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European Journal of Medical Research Nov 2023Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children. Due to drug resistance to radiotherapy and chemotherapy, mainly due to the...
BACKGROUND
Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children. Due to drug resistance to radiotherapy and chemotherapy, mainly due to the existence of cancer stem cells (CSCs), some children still have a poor prognosis. Therefore, researchers have focused their attention on CSCs. Our research group successfully constructed cancer stem cell-like cells named Piwil2-iCSCs by reprogramming human preputial fibroblasts (FBs) with the PIWIL2 gene in the early stage, and Piwil2-iCSCs were confirmed to induce the formation of embryonic tumors. PiRNAs, noncoding small RNAs that interact with PIWI proteins, play important roles in a variety of tumors. Therefore, our study aimed to explore the role of differentially expressed (DE) piRNAs derived from sequencing of Piwil2-iCSCs in NB.
METHODS
The DE piRNAs in Piwil2-iCSCs were screened using high-throughput sequencing and further verified in NB tissues and cells. An unknown piRNA, named piRNA-MW557525, showed obvious downregulation in NB. Thus we studied the effect of piRNA-MW557525 on the biological behavior of NB through in vitro and in vivo experiments. On this basis, we successfully constructed a stably transfected NB cell line overexpressing piRNA-MW557525 and performed transcriptome sequencing to further explore the mechanism of piRNA-MW557525 in NB.
RESULTS
In vitro, piRNA-MW557525 inhibited NB cell proliferation, migration and invasion and induced apoptosis; in vivo, piRNA-MW557525 significantly reduced the volume and weight of tumors and inhibited their proliferation, migration and invasion. piRNA-MW557525 overexpression induced G0/G1 phase arrest in NB cells via activation of the P53-P21-CDK2-Cyclin E signaling pathway thus inhibiting NB growth.
CONCLUSIONS
Our findings show that piRNA-MW557525 functions as a tumor suppressor gene in NB and may serve as an innovative biomarker and possible therapeutic target for NB.
Topics: Child; Humans; Piwi-Interacting RNA; Tumor Suppressor Protein p53; RNA, Small Interfering; Neuroblastoma; G1 Phase; Cell Proliferation; Signal Transduction; Cell Line, Tumor; Argonaute Proteins
PubMed: 37941038
DOI: 10.1186/s40001-023-01493-w -
Nature Communications Nov 2023Argonaute proteins (Agos) bind short nucleic acids as guides and are directed by them to recognize target complementary nucleic acids. Diverse prokaryotic Agos (pAgos)...
Argonaute proteins (Agos) bind short nucleic acids as guides and are directed by them to recognize target complementary nucleic acids. Diverse prokaryotic Agos (pAgos) play potential functions in microbial defense. The functions and mechanisms of a group of full-length yet catalytically inactive pAgos, long-B pAgos, remain unclear. Here, we show that most long-B pAgos are functionally connected with distinct associated proteins, including nucleases, Sir2-domain-containing proteins and trans-membrane proteins, respectively. The long-B pAgo-nuclease system (BPAN) is activated by guide RNA-directed target DNA recognition and performs collateral DNA degradation in vitro. In vivo, the system mediates genomic DNA degradation after sensing invading plasmid, which kills the infected cells and results in the depletion of the invader from the cell population. Together, the BPAN system provides immunoprotection via abortive infection. Our data also suggest that the defense strategy is employed by other long-B pAgos equipped with distinct associated proteins.
Topics: Argonaute Proteins; Prokaryotic Cells; DNA; Plasmids; Nucleic Acids
PubMed: 37914725
DOI: 10.1038/s41467-023-42793-3 -
Journal of Extracellular Vesicles Nov 2023Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein...
Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination.
Topics: Extracellular Vesicles; MicroRNAs; Argonaute Proteins
PubMed: 37885043
DOI: 10.1002/jev2.12366 -
RNA (New York, N.Y.) Dec 2023Increasing evidence suggests mammalian Argonaute (Ago) proteins partition into distinct complexes within cells, but there is still little biochemical or functional...
