Silencing Of Gene Expression Research Articles

The developmentally timed decay of an essential microRNA family is seed-sequence dependent.

SUMMARYMicroRNA (miRNA) abundance is tightly controlled by regulation of biogenesis and decay. Here, we show that the mir-35 miRNA family undergoes selective decay at the transition from embryonic to larval development in C. elegans. The seed sequence of the miRNA is necessary and largely sufficient for this regulation. Sequences outside the seed (3′ end) regulate mir-35 abundance in the embryo but are not necessary for sharp decay at the transition to larval development. Enzymatic modifications of the miRNA 3′ end are neither prevalent nor correlated with changes in decay, suggesting that miRNA 3′ end display is not a core feature of this mechanism and further supporting a seed-driven decay model. Our findings demonstrate that seed-sequence-specific decay can selectively and coherently regulate all redundant members of a miRNA seed family, a class of mechanism that has great biological and therapeutic potential for dynamic regulation of a miRNA family’s target repertoire.

Maternal SMCHD1 regulates Hox gene expression and patterning in the mouse embryo

Parents transmit genetic and epigenetic information to their offspring. Maternal effect genes regulate the offspring epigenome to ensure normal development. Here we report that the epigenetic regulator SMCHD1 has a maternal effect on Hox gene expression and skeletal patterning. Maternal SMCHD1, present in the oocyte and preimplantation embryo, prevents precocious activation of Hox genes post-implantation. Without maternal SMCHD1, highly penetrant posterior homeotic transformations occur in the embryo. Hox genes are decorated with Polycomb marks H2AK119ub and H3K27me3 from the oocyte throughout early embryonic development; however, loss of maternal SMCHD1 does not deplete these marks. Therefore, we propose maternal SMCHD1 acts downstream of Polycomb marks to establish a chromatin state necessary for persistent epigenetic silencing and appropriate Hox gene expression later in the developing embryo. This is a striking role for maternal SMCHD1 in long-lived epigenetic effects impacting offspring phenotype.

Gene Silencing RNAi Technology: Uses in Plants

Ensuring sustainable food production in national and global area depends on the determination of plant species and varieties that can survive under the influence of various stress factors that may occur due to global climate changes and other factors that adversely limit growth and development, and depends on the protection and development of existing ones. It is important to develop new plant varieties that are resistant to abiotic stress factors that have occurred as a result of global climate changes. At this point, modern biotechnological methods have been widely needed in plant breeding in recent years. One of these techniques is RNAi technology. The mechanism of RNA interference (RNAi) is defined as post-transcriptional gene silencing or regulation of gene expression, resulting in the degradation of mRNA chain, which is the complement of double-stranded RNA (dsRNA) entering the cell. RNA interference begins when double-stranded RNA is cut into small inhibitory RNAs (siRNA) by an RNase III enzyme called as Dicer. These siRNAs then bind to the RNA-inducing silencing complex (RISC) which is a multiprotein-RNA nuclease complex. RISC uses siRNAs to find complementary mRNA and cuts the target mRNA endonucleolytically. The resulting decrease in specific mRNA leads to a decrease in available protein(s). Post transcriptional gene silencing, RNA interference and other forms of RNA silencing have been observed particularly in plants. In recent years, RNAi studies, which are among the leading topics in the global area, have shown that non-coding RNAs in plants play a role in the control of tissue differentiation and development, signal transmission, interaction with phytohormones, abiotic (drought, salinity, etc.) and environmental factors such as biotic stress. In this review paper, the basics of RNAi mechanism and the usage of RNAi in plants are explained.

Antitumoral RNA-targeted oligonucleotide therapeutics: The third pillar after small molecule inhibitors and antibodies.

