Silicon Ether Ionizable Lipids Enable Potent mRNA Lipid Nanoparticles with Rapid Tissue Clearance.

The advent of mRNA for nucleic acid (NA) therapeutics has unlocked many diverse areas of research and clinical investigation. However, the shorter intracellular half-life of mRNA compared with other NAs may necessitate more frequent dosing regimens. Because lipid nanoparticles (LNPs) are the principal delivery system used for mRNA, this could lead to tolerability challenges associated with an accumulated lipid burden. This can be addressed by introducing enzymatically cleaved carboxylic esters into the hydrophobic domains of lipid components, notably, the ionizable lipid. However, enzymatic activity can vary significantly with age, disease state, and species, potentially limiting the application in humans. Here we report an alternative approach to ionizable lipid degradability that relies on nonenzymatic hydrolysis, leading to a controlled and highly efficient lipid clearance profile. We identify highly potent examples and demonstrate their exceptional tolerability in multiple preclinical species, including multidosing in nonhuman primates (NHP).

BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts.

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac CaV1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca2+ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

siRNA treatment targeting integrin α11 overexpressed via EZH2-driven axis inhibits drug-resistant breast cancer progression.

BACKGROUND: Breast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets. METHODS: Transcriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target. RESULTS: Integrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours. CONCLUSION: Our findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.

Multiple factors and features dictate the selective production of ct-siRNA in Arabidopsis.

Coding transcript-derived siRNAs (ct-siRNAs) produced from specific endogenous loci can suppress the translation of their source genes to balance plant growth and stress response. In this study, we generated Arabidopsis mutants with deficiencies in RNA decay and/or post-transcriptional gene silencing (PTGS) pathways and performed comparative sRNA-seq analysis, revealing that multiple RNA decay and PTGS factors impede the ct-siRNA selective production. Genes that produce ct-siRNAs often show increased or unchanged expression and typically have higher GC content in sequence composition. The growth and development of plants can perturb the dynamic accumulation of ct-siRNAs from different gene loci. Two nitrate reductase genes, NIA1 and NIA2, produce massive amounts of 22-nt ct-siRNAs and are highly expressed in a subtype of mesophyll cells where DCL2 exhibits higher expression relative to DCL4, suggesting a potential role of cell-specific expression of ct-siRNAs. Overall, our findings unveil the multifaceted factors and features involved in the selective production and regulation of ct-siRNAs and enrich our understanding of gene silencing process in plants.

Resiquimod Induces C-C Motif Chemokine Ligand 2 Via Nuclear Factor-Kappa B in SH-SY5Y Human Neuroblastoma Cells.

Toll-like receptor (TLR) 7 plays an important role in recognizing virus-derived nucleic acids. TLR7 signaling in astrocytes and microglia is critical for activating immune responses against neurotrophic viruses. Neurons express TLR7, similar to glial cells; however, the role of neuronal TLR7 has not yet been fully elucidated. This study sought to determine whether resiquimod, the TLR7/8 agonist, induces the expression of inflammatory chemokines in SH-SY5Y human neuroblastoma cells. Immunofluorescence microscopy revealed that TLR7 was constitutively expressed in SH-SY5Y cells. Stimulation with resiquimod induced C-C motif chemokine ligand 2 (CCL2) expression, accompanied by the activation of nuclear factor-kappa B (NF-κB) in SH-SY5Y cells. Resiquimod increased mRNA levels of C-X-C motif chemokine ligand 8 (CXCL8) and CXCL10, while the increase was slight at the protein level. Knockdown of NF-κB p65 eliminated resiquimod-induced CCL2 production. This study provides novel evidence that resiquimod has promising therapeutic potential against central nervous system viral infections through its immunostimulatory effects on neurons.

Study of the Synergistic Immunomodulatory and Antifibrotic Effects of Dual-Loaded Budesonide and Serpine1 siRNA Lipid-Polymer Nanoparticles Targeting Macrophage Dysregulation in Tendinopathy.

