During the hyperacute phase of intracerebral hemorrhage (ICH), the mass effect and blood components mechanically lead to brain damage and neurotoxicity. Our findings revealed that the mass effect and transferrin precipitate neuronal oxidative stress and iron uptake, culminating in ferroptosis in neurons. M6A (N6-methyladenosine) modification, the most prevalent mRNA modification, plays a critical role in various cell death pathways. The Fto (fat mass and obesity-associated protein) demethylase has been implicated in numerous signaling pathways of neurological diseases by modulating m6A mRNA levels. Regulation of Fto protein levels in neurons effectively mitigated mass effect-induced neuronal ferroptosis. Applying nanopore direct RNA sequencing, we identified voltage-dependent anion channel 3 (Vdac3) as a potential target associated with ferroptosis. Fto influenced neuronal ferroptosis by regulating the m6A methylation of Vdac3 mRNA. These findings elucidate the intricate interplay between Fto, Vdac3, m6A methylation, and ferroptosis in neurons during the hyperacute phase post-ICH and suggest novel therapeutic strategies for ICH.
Ferroptosis/physiology* ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics* ; Animals ; Neurons/metabolism* ; Transferrin/pharmacology* ; Mice ; Methylation ; Mice, Inbred C57BL ; Adenosine/metabolism* ; RNA, Messenger/metabolism* ; Male ; Oxidative Stress/physiology*

Neuropathic pain is frequently comorbidity with cognitive deficits. Neuralized1 (Neurl1)-mediated ubiquitination of CPEB3 in the hippocampus is critical in learning and memory. However, the role of Neurl1 in the cognitive impairment in neuropathic pain remains elusive. Herein, we found that lumbar 5 spinal nerve ligation (SNL) in male rat-induced neuropathic pain was followed by learning and memory deficits and LTP impairment in the hippocampus. The Neurl1 expression in the hippocampal CA1 was decreased after SNL. And this decrease paralleled the reduction of ubiquitinated-CPEB3 level and reduced production of GluA1 and GluA2. Overexpression of Neurl1 in the CA1 rescued cognitive deficits and LTP impairment, and reversed the reduction of ubiquitinated-CPEB3 level and the decrease of GluA1 and GluA2 production following SNL. Specific knockdown of Neurl1 or CPEB3 in bilateral hippocampal CA1 in naïve rats resulted in cognitive deficits and impairment of synaptic plasticity. The rescued cognitive function and synaptic plasticity by the treatment of overexpression of Neurl1 before SNL were counteracted by the knockdown of CPEB3 in the CA1. Collectively, the above results suggest that the downregulation of Neurl1 through reducing CPEB3 ubiquitination and, in turn, repressing GluA1 and GluA2 production and mediating synaptic plasticity impairment in hippocampal CA1 leads to the genesis of cognitive deficits in neuropathic pain.
Animals ; Male ; Neuralgia/metabolism* ; Rats ; Down-Regulation/physiology* ; Ubiquitination/physiology* ; Neuronal Plasticity/physiology* ; Rats, Sprague-Dawley ; CA1 Region, Hippocampal/metabolism* ; Cognitive Dysfunction/metabolism* ; RNA-Binding Proteins/metabolism* ; Receptors, AMPA/metabolism*

Microbial communities such as those residing in the human gut are highly diverse and complex, and many with important implications for health and diseases. The effects and functions of these microbial communities are determined not only by their species compositions and diversities but also by the dynamic intra- and inter-cellular states at the transcriptional level. Powerful and scalable technologies capable of acquiring single-microbe-resolution RNA sequencing information in order to achieve a comprehensive understanding of complex microbial communities together with their hosts are therefore utterly needed. Here we report the development and utilization of a droplet-based smRNA-seq (single-microbe RNA sequencing) method capable of identifying large species varieties in human samples, which we name smRandom-seq2. Together with a triple-module computational pipeline designed for the bacteria and bacteriophage sequencing data by smRandom-seq2 in four human gut samples, we established a single-cell level bacterial transcriptional landscape of human gut microbiome, which included 29,742 single microbes and 329 unique species. Distinct adaptive response states among species in Prevotella and Roseburia genera and intrinsic adaptive strategy heterogeneity in Phascolarctobacterium succinatutens were uncovered. Additionally, we identified hundreds of novel host-phage transcriptional activity associations in the human gut microbiome. Our results indicated that smRandom-seq2 is a high-throughput and high-resolution smRNA-seq technique that is highly adaptable to complex microbial communities in real-world situations and promises new perspectives in the understanding of human microbiomes.
Humans ; Gastrointestinal Microbiome/genetics* ; Bacteriophages/physiology* ; High-Throughput Nucleotide Sequencing ; Sequence Analysis, RNA/methods* ; Bacteria/virology*