Oligonucleotide therapeutics, drugs consisting of 10–50 nucleotide‐long single‐ or double‐stranded DNA or RNA molecules that can bind to specific DNA or RNA sequences or proteins, include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamers, and decoys. These oligonucleotide therapeutics could potentially become the third pillar of drug development. In particular, ASOs and siRNAs are advanced tools that are widely used to silence gene expression. They are used in clinical trials, as they have high specificity for target mRNAs and non‐coding RNAs and limited toxicity. However, their clinical application remains challenging. Although chemotherapy has benefits, it has severe adverse effects in many patients. Therefore, new modalities for targeted molecular therapy against tumors, including oligonucleotide therapeutics, are required, and they should be compatible with diagnosis using next‐generation sequencing. This review provides an overview of the therapeutic uses of ASOs, siRNAs, and miRNAs in clinical studies on malignant tumors. Understanding previous research and development will help in developing novel oligonucleotide therapeutics against malignant tumors.

BcTFIIIA Negatively Regulates Turnip Mosaic Virus Infection through Interaction with Viral CP and VPg Proteins in Pak Choi (Brassica campestris ssp. chinensis)

TFIIIA is a zinc-finger transcription factor that is involved in post-transcriptional regulation during development. Here, the BcTFIIIA gene was isolated from pak choi. Sequence analysis showed that BcTFIIIA encodes 383 amino acids (aa) with an open reading frame (ORF) of 1152 base pairs (bp). We investigated the subcellular location of BcTFIIIA and found the localized protein in the nucleus. BcTFIIIA was suppressed when the pak choi was infected by the turnip mosaic virus (TuMV). The BcTFIIIA mRNA expression level in a resistant variety was higher than that in a sensitive variety, as determined by qRT-PCR analysis. Yeast two hybrid (Y2H) assay and bimolecular fluorescence complementation (BiFC) suggested that BcTFIIIA interacts with TuMV CP and VPg in vivo, respectively, and in vitro. A virus-induced gene silencing (VIGS) experiment showed that the silencing of BcTFIIIA gene expression in pak choi promoted the accumulation of TuMV. These results suggest that BcTFIIIA negatively regulates viral infection through the interaction with TuMV CP and VPg.

The Interaction Network of MicroRNAs with Cytokines and Signaling Pathways in Allergic Asthma.

Allergic asthma is a complicated disease that is affected by many factors. Numerous cytokines and signaling pathways are attributed to the cause of asthma symptoms. MicroRNAs (miRNAs) are a group of small non-coding single-stranded RNA molecules that are involved in gene silencing and posttranscriptional regulation of gene expression by targeting mRNAs. In pathological conditions, altered expression of microRNAs differentially regulates cytokines and signaling pathways and therefore, can be the underlying reason for the pathogenesis of allergic asthma. Indeed, microRNAs participate in airway inflammation via inducing airway structural cells and activating immune responses by targeting cytokines and signaling pathways. Thus, to make a complete understanding of allergic asthma, it is necessary to investigate the communication network of microRNAs with cytokines and signaling pathways which is contributed to the pathogenesis of allergic asthma. Here, we shed light on this aspect of asthma pathology by Summarizing our current knowledge of this topic.

The conserved helicase ZNFX-1 memorializes silenced RNAs in perinuclear condensates

RNA-mediated interference (RNAi) is a conserved mechanism that uses small RNAs (sRNAs) to silence gene expression. In the Caenorhabditis elegans germline, transcripts targeted by sRNAs are used as templates for sRNA amplification to propagate silencing into the next generation. Here we show that RNAi leads to heritable changes in the distribution of nascent and mature transcripts that correlate with two parallel sRNA amplification loops. The first loop, dependent on the nuclear Argonaute HRDE-1, targets nascent transcripts and reduces but does not eliminate productive transcription at the locus. The second loop, dependent on the conserved helicase ZNFX-1, targets mature transcripts and concentrates them in perinuclear condensates. ZNFX-1 interacts with sRNA-targeted transcripts that have acquired poly(UG) tails and is required to sustain pUGylation and robust sRNA amplification in the inheriting generation. By maintaining a pool of transcripts for amplification, ZNFX-1 prevents premature extinction of the RNAi response and extends silencing into the next generation.

