STAR Protocols
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2026
·Vol. 7
·期 2
·104484
·
IF 1.4
Qiuyao Guo,Wanqing Li,Weiqiang Zheng,Meng Qiu,Chengtian Zhao
Summary Here, we present a protocol for gene knockdown in Trichoplax adhaerens using a silica nanoparticle-mediated RNA interference (RNAi) technique. We describe steps for preparing silica nanoparticles, assembling RNAi complexes, and delivering them to live specimens. This protocol enables reproducible suppression of target gene expression in T. adhaerens and achieves transcript knockdown efficiencies of approximately 50%–70%, as assessed by quantitative PCR. For complete details on the use and execution of this protocol, please refer to Li et al. 1
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CHEMISTRY OF MATERIALS
·
2026
·
IF 7.1
Renpeng Xia,Jiarong Guo,Jinwen Zhu,Peng Miao
RNA interference has garnered significant interest as a type of nucleic acid therapeutics. MicroRNA (miRNA)-mediated mRNA modulation and gene therapy have yielded promising progress. However, clinical application is impeded by low delivery efficiency and insufficient targeting specificity. We herein develop a hybrid membrane-based delivery system with a magnetic core and spatially isolated functional nucleic acid strands. Due to the hybrid lipid bilayer, CD47-mediated immune evasion and galectin-3-mediated homologous targeting facilitate effective delivery of nucleic acids and magnetic nanomaterials. The membranes effectively protect these antitumor reagents during transportation. After internalization inside tumor sites, the strand displacement reaction between the nucleic acids successfully releases miR-200c, which stimulates inflammatory cytokine secretion and modulates apoptotic cytokine expression. Meanwhile, the acidic microenvironment dissociates Fe2+ from the magnetic core and initiates the Fenton reaction to amplify cytotoxic oxidative damage. The two pathways thereby confer robust antitumor efficacy with synergistic effects, which have great potential for future clinical translation.
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Journal for ImmunoTherapy of Cancer
·
2025
·Vol. 13
·期 9
·e011893
·
IF 10.6
Zhengning Yang,Zhongxuan Meng,Shangyuan Liu,Yuxin Chen,Xuhong Gan,Zi‑Mei Zhang,Xiaoshan Luo,Jiahong Yan,Huijin Chen,Danqing Huang,Lihong Huang,Xiaomin Zeng,Wenyu Ma,Jun Liu,Linlin Wang,Haotian Huang,Xinyi Yang,Mei Hu,Lin Chen,Hongli Du,Qibin Leng,Li Qiu,Xiaoling Wang
Background Nonsense-mediated messenger RNA decay (NMD) is a highly conserved surveillance system that degrades mRNAs with premature termination codons (PTCs), and regulates the mRNA quantity. Cancer cells hijack NMD to compensate for the imbalanced DNA levels and modulate their antigenicity. We identified the LTO1/YAE1 complex as NMD factors regulating ribosome biogenesis. Our study aimed to investigate their roles in modulating NMD and major histocompatibility complex class I (MHC-I) antigen presentation, which is vital for cancer immunotherapy. Methods Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9)-mediated gene knockouts, overexpression, and mutational analysis were used to explore the role of LTO1 and YAE1 in NMD and MHC-I expression across various tumor cell lines, assayed by fluorescent reporter-based assays, fluorescence-activated cell sorting analysis, reverse transcription-quantitative PCR, mRNA decay assay, and polysome profiling. Transcriptomic analyses were used to assess the expression of LTO1/YAE1 and their correlation with MHC-I molecules in human cancers. T cell receptor (TCR)-T cells and tumor cells coculture were employed to monitor the effect of LTO1 and YAE1 loss on T cell activation. Iron chelators were further tested as NMD inhibitors to enhance cancer immunotherapy using in vitro coculture assay and a mouse tumor model for immune checkpoint blockade (ICB) therapy. Results We demonstrate that deficiency in LTO1, YAE1, or their downstream target ABCE1 impairs NMD, causes the overexpression of key regulators of MHC-I, including NLR family CARD domain containing 5 (NLRC5), interferon regulatory factor 1 (IRF1), and nuclear factor-kappa B (NFκB), which results in enhanced T cell activation and tumor cell killing in TCR-T models. Transcriptomic analyses reveal that the LTO1/YAE1 complex is frequently overexpressed in human cancers, where it negatively regulates MHC-I. Moreover, low doses of iron chelators inhibit NMD and enhance MHC-I expression, leading to improved recognition and activation of CD8 + T cells, thereby promoting antigen-specific killing and increasing the efficacy of TCR-T and ICB therapy. Conclusions Collectively, our findings establish novel roles for the LTO1/YAE1 complex in regulating MHC-I expression via NMD. As MHC-I is crucial for antigen presentation and T cell activation, these results reveal a previously unappreciated link between NMD and tumor immunogenicity, with potential implications for cancer immunotherapies.
