphological changes in histological sections.Results: DAC significantly inhibited the proliferation of CCRF-CEM cells, accelerated apoptosis, and effectively downregulated the expression of PI3K, AKT, 4EBP1, and mTOR while concurrently upregulating PTEN protein expression. Its regulatory efficacy was markedly enhanced by increasing the dosage. Animal experimental results indicated that both DAC and doxorubicin substantially decreased tumor length, width, volume, and mass; however, DAC demonstrated significantly superior efficacy in inhibiting tumor growth compared to doxorubicin.Conclusion: By selectively targeting the regulation of PTEN and 4EBP1, along with their associated downstream signaling pathways, DAC effectively modulated cellular proliferation, facilitated apoptotic processes, and restrained tumor growth, providing a robust theoretical foundation for clinical treatment strategies in T-ALL.
研究领域:
血液学
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肿瘤学
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分子生物学
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药理学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF CONTROLLED RELEASE
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2026
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IF 12.4
Xinchen Zhao,Liyan Qiu
Chemodynamic therapy (CDT) has been widely explored for the treatment of various solid tumors. However, its efficacy is severely limited by insufficient reactive oxygen species (ROS) amplification and epigenetic silencing of key immune-regulatory genes. Herein, we developed a composite nanovesicle (PLMD) that integrates CDT with epigenetic reprogramming to synergistically activate the cGAS-STING pathway and induce pyroptosis. PLMD was constructed via amphiphilic polymer self-assembly and surface-functionalization with sialic acid to enhance tumor targeting, enabling the co-delivery of β-lapachone (Lap), Mn 2+ , and decitabine (Dac) with acid- and carboxylesterase-responsive release in tumor cells. Lap selectively generates H 2 O 2 in NQO1-overexpressing tumor cells, which cooperates with Mn 2+ -mediated Fenton-like reactions to amplify intracellular ROS level, induce mitochondrial damage, and promote cytosolic mitochondrial DNA (mtDNA) release. Mn 2+ further sensitizes cGAS DNA sensing, leading to robust activation of the cGAS-STING signaling pathway. Meanwhile, activation of the intrinsic apoptotic pathway induces caspase-3 cleavage of gasdermin E (GSDME), thereby inducing pyroptosis. Furthermore, Dac epigenetically restores the expression of STING and GSDME via DNA demethylation, markedly augmenting cGAS-STING activation and pyroptosis. As a result, PLMD treatment enhances dendritic cell maturation and T-cell priming, ultimately achieving pronounced tumor growth inhibition and robust antitumor immune responses in a 4T1 tumor model.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Analytical Cellular Pathology
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2023
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IF 3.2
Fan Du,Ting Jin,Li Wang
Objective. Acute myeloid leukemia (AML) is a heterogeneous malignancy with a low long-term survival rate. The aim of this study was to investigate the effects of decitabine (DAC) treatment cell proliferation and apoptosis in AML and role of the expression of LINC00599 and, consequently, miR-135a-5p. Materials and Methods. Human promyelocytic leukemia cells (HL-60) and human acute lymphatic leukemia (CCRF-CEM) cells were treated with various concentrations of DAC. Cell proliferation in each group was detected using the cell counting kit 8. For each group, apoptosis and reactive oxygen species (ROS) levels were detected using flow cytometry. Reverse transcription polymerase chain reaction (RT-PCR) was performed to examine the expression of lncRNA LINC00599. The expression of apoptosis-related proteins was analyzed using western blotting. The regulatory relationship between miR-135a-5p and LINC00599 was verified by constructing miR-135a-5p mimics, miR-135a-5p inhibit, wild type LINC00599 3-untranslated region (UTR), and mutant LINC00599 3-UTR. Ki-67 expression in the tumor tissues of nude mice was detected using immunofluorescent assays. Results. Both DAC and LINC00599 Inhibit groups were able to significantly reduce the proliferation of HL60 and CCRF-CEM cells, increase apoptosis, upregulate