The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex

SummaryThere is an urgent need for a molecular understanding of how SARS-CoV-2 influences the machineries of the host cell. Herein, we focused our attention on the capacity of the SARS-CoV-2 protein NSP2 to bind the human 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression. Using in vitro interaction assays, our data demonstrate that NSP2 physically associates with both 4EHP and a central segment in GIGYF2 in the cytoplasm. We also provide functional evidence showing that NSP2 impairs the function of GIGYF2 in mediating translation repression using reporter-based assays. Collectively, these data reveal the potential impact of NSP2 on the post-transcriptional silencing of gene expression in human cells, pointing out 4EHP-GIGYF2 targeting as a possible strategy of SARS-CoV-2 to take over the silencing machinery and to suppress host defenses.

Infl uence of interference silencing of the gene of translation initiation factors <i>eIF(iso)G</i> and <i>eIF(iso)E</i> on the resistance of the clone stone fruits rootstock 146-2 and commercial plum variety Startovaya to the Plum pox virus

The method of RNA interference gene expression silencing was used to obtain Plum pox virus (PPV) resistant rootstock and commercial variety Startovaya.For this purpose, a vector with self-complementary sequences of the 578 bp eIF(iso)4G and eIF(iso)E genes fragment was created. The eIF(iso)4G and eIF(iso)E genes encodes factors of initiation of translation involved in the life cycle of a Plum pox virus. A strong promoter of the ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBisCo) gene was chosen to drive the expression of RNA interference hairpin in full and truncated variants. Successful genetic transformation of the 146-2 rootstock and variety Startovaya were carried out by A. tumefaciens CBE21 strain. Whole leaves from in vitro cultured shoots were used as an explant source. The nptII and hpt genes coding for neomycin II and hygromycin phosphotransferase were used as a plant-selectable markers. In our experiments, 5 independent transgenic lines of clonal rootstock and variety were obtained and acclimatized to greenhouse conditions. Th eir status was confirmed by PCR and Southern blot analyses. The transformation efficiency was 0.3-0.4 %. One of these lines was grafted with PPV-infected plum buds and its resistance was verified by ELISA. The use of a full-length gene promoter of the small subunit of ribulosobiephosphate carboxylase (RBCS) in the transformation of plants of the Starter variety led to a decrease in plant viability in the case of suppression of the eIF(iso)4E gene and ensured stability at least in the first year after inoculation in the case of suppression of the eIF(iso)4G gene.

Abstract 5851: Negative regulation of p53 by ZnT1

Abstract Rationale and Background: p53 is a powerful tumor suppressor protein that also autorepresses by inducing expression of its own negative regulators, such as MDM2. Here we explore another potential mechanism for p53 autorepression, regulation of zinc metabolism. Proper folding of p53 requires zinc binding and cancer-associated mutations in p53 often lower the affinity of the mutant proteins for zinc. Our previous work showed that ZMC1, a zinc metallochaperone, can reactivate certain p53 mutants by raising intracellular zinc concentrations. Here we show that p53 activates transcription of ZnT1. ZnT1 can then export zinc from the cell, which in turn lowers p53 activity. Methods: To explore the relationship between ZnT1 and p53, we studied several human cancer cell lines under various conditions, including ZnT1 overexpression, ZnT1 silencing, and p53 activation. For silencing gene expression, we used CRISPR-Cas9 with a lentiviral vector to knock out ZnT1 and also employed shRNAs to knockdown expression of p53 or ZnT1. To analyze the relationship between TP53 and ZnT1 genes in clinical samples, data from cBioPortal were examined. Results: ZnT1 transcription increased after p53 was activated in several p53+ cell lines but did not increase in isogenic p53-null cell lines, suggesting that ZnT1 transcription is dependent on p53. p53 also bound to the ZnT1 promoter by chromatin immunoprecipitation assay, and activated transcription of a ZnT1/luciferase promoter fusion. Overexpression of ZnT1 decreased p21 transcription, indicating lowered p53 function; conversely, knockdown of ZnT1 increased p21 transcription. By cBioPortal analysis, ZnT1 gene amplifications were mutually exclusive with TP53 gene mutations in breast and lung tumors. Conclusion: These data suggest that autorepression of p53 occurs in part due to lowered intracellular zinc caused by upregulation of ZnT1. Citation Format: Ben Brik, Kathleen Carino, Wade Narrow, Chris R. Harris, Xin Yu, Darren R. Carpizo. Negative regulation of p53 by ZnT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5851.

