MICROCHIMICA ACTA
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2021
·Vol. 188
·期 8
·1-10
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IF 5.833
Yang Hongmei,Wang Yafen,Tang Jing,Wang Fang,Chen Zilin
A promising electrochemical strategy for assay of N6-methyladenosine (m6A)/N6-methyladenine (6mA) in RNA/DNA is proposed. The key of this strategy is the end-labeling of nucleic acid, which makes it possible to detect methylation level in unknown sequence. Firstly, the end of m6A-RNA or 6mA-DNA was labeled with sulfhydryl group through T4 polynucleotide kinase (T4 PNK) and then directly assembled on a gold nanoparticle–modified glassy carbon electrode (AuNPs/GCE). Secondly, methylation sites in RNA/DNA were specifically recognized by anti-m6A-antibody, and then, horseradish peroxidase–labeled goat anti-rabbit IgG (HRP-IgG) was further conjugated on the antibody. Thirdly, HRP-IgG catalyzed the hydroquinone oxidation reaction to generate amplified current signal which correlates with the amount of m6A/6mA in nucleic acid. This method showed a wide linear range from 0.0001 to 10 nM for m6A-RNA, 0.001 to 100 nM for 6mA-dsDNA, and 0.0001 to 10 nM for 6mA-ssDNA. The method was successfully applied to detection of m6A/6mA in RNA/DNA from HeLa cells and E. coli cells and validation of the decrease of m6A-RNA in HeLa cells after treatment with FTO protein. Graphical abstract
研究领域:
电化学
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生物化学
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分子生物学
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分析化学
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产品关联
DOI
PubMed
高影响因子
CELLULAR SIGNALLING
·
2021
·
IF 4.315
Jinpeng Zhuang,Hua Ning,Maoqing Wang,Wei Zhao,Yongbin Jing,Xiaoqi Liu,Jianing Zu,Pengyu Kong,Xiaoyan Wang,Changhao Sun,Jinglong Yan
During osteoporosis, fat mass and obesity-associated protein (FTO) promotes the shift of bone marrow mesenchymal stem cells to adipocytes and represses osteoblast activity. However, the role and mechanisms of FTO on osteoclast formation and bone resorption remain unknown. In this study, we investigated the effect of FTO on RAW264.7 cells and bone marrow monocytes (BMMs)-derived osteoclasts in vitro and observed the influence of FTO on ovariectomized (OVX) mice model to mimic postmenopausal osteoporosis in vivo. Results found that FTO was up-regulated in BMMs from OVX mice. Double immunofluorescence assay showed co-localization of FTO with tartrate-resistant acid phosphatase (TRAP) in femurs of OVX mice. FTO overexpression enhanced TRAP-positive osteoclasts and F-actin ring formation in RAW264.7 cells upon RANKL stimulation. The expression of osteoclast differentiation-related genes, including nuclear factor of activated T cells c1 (NFATc1) and c-FOS, was upregulated in BMMs and RAW264.7 cells after FTO overexpression. FTO overexpression induced the phosphorylation and nuclear translocation of factor-kappa B (NF-κB) p65 in BMMs and RAW264.7 cells exposed to RANKL. ChIP and dual-luciferase assays revealed that FTO overexpression contributed to RANKL-induced binding of NF-κB to NFATc1 promoter. Rescue experiments suggested that FTO overexpression-mediated osteoclast differentiation was suppressed after intervention with a NF-κB inhibitor pyrrolidine dithiocarbamate . Further in vivo evidence revealed that FTO knockdown increased bone trabecula and bone mineral density, inhibited bone resorption and osteoclastogenesis in osteoporotic mice. Collectively, our research demonstrates that downregulated FTO inhibits bone resorption and osteoclastogenesis through NF-κB inactivation, which provides a novel reference for osteoporosis treatment.
研究领域:
骨骼生物学
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分子生物学
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细胞生物学
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内分泌学
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DOI
PubMed
高影响因子
CELLULAR SIGNALLING
·
2024
·
IF 4.4
Wentao Hu,Menghan Wang,Guifang Sun,Limin Zhang,Hong Lu
Parkinson's disease (PD) is a gradually debilitating neurodegenerative syndrome. Here, we analyzed GSE7621 chip data obtained from the Gene Expression Omnibus (GEO) database to explore the pathogenesis of PD. Early B Cell Factor 3 (EBF3), a member of the highly evolutionarily conserved EBF-transcription factor family, is involved in neuronal development. EBF3 expression is low in the substantia nigra of patients with PD. However, whether EBF3 is implicated in dopaminergic neuron death during PD has not yet been investigated. Therefore, we aimed to reveal the potential anti-apoptotic effect and molecular mechanism of EBF3 in PD. We established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model in vivo and a 1-methyl-4-phenylpyridine (MPP + )-induced SH-SY5Y cell model in vitro . EBF3 was downregulated in the substantia nigra of PD mice and SH-SY5Y cells treated with MPP + , and the m6A methylation modification level was low. Fat mass and obesity-associated protein (FTO) siRNA upregulated m6A methylation modification of EBF3 and extended the EBF3 mRNA half-life. Functionally, as demonstrated by the results of the open-field test, pole test and gait analysis, EBF3 overexpression ameliorated MPTP-induced behavioral disorder. Further, EBF3 overexpression suppressed neuronal apoptosis in vivo , as evidenced by decreased TUNEL + cells, and the increased activation of caspase-3 and caspase-9. Similar results were obtained in vitro , as reflected by increased cell viability, decreased LDH activity and restored mitochondrial function, collectively protecting SH-SY5Y cells from MPP + -induced apoptosis. Mechanistically, the results of luciferase reporter, ch-IP and DNA pull-down assays confirmed that, as a transcription factor, EBF3 bound to the promoter of CNTNAP4 (a protein associated with neuronal differentiation) and directly regulated CNTNAP4 transcription. Strikingly, CNTNAP4 knockdown markedly abolished the effect of EBF3 on cell apoptosis, thus aggravating PD. In conclusion, the low level of m6A methylation modification may contribute to the low expression of EBF3 during PD. Additionally, EBF3 attenuates PD by activating CNTNAP4 transcription, suggesting that EBF3 may be a novel therapeutic target in PD.
研究领域:
神经科学
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分子生物学
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转化医学
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产品关联
DOI
PubMed
高影响因子
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