Increasing evidence suggests mammalian Argonaute (Ago) proteins partition into distinct complexes within cells, but there is still little biochemical or functional understanding of the miRNAs differentially associated with these complexes. In naïve T cells, Ago2 is found almost exclusively in low molecular weight (LMW) complexes which are associated with miRNAs but not their target mRNAs. Upon T-cell activation, a proportion of these Ago2 complexes move into a newly formed high molecular weight (HMW) RNA-induced silencing complex (RISC), which is characterized by the presence of the GW182 protein that mediates translational repression. Here, we demonstrate distinct partitioning of miRNAs and isomiRs in LMW versus HMW RISCs upon antigen-mediated activation of CD8 T cells. We identify miR-7 as highly enriched in HMW RISC and demonstrate that miR-7 inhibition leads to increased production of IL-2 and up-regulation of the IL-2 receptor, the transferrin receptor, CD71 and the amino acid transporter, CD98. Our data support a model where recruitment of miR-7 to HMW RISC restrains IL-2 signaling and the metabolic processes regulated by IL-2.
Topics: Animals; RNA-Induced Silencing Complex; Interleukin-2; CD8-Positive T-Lymphocytes; Molecular Weight; MicroRNAs; Argonaute Proteins; Mammals
PubMed: 37879863
DOI: 10.1261/rna.079030.121 -
Genetics Jan 2024The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological...
The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, the efficacy of exogenous RNA interference (RNAi) varies dramatically within the Caenorhabditis elegans natural population, raising questions about our understanding of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some wild strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others, the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains but dampens it further in others. Among wild strains, genes known to mediate RNAi exhibited very high expression variation relative to other genes in the genome as well as allelic divergence and strain-specific instances of pseudogenization at the sequence level. Our results demonstrate functional diversification in the small RNA pathways in C. elegans and suggest that RNAi processes are evolving rapidly and dynamically in nature.
Topics: Humans; Animals; RNA Interference; Caenorhabditis elegans; Caenorhabditis elegans Proteins; RNA, Small Interfering; RNA, Double-Stranded; Germ Cells
PubMed: 37865119
DOI: 10.1093/genetics/iyad191 -
International Journal of Molecular... Oct 2023The recent pandemic of SARS-CoV-2 has underscored the critical need for rapid and precise viral detection technologies. Point-of-care (POC) technologies, which offer... (Review)
Review
The recent pandemic of SARS-CoV-2 has underscored the critical need for rapid and precise viral detection technologies. Point-of-care (POC) technologies, which offer immediate and accurate testing at or near the site of patient care, have become a cornerstone of modern medicine. Prokaryotic Argonaute proteins (pAgo), proficient in recognizing target RNA or DNA with complementary sequences, have emerged as potential game-changers. pAgo present several advantages over the currently popular CRISPR/Cas systems-based POC diagnostics, including the absence of a PAM sequence requirement, the use of shorter nucleic acid molecules as guides, and a smaller protein size. This review provides a comprehensive overview of pAgo protein detection platforms and critically assesses their potential in the field of viral POC diagnostics. The objective is to catalyze further research and innovation in pAgo nucleic acid detection and diagnostics, ultimately facilitating the creation of enhanced diagnostic tools for clinic viral infections in POC settings.
Topics: Humans; Point-of-Care Systems; Argonaute Proteins; Prokaryotic Cells; Point-of-Care Testing; Nucleic Acids; CRISPR-Cas Systems
PubMed: 37834437
DOI: 10.3390/ijms241914987 -
Disease Markers 2023Compelling evidence indicates the regulatory role of circular RNAs in cancers, including hepatocellular carcinoma (HCC). Our study aimed to elucidate the regulatory...
Compelling evidence indicates the regulatory role of circular RNAs in cancers, including hepatocellular carcinoma (HCC). Our study aimed to elucidate the regulatory function of circ_0129047 in HCC progression. A reverse transcription-quantitative polymeric chain reaction was conducted to detect the expression of circ_0129047, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1), and miR-492 in HCC tissues and cells. The characteristics of circ_0129047 were determined by evaluating the nuclear and cytoplasmic fractions and by RNase R digestion assays. The cell counting kit-8 assay, scratch wound, and transwell invasion assays were used to examine the effects of circ_0129047 overexpression, miR-492 mimic, and LYVE1 overexpression on the proliferation, migration, and invasion abilities of HCC cells in vitro. A mouse xenograft model was also established. The relationship between miR-492 and circ_0129047 or LYVE1 was clarified using luciferase reporter and Argonaute-2 RNA immunoprecipitation assays. We found that circ_0129047 and LYVE1 were poorly expressed in HCC tissues and cells, whereas miR-492 was upregulated. Overexpression of circ_0129047 inhibits HCC cell proliferation, migration, and invasion and delays in vivo tumor growth. Furthermore, circ_0129047 sponged miR-492, and 3'UTR LYVE1 was a direct target of miR-492. Additionally, LYVE1 overexpression reduced the oncogenic activity of the miR-492 mimic, whereas the miR-492 mimic abolished the antimigratory, antiproliferative, and anti-invasive effects of circ_0129047 overexpression in HCC cells. These data suggest that circ_0129047 exerts a tumor-suppressive role in HCC by sponging miR-492 away from LYVE1 and that the circ_0129047/miR-492/LYVE1 axis may be a promising target for HCC treatment.