Laodelphax striatellus saliva mucin enables the formation of stylet sheathes to facilitate its feeding and rice stripe virus transmission.

Laodelphax striatellus transmits rice stripe virus (RSV) during sap feeding on the rice plant. The insect saliva proteins have direct and indirect roles in mediating RSV transmission; however, the function of most saliva proteins remains unclear. In this study, we sequenced L. striatellus saliva proteins using shotgun liquid chromatography-electrospray ionization-tandem mass spectrometry. We identified 41 secreted saliva proteins, among which a saliva mucin-like protein, designated LssaMP, was the most abundant. In silico analysis revealed the sequence conservation among planthoppers. We revealed that the LssaMP gene is specifically expressed in the salivary glands and the protein is secreted as a component of gel saliva. Using LssaMP-specific double-stranded RNA (dsRNA) to silence gene expression, we revealed that LssaMP is required for formation of the salivary sheath, an important structure for sap feeding. Disrupting LssaMP expression resulted in inefficient formation of the feeding structure, thereby stopping insects from secreting watery saliva and acquiring sufficient nutrients from the phloem sap. We confirmed that RSV is mainly released via the watery saliva, which passes through the salivary sheathes into the plant phloem. An insufficient feeding structure results in decreased release of watery saliva, as well as the arboviruses. This study clarified the function of an insect saliva protein in mediating insect feeding, as well as arbovirus transmission. © 2022 Society of Chemical Industry.

ETMR-11. Transcriptional changes upon knockdown of alteredBCOR/BCORL1 transcripts in preclinical models of CNS embryonal tumors with BCOR-related alterations

BCL-6 transcriptional corepressor (BCOR) is an epigenetic regulator that silences gene expression mainly via the polycomb repressive complex 1.1 (PRC1.1). BCOR genomic alterations are found in a variety of different tumors and recently central nervous system (CNS) tumors with BCOR internal tandem duplication (ITD) were classified as a distinct molecular subgroup. We established and characterized two cell models derived from BCOR altered CNS tumor patients. One model is characterized by a frameshift mutation in the BCOR gene resulting in the expression of a truncated protein lacking the C-terminal PUFD domain required for correct assembly of the PRC1.1. Additionally, this model harbors a translocation of the BCOR homologue BCORL1. The second model has a characteristic internal tandem duplication (ITD) within the BCOR gene. To study the effects of mutated BCOR/BCORL1 on gene expression, we performed siRNA mediated knockdown of altered BCOR/BCORL1 transcripts in both models and analyzed transcriptional changes by mRNA expression array. Differentially expressed genes in BCOR/BCORL1 knockdown versus wild type conditions were enriched for signaling pathways involved in cell cycle progression, cell growth, DNA replication and cancer. This suggests that the alterations in BCOR/BCORL1 might have pro-oncogenic effects and thereby contribute to the aggressive phenotype of this disease. Especially in the BCOR ITD model knockdown of BCOR led to transcriptional downregulation of genes associated with the development of brain tumors such as FGF18, PDGFA and PDGFRA. Our results indicate that specific BCOR/BCORL1 alterations might impair its endogenous function as transcriptional repressor and deregulate the expression of multiple PRC1.1 target genes. An in depth characterization of epigenetic and transcriptional changes in BCOR/BCORL1 altered CNS tumors could lead to the identification of critical downstream effectors and ultimately reveal new therapeutic vulnerabilities.

