Nature Communications
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2023
·Vol. 14
·期 1
·1-20
·
IF 16.6
Han Xiao,Liu Lijuan,Huang Saihua,Xiao Wenfeng,Gao Yajing,Zhou Weitao,Zhang Caiyan,Zheng Hongmei,Yang Lan,Xie Xueru,Liang Qiuyan,Tu Zikun,Yu Hongmiao,Fu Jinrong,Wang Libo,Zhang Xiaobo,Qian Liling,Zhou Yufeng
N 6 -methyladenosine (m 6 A), the most prevalent mRNA modification, has an important function in diverse biological processes. However, the involvement of m 6 A in allergic asthma and macrophage homeostasis remains largely unknown. Here we show that m 6 A methyltransferases METTL3 is expressed at a low level in monocyte-derived macrophages from childhood allergic asthma patients. Conditional knockout of Mettl3 in myeloid cells enhances Th2 cell response and aggravates allergic airway inflammation by facilitating M2 macrophage activation. Loss and gain functional studies confirm that METTL3 suppresses M2 macrophage activation partly through PI3K/AKT and JAK/STAT6 signaling. Mechanistically, m 6 A-sequencing shows that loss of METTL3 impairs the m 6 A-YTHDF3-dependent degradation of PTX3 mRNA, while higher PTX3 expression positively correlates with asthma severity through promoting M2 macrophage activation. Furthermore, the METTL3/YTHDF3-m 6 A/PTX3 interactions contribute to autophagy maturation in macrophages by modulating STX17 expression. Collectively, this study highlights the function of m 6 A in regulating macrophage homeostasis and identifies potential targets in controlling allergic asthma.
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HUMAN GENE THERAPY
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2020
·
IF 4.51
Kaihua Zhai,Boyu Liu,Lin Gao
Long-noncoding RNA taurine upregulated gene 1 (TUG1) participates in nervous system diseases, but its function in Parkinson's disease (PD) remains unclear. This study explored the function and mechanism of TUG1 in PD. A PD model was constructed using SH-SY5Y cells induced by 1-methyl-4-phenylpyridinium (MPP+) in vitro and mice treated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in vivo. The expressions of TUG1, miR-152-3p, phosphatase and tensin homologue (PTEN), tyrosine hydroxylase (TH), and Bcl-2, and cleaved caspase-3 expressions were determined by quantitative reverse transcription-PCR and Western blotting. The viability, apoptosis, reactive oxygen species, and release of inflammatory factors from SH-SY5Y cells and substantia nigra tissues were detected by commercial kits. The interaction between TUG1 and miR-152-3p was analyzed by dual-luciferase reporter assay. Hematoxylin/eosin and immunohistochemical staining was performed for assessing the pathological damage and proportion of TH-positive cells. In PD cell model and mice model, TUG1 expression was upregulated and that of miR-152-3p was downregulated. Further research showed that TUG1 sponged and regulated miR-152-3p expression. Silencing of TUG1 not only protected SH-SY5Y cells against cell apoptosis, oxidative stress, and neuroinflammation in vitro, pathological damage and neuroinflammation in vivo, but also suppressed the expressions of PTEN and cleaved caspase-3, and increased the expressions of TH and Bcl-2 in MPP+-treated SH-SY5Y cells. However, the protective role of siTUG1 in SH-SY5Y cells was significantly inhibited by the miR-152-3p inhibitor. Thus, knocking down TUG1 might have a protective effect on PD through the miR-152-3p/PTEN pathway.