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Nature Communications
·
2026
·Vol. 17
·期 1
·2565
·
IF 18.1
He Mingbin,Wang Weiwei,Zhou Haiwu,Liu Cong,Zhao Chunbei,Li Jian,Han Yuewen,Qin Yali,Chen Mingzhou
Pyroptosis plays a crucial role in immune defense against infections and endogenous threats by eliminating harmful cells and modulating the immune response through inflammation. However, the natural activation of pyroptosis involves intricate signaling pathways, posing significant challenges for its artificial manipulation in research and therapies. Here, we present DAMAGE ( D e a th Ma nipulation Ge ne), an innovative system that integrates gasdermins within the type III-E CRISPR framework, enabling the specific recognition of target RNA (tgRNA) and triggering pyroptosis. This mechanism allows DAMAGE to selectively identify and eliminate virus-infected, cancerous, and senescent cells, all of which exhibit altered RNA transcriptomes. Additionally, DAMAGE exhibits considerable promise as a platform for mRNA-LNP therapy. Our study highlights the potential of this CRISPR-based system in the controllable induction of pyroptosis, offering an innovative therapeutic strategy for treating RNA-heterogeneous diseases.
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PLANT PHYSIOLOGY
·
2026
·
IF 8.2
Liu Yuhan,Zheng Yutong,Jiang Deyuan,Shi Yafei,He Kaixiang,Ke Xiangsheng,Wang Yetao,Liu Xinlian,Hou Xin
Chloroplast-to-nucleus retrograde signaling and plastid RNA editing are both essential for chloroplast biogenesis and plant development, but the underlying mechanism linking these two processes remains unclear. Here, we identify the mitochondrial transcription termination factor mTERF3/Seedling Lethal 1 (SL1), previously characterized as a plastid-encoded RNA polymerase (PEP)-associated protein, as a key regulator connecting RNA editing to retrograde signaling. SL1 directly interacts with GUN1 and MORF2 and is indispensable for 31 out of 34 plastid RNA editing sites in Arabidopsis. Loss of SL1 function results in a strong genome uncoupled (gun) molecular phenotype under norflurazon (NF) treatment, accompanied by defective RNA editing and complete loss of the NDH complex. Mechanistically, SL1 assembles the editosome by recruiting canonical and atypical PPR-DYW proteins (CRR28, RARE1, DYW1, and DYW2) together with multiple non-PPR editing factors, while its strong affinity to MORF2 ensures appropriate editosome stoichiometry. SL1 also colocalizes with the PEP complex, suggesting a physical coupling between transcription and RNA editing in plastid nucleoids. Furthermore, SL1 modulates RNA editing profiles and regulates GLK1/2 expression during NF-induced retrograde signaling. Our findings expand the functional repertoire of mTERF proteins and uncover a molecular mechanism that connects RNA editing with retrograde signaling through SL1.
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International Journal of Electrochemical Science
·
2023
·
IF 1.5
Yang Liu,Yan Huang
MicroRNAs (miRs) are believed to be associated with cancer and serve as a marker of its presence. Detecting miR in body fluid with a high level of sensitivity is a significant factor for disease screening. A point-of-care technology (POCT) based electrochemiluminescence (ECL) biosensor was created with a higher level of sensitivity for the recognition of miR-126. Glutathione-stabilized gold nanoclusters (GSH-Au NCs) were employed as ECL reagents in this study. Potassium persulfate (K 2 S 2 O 8 ) was chosen as the co-reaction reagent. Consequently, we chose the catalytic hairpin self-assembly strategy to attain accurate detection of miR-126. The prepared sensor is able to detect miR-126 linearly from 10 aM to 0.1 nM, and the detection limit is as low as 3.17 aM.