A transcriptomic atlas underlying developmental plasticity of seasonal forms of Bicyclus anynana butterflies.

Organisms residing in regions with alternating seasons often develop different phenotypes, or forms, in each season. These forms are often adaptations to each season and result from an altered developmental response to specific environmental cues such as temperature. Although multiple studies have examined form-specific gene expression profiles in a diversity of species, little is known about how environments and developmental transitions, cued by hormone pulses, alter post-transcriptional patterns. In this study, we examine how gene expression, alternative splicing, and miRNA-mediated gene silencing in Bicyclus anynana butterfly hindwing tissue, varies across two rearing temperatures at four developmental timepoints. These timepoints flank two temperature-sensitive periods that coincide with two pulses of the insect hormone 20E. Our results suggest that developmental transitions, coincident with 20E pulses, elicit a greater impact on all these transcriptomic patterns than rearing temperatures per se. More similar transcriptomic patterns are observed pre-20E pulses than those observed post-20E pulses. We also found functionally distinct sets of differentially expressed genes (DEGs) and differentially spliced genes (DSGs) in the seasonal forms. Furthermore, around 10% of DEGs are predicted to be direct targets of, and regulated by, differentially expressed miRNAs (DEmiRs) between the seasonal forms. Many DEGs, DEmiRs, or DSGs potentially regulate eyespot size plasticity, and we validated the differential splicing pattern of one such gene, daughterless. We present a comprehensive and interactive transcriptomic atlas of the hindwing tissue of both seasonal forms of B. anynana throughout development, a model organism of seasonal plasticity.

Milk-Derived miR-22–3p Promotes Proliferation of Human Intestinal Epithelial Cells (HIECs) by Regulating Gene Expression

ObjectivesMicroRNAs (miRNAs) are small non-coding RNA involved in RNA silencing and post-transcriptional regulation of gene expression. Milk exosomes are microvesicles containing miRNAs, and miR-22–3p (miR-22) abundantly appears in human milk exosomes. Both human milk exosomes and miR-22 survive in vitro gastrointestinal digestion. The aim of the current study was to evaluate effects of milk derived miR-22 on intestinal epithelial cells in early life. MethodsTo evaluate effects of miR-22 on intestinal epithelial cells and mimic uptake of miR-22 in exosomes by intestinal epithelial cells, miR-22 was transfected to human intestinal epithelial cells (HIECs) using Lipofectamine, and then total RNA was isolated after 24 h for microarray assay. After microarray results were verified by qRT-PCR, microarray they were analyzed using the Bioconductor package limma and compared with miR-22 predicted target genes to identify a direct target gene for miR-22 in HIECs. ResultsTotally, 608 genes were markedly regulated by miR-22. Based on Gene Ontology terms, the roles of miR-22 in HIECs include promotion of proliferation and regulation of immune functions. A proliferation assay (BrdU) was subsequently conducted to confirm the effect of miR-22 on promotion of proliferation of HIECs. Based on analysis of the microarray results and miR-22 predicted target genes, C/EBPδ, a transcription factor, may be a direct target for miR-22. In miR-22 transfected HIECs, transcription and translation of the C/EBPδ gene was significantly inhibited, and cell proliferation was increased when the C/EBPδ gene was silenced by siRNA. A luciferase assay showed that miR-22 specifically bound to the 3’-untranslated region of the C/EBPδ gene. ConclusionsMilk-derived miR-22 promotes intestinal proliferation by modifying gene expression, and C/EBPδ may be an important direct target for miR-22 in human intestinal epithelial cells. Funding SourcesIntramural funds.