Topics: Humans; Animals; Mice; Carcinoma, Hepatocellular; Liver Neoplasms; 3' Untranslated Regions; Cell Count; Disease Models, Animal; MicroRNAs; Cell Proliferation; Cell Line, Tumor; Vesicular Transport Proteins
PubMed: 37810197
DOI: 10.1155/2023/6978234 -
Frontiers in Bioscience (Landmark... Sep 2023One of the crucial processes for small RNA synthesis and plant disease resistance is RNA interference (RNAi). Dicer-like (DCL), RNA-dependent RNA polymerase (RDR),...
BACKGROUND
One of the crucial processes for small RNA synthesis and plant disease resistance is RNA interference (RNAi). Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), double-stranded RNA binding (DRB), and Argonaute are important proteins implicated in RNAi (AGO). Numerous significant woody plants belong to the Juglandaceae; walnut is one of the four groups of woody plants on earth and one of the four groups of dried fruits.
METHODS
In order to correlate walnuts and their homologues, this work integrated numerous web resources from structural analysis and transcriptome data collected from gene families in order to elucidate the evolution and functional differentiation of RNA-related proteins in the walnut () genome.
RESULTS
5 genes, 13 genes, 15 genes, and 15 genes are found in the walnut genome and encode conserved protein domains and motifs with similar subcellular distribution.There are three classes and seven subclasses of walnut AGO proteins. RDRS are primarily split into four categories, whereas DRBs can be divided into six. DCLs are separated into four groups. The walnut RDR1 copy number of 9 is the exception, with 7 of those copies being dispersed in clusters on chromosome 16. Proteins are susceptible to various levels of purification selection, but in walnut, purification selection drives gene creation. These findings also indicated some resemblance in other plants belonging to the walnut family. Under various tissues and stresses, many RNA-related genes in walnut produced abundant, selective expression.
CONCLUSIONS
In this study, the genome of the Juglandaceae's , , , and gene families were discovered and analysed for the first time. The evolution, structure, and expression characteristics of these families were also preliminary studied, offering a foundation for the development and breeding of the walnut RNAi pathway.
Topics: RNA Interference; Juglandaceae; Plants; RNA; RNA-Dependent RNA Polymerase; Gene Expression Regulation, Plant; Phylogeny
PubMed: 37796691
DOI: 10.31083/j.fbl2809218 -
MBio Oct 2023is an important cyanobacterial model organism for the study of its prokaryotic circadian clock, photosynthesis, and other biological processes. It is also widely used...
is an important cyanobacterial model organism for the study of its prokaryotic circadian clock, photosynthesis, and other biological processes. It is also widely used for genetic engineering to produce renewable biochemicals. Our findings reveal an SeAgo-based defense mechanism in against the horizontal transfer of genetic material. We demonstrate that deletion of the gene facilitates genetic studies and genetic engineering of .
Topics: Synechococcus; Plasmids; Circadian Clocks; Genetic Engineering; Bacterial Proteins
PubMed: 37791787
DOI: 10.1128/mbio.01843-23 -
Nature Oct 2023Transposable elements are genomic parasites that expand within and spread between genomes. PIWI proteins control transposon activity, notably in the germline. These...
Transposable elements are genomic parasites that expand within and spread between genomes. PIWI proteins control transposon activity, notably in the germline. These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (mG-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors, and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.
Topics: Animals; Argonaute Proteins; Caenorhabditis elegans; Caenorhabditis elegans Proteins; DNA Transposable Elements; Endoribonucleases; Holoenzymes; Piwi-Interacting RNA; RNA Cap Analogs
PubMed: 37758951
DOI: 10.1038/s41586-023-06588-2