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Science China-Chemistry
·
2020
·Vol. 63
·期 4
·546-553
·
IF 6.356
Li Junwei,Yuan Dandan,Zheng Xiangjiang,Zhang Xinyue,Li Xuemei,Zhang Shusheng
Conventional cancer combination therapy usually involves systemic delivery of anticancer drugs which may lead to the destruction of normal cells and physiological toxicity due to the lack of targeting ability and toxicity of drug carriers. In the present study, a triple combination nanosystem of gene therapy, chemotherapy and phototherapy delivered by multifunctional RNA nanohydrogels (RNA NHs) was established. By taking the advantages of DNA nanotechnology and rolling circle transcription (RCT), three lung cancer inhibitor microRNA (let-7a, microRNA 34a, microRNA 145) hairpins were integrated in one RNA NH nanoparticle, leading to the simultaneous silencing of three targeted mRNAs. Meanwhile, RNA NH carried doxorubicin (DOX, a chemotherapy drug) as well as 5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4, a photosensitizer) and delivered these drugs to cancer cells. It was demonstrated that lung cancer inhibitor microRNAs integrated in RNA NHs, DOX and TMPyP4 could play a synergistic anti-cancer role in multi-drug resistance cancer cells. Under the action of aptamer sequence S6 that was modified with cholesterol, the resulting RNA NHs were condensed to feasible size without the assistance of polyelectrolyte condensation reagents and showed cancer-specific cellular targeting. Subsequently, thousands of copies of miRNA together with chemotherapy drug as well as photosensitizer were delivered to cancer cells specifically, and an ideal synergistic treatment effect was achieved in vivo , thus playing a combined role of gene therapy, chemotherapy and phototherapy. Through this study, it can be concluded that the triple combination therapy nanosystem can overcome the multidrug resistance caused by the malfunction of genes in chemotherapy and shows a great potential in the field of multifunctional synergistic cancer treatment.
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ACS Chemical Neuroscience
·
2021
·
IF 4.418
Bei Sun,Hao Ou,Fei Ren,Yujiao Guan,Ye Huan,Hongwei Cai
Cerebral ischemia-reperfusion (CI/R) injury is a serious central nervous system disease. Propofol (PPF) exerts a neuroprotective effect in CI/R injury; the underlying cause is still unclear. Here, we cultured mouse hippocampal neuron (HT22 cells) in oxygen-glucose deprivation/reoxygenation (OGD/R) conditions to mimic CI/R injury in vitro. PPF treatment promoted cell viability and reduced apoptotic cells in the OGD/R-treated HT22 cells, which was effectively abrogated by SNHG14 overexpression. Moreover, we constructed a CI/R injury mouse model on C57BL/6J mice by middle cerebral artery occlusion/reperfusion (MCAO/R), followed by administration of PPF. PPF reduced neuronal damage and loss, enhanced glial cell hyperplasia, and ameliorated cerebral cortex tissue damage and brain infarct in MCAO/R-induced mice. SNHG14 overexpression aggravated MCAO/R-induced CI/R injury in mice. Furthermore, SNHG14 promoted the expression of Atg5 and Beclin 1 via competitively binding miR-30b-5p, which contributed to activate autophagy and apoptosis in HT22 cells. In addition, the levels of p-p38 and p-SP1 were reduced in the OGD/R-treated HT22 cells in the presence of PPF. SP1 interacted with the promoter of SNHG14 and elevated the expression of SNHG14. PPF treatment inhibited the SP1-mediated up-regulation of SNHG14. In conclusion, this work demonstrates that PPF inhibits SNHG14 expression though the p38 MAPK signaling pathway. SNHG14 promotes Atg5 and Beclin 1 expression by sponging miR-30b-5p and thus activates autophagy and aggravates CI/R injury.