Maize resistance to insect herbivory is enhanced by silencing expression of genes for jasmonate‐isoleucine degradation using sugarcane mosaic virus

AbstractPreviously, sugarcane mosaic virus (SCMV) was developed as a vector for transient expression of heterologous genes in Zea mays (maize). Here, we show that SCMV can also be applied for virus‐induced gene silencing (VIGS) of endogenous maize genes. Comparison of sense and antisense VIGS constructs targeting maize phytoene desaturase (PDS) showed that antisense constructs resulted in a greater reduction in gene expression. In a time course of gene expression after infection with VIGS constructs targeting PDS, lesion mimic 22 (Les22), and Iodent japonica 1 (Ij1), efficient expression silencing was observed 2, 3, and 4 weeks after infection with SCMV. However, at Week 5, expression of Les22 and Ij1 was no longer significantly reduced compared with control plants. The defense signaling molecule jasmonate‐isoleucine (JA‐Ile) can be inactivated by 12C‐hydroxylation and hydrolysis, and knockout of these genes leads to herbivore resistance. JA‐Ile hydroxylases and hydrolases have been investigated in Arabidopsis, rice, and Nicotiana attenuata. To determine whether the maize homologs of these genes function in plant defense, we silenced expression of ZmCYP94B1 (predicted JA‐Ile hydroxylase) and ZmJIH1 (predicted JA‐Ile hydrolase) by VIGS with SCMV, which resulted in elevated expression of two defense‐related genes, Maize Proteinase Inhibitor (MPI) and Ribosome Inactivating Protein 2 (RIP2). Although ZmCYP94B1 and ZmJIH1 gene expression silencing increased resistance to Spodoptera frugiperda (fall armyworm), Schistocerca americana (American birdwing grasshopper), and Rhopalosiphum maidis (corn leaf aphid), there was no additive effect from silencing the expression of both genes. Further work will be required to determine the more precise functions of these enzymes in regulating maize defenses.

Sirtuins in Epigenetic Silencing and Control of Gene Expression in Model and Pathogenic Fungi.

Fungi, including yeasts, molds, and mushrooms, proliferate on decaying matter and then adopt quiescent forms once nutrients are depleted. This review explores how fungi use sirtuin deacetylases to sense and respond appropriately to changing nutrients. Because sirtuins are NAD+-dependent deacetylases, their activity is sensitive to intracellular NAD+ availability. This allows them to transmit information about a cell's metabolic state on to the biological processes they influence. Fungal sirtuins are primarily known to deacetylate histones, repressing transcription and modulating genome stability. Their target genes include those involved in NAD+ homeostasis, metabolism, sporulation, secondary metabolite production, and virulence traits of pathogenic fungi. By targeting different genes over evolutionary time, sirtuins serve as rewiring points that allow organisms to evolve novel responses to low NAD+ stress by bringing relevant biological processes under the control of sirtuins.

Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL

AbstractAllogeneic chimeric antigen receptor T-cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CARTs have been created using gene editing techniques that induce DNA double-stranded breaks (DSBs), resulting in unintended on-target editing outcomes with potentially unforeseen consequences. Cytosine base editors (CBEs) install C•G to T•A point mutations in T cells, with between 90% and 99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using 4 simultaneous base edits. We show that CBE, unlike CRISPR-Cas9, does not impact T-cell proliferation, lead to aberrant DNA damage response pathway activation, or result in karyotypic abnormalities following multiplexed editing. We demonstrate 7CAR8 to be highly efficacious against T-cell acute lymphoblastic leukemia (T-ALL) using multiple in vitro and in vivo models. Thus, CBE is a promising technology for applications requiring multiplexed gene editing and can be used to manufacture quadruple-edited 7CAR8 cells, with high potential for clinical translation for relapsed and refractory T-ALL.

M6A-mediated upregulation of circMDK promotes tumorigenesis and acts as a nanotherapeutic target in hepatocellular carcinoma.