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ACS Nano
·
2023
·
IF 17.1
Dou Wang,Xuedong Wang,Feidi Ye,Jin Zou,Jiuxin Qu,Xingyu Jiang
Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a “sample-in-answer-out” single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.
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PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES
·
2023
·Vol. 22
·期 4
·905-917
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IF 3.1
Wen Ying,Zeng Liyun,Chen Qitong,Li Yitong,Fu Mengdie,Wang Zixin,Liu Hong,Li Xiejia,Huang Peng,Wu Wei,Zou Qiongyan,Yi Wenjun
Background The aim of this study was to identify changes in gene expression before and after 5-aminolevulinic acid-mediated photodynamic therapy (5-ALA-PDT) and to investigate the potential mechanism of 5-ALA-PDT based on ribonucleic acid sequencing (RNA-Seq) analysis. Methods Secondary hyperparathyroidism (SHPT) primary cells were isolated from surgically excised specimens and exposed to laser light. The transcription profiles of SHPT primary cells were identified through RNA-Seq. Differentially expressed genes (DEGs) were identified. Enrichment of functions and signaling pathway analysis were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Quantitative real-time polymerase chain reaction (RT-qPCR) and western blot analysis were used to validate genes based on RNA-Seq results. Results In total, 1320 DEGs were identified, of which 1019 genes were upregulated and 301 genes were downregulated. GO and KEGG pathway analyses identified significantly enriched pathways in DEGs, including TGF beta in extracellular matrix (ECM), negative regulation of triglyceride biosynthetic process, protein heterodimerization activity, systemic lupus erythematosus, ECM–receptor interaction, focal adhesion and protein digestion and absorption. Protein–protein interaction (PPI) network analyses identified potential heat shock protein (HSP) interactions among the DEGs. Eight HSP genes were also identified that were most likely involved in 5-ALA-PDT, which were further validated by RT-qPCR and western blotting. Conclusions The findings of this descriptive study reveal changes in the transcriptome profile during 5-ALA-PDT, suggesting that gene expression and mutation, signaling pathways, and the molecular network are altered in SHPT primary cells. The above findings provide new insight for further studies on the mechanisms underlying 5-ALA-PDT in SHPT. Graphical abstract
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Molecular Cancer
·
2023
·Vol. 22
·期 1
·1-20
·
IF 37.3
Song Runjie,Ma Shuoqian,Xu Jiajia,Ren Xin,Guo Peilan,Liu Huijiao,Li Peng,Yin Fan,Liu Mei,Wang Qiang,Yu Lei,Liu Jiali,Duan Binwei,Rahman Nafis A.,Wołczyński Sławomir,Li Guangming,Li Xiangdong
Background hsa_circ_0001727 (circZKSCAN1) has been reported to be a tumor-associated circRNA by sponging microRNAs. Intriguingly, we found that circZKSCAN1 encoded a secretory peptide (circZKSaa) in the liver. The present study aims to elucidate the potential role and molecular mechanism of circZKSaa in the regulation of hepatocellular carcinoma (HCC) progression.Methods The circRNA profiling datasets (RNA-seq data GSE143233 and GSE140202) were reanalyzed and circZKSCAN1 was selected for further study. Mass spectrometry, polysome fractionation assay, dual-luciferase reporter, and a series of experiments showed that circZKSCAN1 encodes circZKSaa. Cell proliferation, apoptosis, and tumorigenesis in nude mice were examined to investigate the functions of circZKSaa. Mechanistically, the relationship between the circZKSaa and mTOR in HCC was verified by immunoprecipitation analyses, mass spectrometry, and immunofluorescence staining analyses.Results Receiver operating characteristic (ROC) analysis demonstrated that the secretory peptide circZKSaa encoded by circZKSCAN1 might be the potential biomarker for HCC tissues. Through a series of experiments, we found that circZKSaa inhibited HCC progression and sensitize HCC cells to sorafenib. Mechanistically, we found that the sponge function of circZKSCAN1 to microRNA is weak in HCC, while overexpression of circZKSaa promoted the interaction of FBXW7 with the mammalian target of rapamycin (mTOR) to promote the ubiquitination of mTOR, thereby inhibiting the PI3K/AKT/mTOR pathway. Furthermore, we found that the high expression of cicZKSCAN1 in sorafenib-treated HCC cells was regulated by QKI-5.Conclusion sThese results reveal that a novel circZKSCAN1-encoded peptide acts as a tumor suppressor on PI3K/AKT/mTOR pathway, and sensitizes HCC cells to sorafenib via ubiquitination of mTOR. These findings demonstrated that circZKSaa has the potential to serve as a therapeutic target and biomarker for HCC treatment.