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Viruses-Basel
·
2019
·Vol. 11
·期 4
·314
·
IF 3.811
Gaobo Zhang,Jian Yang,Fujun Qin,Congrui Xu,Jia Wang,Chengfeng Lei,Jia Hu,Xiulian Sun
Dendrolimus punctatuscypovirus (DpCPV), belonging to the genusCypoviruswithin the familyReoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed avp80-knockoutAutographa californicamultiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of aHindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing anegfpgene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infectSpodopetra exigualarvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.Keywords:DpCPV;reverse genetics system;vp80-knockout;foreign protein
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NANOTECHNOLOGY
·
2019
·Vol. 31
·期 9
·095501
·
IF 3.399
Jun Cai,Liyun Ding,Panfeng Gong,Jun Huang
For the early diagnosis of gastric cancer, microRNA-148a (miRNA-148a) as a promising biomarker is measured by a simple colorimetric biosensor due to its unique surface plasmon resonance (SPR) absorption of gold nanoparticles (AuNPs). In the assay system, the sensing probes are facilitated by the conjugation of AuNPs with RNA probes (RNAP) via Au–S bonds, which align in a tail-to-tail fashion onto the target RNA. When miRNA-148a is introduced, a sandwich hybridization reaction is triggered between the AuNP–RNAP conjugates and targets, resulting in changes in the SPR absorption band, microscopic distribution and macroscopic color of the AuNP solution. Following this principle, this colorimetric method is able to quantitatively detect miRNA-148a at nanomolar level with a limit of ∼1.9 nM, and exhibits high sensitivity and selectivity by a low-cost UV–vis spectrometer or even the naked eye. Moreover, the AuNP network materials with a characteristic sharp 'melting transition' provide significant guidance for the reusability of DNA or RNA biosensors.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2017
·
IF 5.401
Liang Qi,Yao-Yao Fan,He Wei,Dan Zhang,Zhi-Qi Zhang
Proflavine could recognise HIV RRE (Rev response element) RNA, thus inhibiting its interaction with Rev peptide. Owing to the non-significant difference in fluorescence polarization (FP) signal between the proflavine and proflavine-RRE complex, it is not practicable for the identification of antagonists of HIV Rev peptide using proflavine as a FP probe. In the present study, graphene oxide (GO) was introduced to amplify the FP signal to enhance the sensitivity for the identification of Rev antagonists, thereby enabling the development of a primary GO-enhanced FP biosensor for the discovery of Rev antagonists. The effect of the amount of GO on the FP changes was investigated via displacement assay using a known Rev antagonist, neomycin B. The mechanism underlying the changes in FP caused by the addition of neomycin B was explored in the presence of GO. Finally, the performance of this biosensor was further evaluated using other 4 test drugs. The introduction of GO to this interaction system increased the practicability for the discovery of Rev antagonists using proflavine as a probe, and this label-free method does not suffer from limitations caused by fluorescent covalent labeling, thus providing a novel strategy for the characterization of ligand-RNA binding interactions.
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Frontiers in Bioengineering and Biotechnology
·
2024
·
IF 4.3
He Wei,Zhang Xinya,Zou Yangxiaoyu,Li Ji,Chang Le,He Yu-Cai,Jin Qiuheng,Ye Jianren
Introduction: Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing to their stable structure and ability to treat complicated diseases. Our objective was to create a one-step reaction for circRNA synthesis using wild-type T7 RNA polymerase as the catalyst. However, T7 RNA polymerase is thermally unstable, and we streamlined circRNA synthesis via consensus and folding free energy calculations for hotspot selection. Because of the thermal instability, the permuted intron and exon (PIE) method for circRNA synthesis is conducted via tandem catalysis with a transcription reaction at a low temperature and linear RNA precursor cyclization at a high temperature.Methods: To streamline the process, a multisite mutant T7 RNA polymerase (S430P, N433T, S633P, F849I, F880Y, and G788A) with significantly improved thermostability was constructed, and G788A was used.Results: The resulting mutant exhibited stable activity at 45°C for over an hour, enabling the implementation of a one-pot transcription and cyclization reaction. The simplified circRNA production process demonstrated an efficiency comparable to that of the conventional two-step reaction, with a cyclization rate exceeding 95% and reduced production of immunostimulatory dsRNA byproducts.
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ACS Synthetic Biology
·
2024
·
IF 3.7
Qing Wang,Jiajia You,Yichen Li,Jie Zhang,Yi Wang,Meijuan Xu,Zhiming Rao
In vivo targeted mutagenesis technologies are the basis for the continuous directed evolution of specific proteins. Here, an efficient mutagenesis system (CgMutaT7) for continuous evolution of the targeted gene in Corynebacterium glutamicum was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers to build the CgMutaT7 system, which introduces mutations in targeted regions controlled by the T7 promoter. After a series of optimizations, the resulting targeted mutagenesis system (CgMutaT74) can increase the mutant frequency of the target gene by 1.12 × 104-fold, with low off-target mutant frequency. Subsequently, high-throughput sequencing further revealed that the CgMutaT74 system performs efficient and uniform C → T transitions in at least a 1.8 kb DNA region. Finally, the xylose isomerase was successfully continuously evolved by CgMutaT74 to improve the xylose utilization, indicating that the CgMutaT7 system has great potential for applications in the continuous evolution of protein function and expression components.