OBJECTIVE: To identify prognostic genes associated with lysosome-dependent cell death (LDCD) in patients with gastric cancer (GC). METHODS: Differentially expressed genes (DEGs) were identified using The Cancer Genome Atlas - Stomach Adenocarcinoma. Weighted gene co-expression network analysis was performed to identify the key module genes associated with LDCD score. Candidate genes were identified by DEGs and key module genes. Univariate Cox regression analysis, and least absolute shrinkage and selection operator regression and multivariate Cox regression analyses were performed for the selection of prognostic genes, and risk module was established. Subsequently, key cells were identified in the single-cell dataset (GSE183904), and prognostic gene expression was analyzed. Cell proliferation and migration were assessed using the Cell Counting Kit-8 assay and the wound healing assay. RESULTS: A total of 4,465 DEGs, 95 candidate genes, and 4 prognostic genes, including C19orf59, BATF2, TNFAIP2, and TNFSF18, were identified in the analysis. Receiver operating characteristic curves indicated the excellent predictive power of the risk model. Three key cell types (B cells, chief cells, and endothelial/pericyte cells) were identified in the GSE183904 dataset. C19orf59 and TNFAIP2 exhibited predominant expression in macrophage species, whereas TNFAIP2 evolved over time in endothelial/pericyte cells and chief cells. Functional experiments confirmed that interfering with C19orf59 inhibited proliferation and migration in GC cells. CONCLUSION C19orf59, BATF2, TNFAIP2, and TNFSF18 are prognostic genes associated with LDCD in GC. Furthermore, the risk model established in this study showed robust predictive power.
Stomach Neoplasms/pathology* ; Humans ; Prognosis ; Lysosomes/physiology* ; RNA-Seq ; Cell Death ; Single-Cell Analysis ; Gene Expression Regulation, Neoplastic ; Cell Proliferation ; Single-Cell Gene Expression Analysis

N⁶-methyladenosine (m⁶A) modification plays a critical role in cell cycle regulation, while the mechanism of m⁶A in regulating mitosis remains underexplored. Here, we found that the total m⁶A modification level in cells increased during mitosis by the liquid chromatography-mass spectrometry/mass spectrometry and m⁶A dot blot assays. Silencing methyltransferase-like 3 (METTL3) or METTL14 results in delayed mitosis, abnormal spindle assembly, and chromosome segregation defects by the immunofluorescence. By analyzing transcriptome-wide m⁶A targets in HeLa cells, we identified polo-like kinase 1 (PLK1) as a key gene modified by m⁶A in regulating mitosis. Specifically, through immunoblotting and RNA pulldown, m⁶A modification inhibits PLK1 translation via YTH N⁶-methyladenosine RNA binding protein 1, thus mediating cell cycle homeostasis. Demethylation of PLK1 mRNA leads to significant mitotic abnormalities. These findings highlight the critical role of m⁶A in regulating mitosis and the potential of m⁶A as a therapeutic target in proliferative diseases such as cancer.
Humans ; Polo-Like Kinase 1 ; Cell Cycle Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Mitosis/physiology* ; HeLa Cells ; Adenosine/genetics* ; Methyltransferases/metabolism* ; RNA, Messenger/metabolism* ; RNA-Binding Proteins/metabolism*