BackgroundEmerging evidence suggest the critical role of circular RNAs (circRNAs) in disease development especially in various cancers. However, the oncogenic role of circRNAs in hepatocellular carcinoma (HCC) is still largely unknown.MethodsRNA sequencing was performed to identify significantly upregulated circRNAs in paired HCC tissues and non-tumor tissues. CCK-8 assay, colony formation, transwell, and xenograft mouse models were used to investigate the role of circRNAs in HCC proliferation and metastasis. Small interfering RNA (siRNA) was used to silence gene expression. RNA immunoprecipitation, biotin pull-down, RNA pull-down, luciferase reporter assay and western blot were used to explore the underlying molecular mechanisms.ResultsHsa_circ_0095868, derived from exon 5 of the MDK gene (named circMDK), was identified as a new oncogenic circRNA that was significantly upregulated in HCC. The upregulation of circMDK was associated with the modification of N6-methyladenosine (m6A) and poor survival in HCC patients. Mechanistically, circMDK sponged miR-346 and miR-874-3p to upregulate ATG16L1 (Autophagy Related 16 Like 1), resulting to the activation of PI3K/AKT/mTOR signaling pathway to promote cell proliferation, migration and invasion. Poly (β-amino esters) (PAEs) were synthesized to assist the delivery of circMDK siRNA (PAE-siRNA), which effectively inhibited tumor progression without obvious adverse effects in four liver tumor models including subcutaneous, metastatic, orthotopic and patient-derived xenograft (PDX) models.ConclusionsCircMDK could serve as a potential tumor biomarker that promotes the progression of HCC via the miR-346/874-3p-ATG16L1 axis. The PAE-based delivery of siRNA improved the stability and efficiency of siRNA targeting circMDK. The PAE-siRNA nanoparticles effectively inhibited HCC proliferation and metastasis in vivo. Our current findings offer a promising nanotherapeutic strategy for the treatment of HCC.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-022-01575-z.

Determining The mRNA Which microRNA‐379‐5p Inhibitor Is Targeting in Human Lung A549 Cells

MicroRNAs are single‐stranded, non‐coding RNA that are complimentary to messenger RNA and play a role in post‐transcription gene expression and RNA silencing. Understanding the specific mRNA targets of miRNA that are elevated in tumors can provide insight into how cancer develops. The purpose of this experiment was to utilize miR‐379‐5p inhibitor to determine its target genes in human lung cells. Ten target genes for miRNA‐379‐5p were selected from Targetscan and miRDB databases, of which nine were novel and one, Mdm2, was a known target gene used as a positive control. QRT‐PCR primers for target genes were designed. A549 cells were transfected with the miR‐379‐5p inhibitor or a negative control, and then putative target mRNA levels were measured by qRT‐PCR. The Mdm2 positive control gene demonstrated increased levels in the presence of the miR‐379‐5p inhibitor, suggesting the assay was working properly. The putative target gene INSR also showed an increase in mRNA levels in the presence of miR‐379‐5p inhibitor, but a western blot did not show increased expression at the protein level. Other putative target genes examined did not show an increase in mRNA levels in the presence of the miR‐379‐5p inhibitor.

The transcription factor Atf1 lowers the transition barrier for nucleosome-mediated establishment of heterochromatin.

Transcription factors can exert opposite effects depending on the chromosomal context. The fission yeast transcription factor Atf1 both activates numerous genes in response to stresses and mediates heterochromatic gene silencing in the mating-type region. Investigating this context dependency, we report here that the establishment of silent heterochromatin in the mating-type region occurs at a reduced rate in the absence of Atf1 binding. Quantitative modeling accounts for the observed establishment profiles by a combinatorial recruitment of histone-modifying enzymes: locally by Atf1 at two binding sites and over the whole region by dynamically appearing heterochromatic nucleosomes, a source of which is the RNAi-dependent cenH element. In the absence of Atf1 binding, the synergy is lost, resulting in a slow rate of heterochromatin formation. The system shows how DNA-binding proteins can influence local nucleosome states and thereby potentiate long-range positive feedback on histone-modification reactions to enable heterochromatin formation over large regions in a context-dependent manner.