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Cell Reports
·
2025
·
IF 6.9
Yixuan Feng,Yiming Wang,Tai Wang,Lingtong Liu
Plant-unique RNA polymerase IV (RNA Pol IV) governs the establishment of small RNA (sRNA)-directed DNA methylation (RdDM). In dicotyledon, elevated RdDM activity is often associated with embryogenesis; however, the loss of RdDM frequently results in indiscernible phenotypes. Here, we report that the absence of SlNRPD1 , encoding the largest subunit of RNA Pol IV, leads to diminished RdDM and abnormal embryogenesis in tomato ( Solanum lycopersicum ). Hypermethylation at pericentric transposable elements (TEs) and a burst of 21/22-nt siRNA from the distal and pericentric genes are observed in slnrpd1 embryos. The specific activation of endoribonuclease Dicer-like 2 ( SlDCL2b / c / d ) is correlated with 21/22-nt sRNA accumulation. Auxin and WUSCHEL-related homeobox (WOX) signaling gene expression is altered by mCHH hypomethylation, which may lead to defective embryos. Due to improper maturation, the slnrpd1 embryos cannot withstand post-harvest desiccation. This study provides insights into how DNA methylation regulates plant embryogenesis.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
·
IF 5.7
Yan He,Zihao Cheng,Jingyu Zhang,Yu Chen,Guode Zhao,Hong Tang,Yufen Liao,Tingmin Ye,Ying Peng,Weiwei Li,Jiang Zheng
Myristicin (MYR) mainly occurs in nutmeg and belongs to alkoxy-substituted allylbenzenes, a class of potentially toxic natural chemicals. RNA interaction with MYR metabolites in vitro and in vivo has been investigated in order to gain a better understanding of MYR toxicities. We detected two guanosine adducts (GA1 and GA2), two adenosine adducts (AA1 and AA2), and two cytosine adducts (CA1 and CA2) by LC-MS/MS analysis of total RNA extracts from cultured primary mouse hepatocytes and liver tissues of mice after exposure to MYR. An order of nucleoside adductions was found to be GAs > AAs > CAs, and the result of density functional theory calculations was in agreement with that detected by the LC-MS/MS-based approach. In vitro and in vivo studies have shown that MYR was oxidized by cytochrome P450 enzymes to 1′-hydroxyl and 3′-hydroxyl metabolites, which were then sulfated by sulfotransferases (SULTs) to form sulfate esters. The resulting sulfates would react with the nucleosides by SN1 and/or SN2 reactions, resulting in RNA adduction. The modification may alter the biochemical properties of RNA and disrupt RNA functions, perhaps partially contributing to the toxicities of MYR.
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iScience
·
2024
·
IF 4.6
Hui Han,Yucong Sun,Wei Wei,Zixin Huang,Maosheng Cheng,Hongshen Qiu,Juan Wang,Siyi Zheng,Lianlian Liu,Qiang Zhang,Canfeng Zhang,Jieyi Ma,Siyao Guo,Zhaoyu Wang,Zhenpeng Li,Xu Jiang,Shuibin Lin,Qianwen Liu,Shuishen Zhang
Summary Emerging studies have demonstrated the link between RNA modifications and various cancers, while the predictive value and functional mechanisms of RNA modification-related genes (RMGs) in esophageal squamous cell carcinoma (ESCC) remain unclear. Here we established a prognostic signature for ESCC based on five RMGs. The analysis of ESCC clinical samples further verified the prognostic power of the prognostic signature. Moreover, we found that the knockdown of NSUN6 promotes ESCC progression in vitro and in vivo , whereas the overexpression of NSUN6 inhibits the malignant phenotype of ESCC cells. Mechanically, NSUN6 mediated tRNA m 5 C modifications selectively enhance the translation efficiency of CDH1 mRNA in a codon dependent manner. Rescue assays revealed that E-cadherin is an essential downstream target that mediates NSUN6’s function in the regulation of ESCC progression. These findings offer additional insights into the link between ESCC and RMGs, as well as provide potential strategies for ESCC management and therapy.
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