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CELL
·
2022
·
IF 66.85
Liming Yan,Yucen Huang,Ji Ge,Zhenyu Liu,Pengchi Lu,Bo Huang,Shan Gao,Junbo Wang,Liping Tan,Sihan Ye,Fengxi Yu,Weiqi Lan,Shiya Xu,Feng Zhou,Lei Shi,Luke W. Guddat,Yan Gao,Zihe Rao,Zhiyong Lou
Summary Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5′ end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown “Nuc-pocket” in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an “induce-and-lock” mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.
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Frontiers in Bioengineering and Biotechnology
·
2022
·
IF 6.064
Huang Zihua,Xie Luoyijun,Zhang Jifan,Li Qiyan,Liu Yulin,Fu Xuemei,Yuan Miaomiao,Li Qingjiao
Mesoporous polydopamine nanoparticles (MPDA NPs) are promising nanomaterials that have the prospect of clinical application for multi-strategy antitumor therapy, while the biosecurity of MPDA NPs remains indistinct. Here, transcriptome sequencing (RNA-Seq) was performed to systematically reveal the toxicity of MPDA NPs to five categories of organs after three different exposure routes, including intravenous injection, intramuscular injection, and intragastric administration. Our results uncovered that MPDA NPs could be deposited in various organs in small amounts after intravenous administration, not for the other two exposure routes. The number of differentially expressed genes (DEGs) identified in the heart, liver, spleen, lung, and kidney from the intragastric administration group was from 22 to 519. Similarly, the corresponding number was from 23 to 64 for the intramuscular injection group and was from 11 to 153 for the intravenous injection group. Functional enrichment analyses showed 6, 39, and 4 GO terms enriched for DEGs in intragastric administration, intramuscular injection, and intravenous injection groups, respectively. One enriched pathway was revealed in intragastric administration group, while no enriched pathway was found in other groups. Our results indicated that MPDA NPs produced only slight changes at the transcriptome level in mice, which provided new insights for further clinical application of MPDA NPs.
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ACS Applied Materials & Interfaces
·
2022
·
IF 10.383
Tongtong Guo,Yuanhang Xiang,Hao Lu,Minmin Huang,Fengfei Liu,Min Fang,Jia Liu,Yujin Tang,Xinchun Li,Fan Yang
Interfacial DNA self-assembly is fundamental to solid nucleic acid biosensors, whereas how to improve the signal-to-noise ratio has always been a challenge, especially in the charge-based electrochemical DNA sensors because of the large noise from the negatively charged DNA capture probes. Here, we report a DNA framework-reversed signal-gain strategy through background-to-signal transition for ultrasensitive and highly specific electrical detection of microRNAs (miRNAs) in blood. By using a model of enzyme-catalyzed deposition of conductive molecules (polyaniline) targeting to DNA, we observed the highest signal contribution per unit area by the highly charged three-dimensional (3D) tetrahedral DNA framework probe, relative to the modest of two-dimensional (2D) polyA probe and the lowest of one-dimensional (1D) single-stranded (ss)DNA probe, suggesting the positive correlation of background DNA charge with signal enhancement. Using such an effective signal-transition design, the DNA framework-based electrochemical sensor achieves ultrasensitive miRNAs detection with sensitivity up to 0.29 fM (at least 10-fold higher than that with 1D ssDNA or 2D polyA probes) and high specificity with single-base resolution. More importantly, this high-performance sensor allows for a generalized sandwich detection of tumor-associated miRNAs in the complex matrices (multiple cell lysates and blood serum) and further distinguishes the tumor patients (e.g., breast, lung, and liver cancer) from the normal individuals. These advantages signify the promise of this miRNA sensor as a versatile tool in precision diagnosis.
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