Small nuclear RNAs (snRNAs) refer to a class of highly abundant and functionally important non-coding small RNAs that are localized in the eukaryotic nucleus. These snRNAs are highly conserved in different eukaryotes during evolution and form complexes with specific chaperones to fulfill critical biological functions, including precursor messenger RNA (pre-mRNA) splicing and ribosomal RNA (rRNA) modification. Consequently, the regulation of snRNA gene expression is a crucial biological process for plants. In plants, the transcription and processing of snRNAs are regulated by RNA polymerase (Pol), snRNA-activating protein complex (SNAPc), defective in snRNA processing (DSP), and specific cis-elements in the snRNA promoter regions. Proper regulation of snRNA expression is essential for normal plant growth, development, and stress responses. This review summarizes the classification, structures, transcriptional regulation, and biological functions of plant snRNA genes, while outlining future research directions for snRNAs.
RNA, Small Nuclear/physiology* ; Gene Expression Regulation, Plant ; Transcription, Genetic ; Plants/metabolism* ; RNA, Plant/genetics*

The study aims to investigate the impacts of prolyl oligopeptidase (POP) on the replication of foot-and-mouth disease virus (FMDV) in BHK-21 cells. Firstly, the effects of FMDV replication on POP expression in BHK-21 cells were analyzed by Western blotting and Real-time reverse transcription polymerase chain reaction (RT-qPCR). Secondly, a eukaryotic expression plasmid for POP was constructed, and the effects of POP overexpression on the replication of two different serotypes of FMDV were assessed by Western blotting, RT-qPCR, and virus titer assays. Thirdly, specific small interfering RNAs (siRNAs) targeting POP were synthesized, and their efficiency in interfering with endogenous POP expression was identified by RT-qPCR. The impacts of downregulating endogenous POP expression on FMDV replication were further evaluated by Western blotting, RT-qPCR, and virus titer assays. The results indicated that FMDV infection did not significantly affect POP expression in BHK-21 cells. Overexpression of POP dose-dependently enhanced the replication of both FMDV/O and FMDV/A serotypes. Conversely, siRNA-mediated downregulation of endogenous POP expression markedly suppressed FMDV/O replication. This study is the first to demonstrated that the role of the host POP protein in promoting FMDV replication in BHK-21 cells, thereby providing a critical theoretical foundation and potential molecular targets for developing efficient candidate cell strains for foot-and-mouth disease inactivated vaccines.
Foot-and-Mouth Disease Virus/genetics* ; Virus Replication/genetics* ; Prolyl Oligopeptidases ; Serine Endopeptidases/physiology* ; Animals ; Cell Line ; RNA, Small Interfering/genetics* ; Foot-and-Mouth Disease/virology* ; Cricetinae

Objective To explore the mechanism of lncRNA-BC200 (BC200) targeting the ubiquitination of Beta-site APP cleaving enzyme 1 (BACE1) and regulating the repair of nerve cell injury. Methods Mouse hippocampal neuron cell line HT22 was divided into four groups: control group, oxygen-glucose deprivation/reoxygenation(OGD/R) group, OGD/R+si-NC group and OGD/R+si-BC200 group. In order to further explore the relationship between BC200 and BACE1, HT22 cells were divided into four groups: OGD/R group, OGD/R+si-BC200 group, OGD/R+si-BC200+NC group and OGD/R+si-BC200+ BACE1 group. Twenty male C57BL/6J mice were randomly assigned to the following four groups: control group, middle cerebral artery occlusion (MCAO) group, MCAO+si-BC200 group and MCAO+si-BC200+BACE1 group. The mRNA expression levels of BC200 and BACE1 in cells were measured by real-time quantitative reverse transcription polymerase chain reaction. The expressions of c-caspase-3, B-cell lymphoma 2 (Bcl2), Bcl2 associated X protein(BAX) and BACE1 were detected by western blot, and the apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. Results Compared with the control group, the activity of HT22 cells in OGD/R group decreased significantly, and the percentage of apoptotic cells increased significantly. Compared with OGD/R+si-NC group, the activity of HT22 cells in OGD/R+si-BC200 group increased significantly, and the percentage of apoptotic cells decreased significantly. Compared with the control group, the expression of BACE1 protein in HT22 cells in OGD/R group was significantly enhanced. Compared with OGD/R+si-NC group, the expression of BACE1 protein in HT22 cells in OGD/R+si-BC200 group decreased significantly. It was observed that after OGD/R treatment, the ubiquitination level of BACE1 decreased significantly and the expression of BACE1 protein increased significantly. After transfection with si-BC200, the ubiquitination level of BACE1 protein increased significantly, while the expression of BACE1 protein decreased significantly. Compared with OGD/R+si-BC200+NC group, the percentage of apoptotic cells, the expression of c-caspase-3 and Bax protein in HT22 cells in OGD/R+si-BC200+BACE1 group increased significantly, and the expression of Bcl2 protein decreased significantly. Compared with the control group, the number of cerebral infarction areas and TUNEL positive cells in MCAO group increased significantly, and the survival number of neurons decreased significantly. Compared with the MCAO group, the number of cerebral infarction areas and TUNEL positive cells in MCAO+si-BC200 group decreased significantly, and the survival number of neurons increased significantly, while the addition of BACE1 reversed the improvement of si-BC200 transfection. Conclusion The combination of BC200 and BACE1 inhibit the ubiquitination of BACE1, and participate in mediating the expression enhancement of BACE1 induced by OGD/R. Specific blocking of BC200/BACE1 axis may be a potential therapeutic target to protect neurons from apoptosis induced by cerebral ischemia/reperfusion.
Animals ; Amyloid Precursor Protein Secretases/genetics* ; RNA, Long Noncoding/physiology* ; Aspartic Acid Endopeptidases/genetics* ; Male ; Neurons/pathology* ; Mice ; Mice, Inbred C57BL ; Apoptosis/genetics* ; Ubiquitination ; Cell Line ; Hippocampus/metabolism* ; bcl-2-Associated X Protein/genetics* ; Caspase 3/genetics* ; Infarction, Middle Cerebral Artery/metabolism*

Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

Objective To investigate the effect and mechanism of Efferocytosis Relatived LncRNA (EFRL) on homocysteine-induced atherosclerosis in macrophage efferocytosis. Methods RAW264.7 cells were cultured in vitro, and the Control group (0 μmol/L Hcy) and Hcy intervention group (100 μmol/L Hcy) were set up. After GapmeR transfection of macrophages with Hcy intervention, EFRL knockdown negative control group (Hcy combined with LNA-NC) and EFRL knockdown group (Hcy combined with LNA-EFRL) were set up. High-throughput sequencing was applied for different expression of LncRNA MSTRG. 88917.16 (EFRL), UCSC was used to analyze its conservation, CPC and CPAT were used to analyze its ability to encode proteins, and GO and KEGG were used to analyze related biological functions. The localization of LncRNA EFRL in macrophages was analyzed by nucleoplasmic separation and RNA-FISH. Quantitative real-time PCR was used to detect the expression levels of LncRNA EFRL and its target gene SPAST in Hcy-treated macrophages. The apoptosis rate of Jurkat cells induced by UV was detected by flow cytometry. In vitro efferocytosis assay combined with immunofluorescence technique was used to analyze macrophage efferocytosis. ELISA was used to detect the levels of interleukin 1β(IL-1β) and IL-18. Results The new LncRNA MSTRG.88917.16 was identified and named EFRL(Efferocytosis Relatived LncRNA). UCSC, CPC and CPAT analyses showed that LncEFRL is highly conserved and does not have the ability to encode proteins. GO and KEGG analyses suggested that LncEFRL may be involved in macrophage efferocytosis. LncRNA EFRL was localized in the nucleus of macrophages as determined by nucleoplasmic separation and RNA-FISH. In comparison to the Control group, the expression levels of LncRNA EFRL and its target gene SPAST in the Hcy group were increased. In comparison to the Control group (0 min), the apoptosis rate of the experimental group (15, 30 min) Annexin V is more than 85%. Compared with Hcy combined with LNA-NC group, Hcy combined with LNA-EFRL group had enhanced macrophage efferocytosis and reduced levels of inflammatory factors. Compared with Hcy combined with LNA-NC group, the expression level of SPAST in Hcy combined with LNA-EFRL group was decreased. Conclusion Inhibition of EFRL expression can alleviate the process of Hcy inhibiting macrophage efferocytosis, and the mechanism is related to the regulation of the downstream target gene SPAST by EFRL.
RNA, Long Noncoding/physiology* ; Animals ; Homocysteine ; Mice ; Macrophages/drug effects* ; Humans ; RAW 264.7 Cells ; Atherosclerosis/chemically induced* ; Apoptosis/genetics* ; Phagocytosis/genetics* ; Jurkat Cells ; Interleukin-1beta/genetics* ; Efferocytosis