the expression of Bad, cleaved caspase-3, and miR-135a-5p, downregulate the expression of Bcl-2, and elevate ROS levels in cells, with these effects being more pronounced after combined treatment with DAC and LINC00599 Inhibit. In comparison to mimic NC, the miR-135a-5p mimic group significantly decreased the relative fluorescence activity ratio of LINC00599 3-UTR wild-type CCRF-CEM cells. The LINC00599 Inhibit and miR-135a-5p mimic groups exhibited substantially reduced proliferation of HL60 and CCRF-CEM cells, increased apoptosis, upregulated Bad, cleaved caspase-3, and miR-135a-5p expression, along with downregulated Bcl-2 and LINC00599 expression and increased ROS levels in cells; these effects were more pronounced after LINC00599 Inhibit was combined with miR-135a-5p mimics. In vivo experiments revealed that both DAC and LINC00599 Inhibit were able to considerably reduce the long diameter, short meridian, volume, and mass of tumors, increase miR-135a-5p expression, and decrease LINC00599 and ki-67 expression in tumor tissues of nude mice. This effect was more pronounced when the DAC and LINC00599 Inhibit were used in combination. Conclusion. DAC regulates the expression of miR-135a-5p by regulating the expression of LINC00599, which in turn affects cell proliferation, apoptosis, and tumor proliferation. Our findings provide a theoretical basis for improving the clinical outcome of AML.
研究领域:
肿瘤学
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血液学
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分子生物学
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药理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF PHARMACEUTICS
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2018
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IF 3.862
Wenhui Tao,Dongyang Zhao,Mengchi Sun,Ziyu Wang,Bin Lin,Yu Bao,Yingying Li,Zhonggui He,Yinghua Sun,Jin Sun
Decitabine (DAC), a potent DNA methyltransferase (DNMT) inhibitor, has a limited oral bioavailability. Its 5′-amino acid ester prodrugs could improve its oral delivery but the specific absorption mechanism is not yet fully understood. The aim of this present study was to investigate the in vivo absorption and activation mechanism of these prodrugs using in situ intestinal perfusion and pharmacokinetics studies in rats. Although PEPT1 transporter is pH dependent, there appeared to be no proton cotransport in the perfusion experiment with a preferable transport at pH 7.4 rather than pH 6.5. This suggested that the transport was mostly dependent on the dissociated state of the prodrugs and the proton gradient might play only a limited role. In pH 7.4 HEPES buffer, an increase in P eff was observed for L-val-DAC, D-val-DAC, L-phe-DAC and L-trp-DAC (2.89-fold, 1.2-fold, 2.73-fold, and 1.90-fold, respectively), compared with the parent drug. When co-perfusing the prodrug with Glysar, a known substrate of PEPT1, the permeabilities of the prodrugs were significantly inhibited compared with the control. To further investigate the absorption of the prodrugs, L-val-DAC was selected and found to be concentration-dependent and saturable, suggesting a carrier-mediated process (intrinsic K m : 7.80 ± 2.61 mM) along with passive transport. Determination of drug in intestinal homogenate after perfusion further confirmed that the metabolic activation mainly involved an intestinal first-pass effect. In a pharmacokinetic evaluation, the oral bioavailability of L-val-DAC, L-phe-DAC and L-trp-DAC were nearly 1.74-fold, 1.69-fold and 1.49-fold greater than that of DAC. The differences in membrane permeability and oral bioavailability might be due to the different stability in the intestinal lumen and the distinct PEPT1 affinity which is mainly caused by the stereochemistry, hydrophobicity and steric hindrance of the side chains. In summary, the detailed investigation of the absorption mechanism by in vivo intestinal perfusion and pharmacokinetic studies showed that the prodrugs of DAC exhibited excellent permeability and oral bioavailability, which might be attributed to a hybrid (partly PEPT1-mediated and partly passive) transport mode and a rapid activation process in enterocytes.
研究领域:
药理学
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药物代谢动力学
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分子生物学
✓ 已验证
DOI
PubMed
高影响因子
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