Musculoskeletal diseases involving tissue injury comprise tendon, ligament, and muscle injury. Recently, macrophages have been identified as key players in the tendon repair process, but no therapeutic strategy involving dual drug delivery and gene delivery to macrophages has been developed for targeting the two main dysregulated aspects of macrophages in tendinopathy, i.e., inflammation and fibrosis. Herein, the anti-inflammatory and antifibrotic effects of dual-loaded budesonide and serpine1 siRNA lipid-polymer hybrid nanoparticles (LPNs) are evaluated in murine and human macrophage cells. The modulation of the gene and protein expression of factors associated with inflammation and fibrosis in tendinopathy is demonstrated by real time polymerase chain reaction and Western blot. Macrophage polarization to the M2 phenotype and a decrease in the production of pro-inflammatory cytokines are confirmed in macrophage cell lines and primary cells. The increase in the activity of a matrix metalloproteinase involved in tissue remodelling is proven, and studies evaluating the interactions of LPNs with T cells proved that dual-loaded LPNs act specifically on macrophages and do not induce any collateral effects on T cells. Overall, these dual-loaded LPNs are a promising combinatorial therapeutic strategy with immunomodulatory and antifibrotic effects in dysregulated macrophages in the context of tendinopathy.

Functional analysis of ESM1 by shRNA-mediated knockdown of its expression in papillary thyroid cancer cells.

OBJECTIVE: Endothelial specific molecule-1 (ESM1) is implicated as an oncogene in multiple human cancers. However, the function of ESM1 in papillary thyroid cancer (PTC) is not well understood. The current study aimed to investigate the effect of ESM1 on the growth, migration, and invasion of PTC to provide a novel perspective for PTC treatment. METHODS: The expression levels of ESM1 in PTC tissues form 53 tumor tissue samples and 59 matching adjacent normal tissue samples were detected by immunohistochemical analysis. Knockdown of ESM1 expression in TPC-1 and SW579 cell lines was established to investigate its role in PTC. Moreover, cell proliferation, apoptosis, wound healing, and transwell assays were conducted in vitro to assess cell proliferation, migration and invasion. RESULTS: The findings revealed that ESM1 expression was significantly higher in PTC tissues than that found in paraneoplastic tissues (P<0.0001). Knockdown of ESM1 expression inhibited the proliferation, migration, and invasion of TPC-1 and SW579 cells in vitro. Compared with the control group, the mRNA and protein levels of ESM1 in PTC cells were significantly reduced following knockdown of its expression (P<0.01). In addition, ESM1-knockdown cells indicated decreased proliferation and decreased migratory and invasive activities (P<0.01, P<0.01, P<0.001, respectively). CONCLUSIONS: ESM1 was identified as a major gene in the occurrence and progression of PTC, which could increase the proliferation, migration, and invasion of PTC cells. It may be a promising diagnostic and therapeutic target gene.

Advances in the mechanisms and applications of RNA silencing in crop protection.

RNA silencing (or RNA interference, RNAi) is a conserved mechanism for regulating gene expression in eukaryotes, which plays vital roles in plant development and response to biotic and abiotic stresses. The discovery of trans-kingdom RNAi and interspecies RNAi provides a theoretical basis for exploiting RNAi-based crop protection strategies. Here, we summarize the canonical RNAi mechanisms in plants and review representative studies associated with plant-pathogen interactions. Meanwhile, we also elaborate upon the principles of host-induced gene silencing, spray-induced gene silencing and microbe-induced gene silencing, and discuss their applications in crop protection, thereby providing help to establish novel RNAi-based crop protection strategies.

Genome-wide view and characterization of natural antisense transcripts in Cannabis Sativa L.

Natural Antisense Transcripts (NATs) are a kind of complex regulatory RNAs that play crucial roles in gene expression and regulation. However, the NATs in Cannabis Sativa L., a widely economic and medicinal plant rich in cannabinoids remain unknown. In this study, we comprehensively predicted C. sativa NATs genome-wide using strand-specific RNA sequencing (ssRNA-Seq) data, and validated the expression profiles by strand-specific quantitative reverse transcription PCR (ssRT-qPCR). Consequently, a total of 307 NATs were predicted in C. sativa, including 104 cis- and 203 trans- NATs. Functional enrichment analysis demonstrated the potential involvement of the C. sativa NATs in DNA polymerase activity, RNA-DNA hybrid ribonuclease activity, and nucleic acid binding. Finally, 18 cis- and 376 trans- NAT-ST pairs were predicted to produce 621 cis- and 5,679 trans- small interfering RNA (nat-siRNAs), respectively. These nat-siRNAs were potentially involved in the biosynthesis of cannabinoids and cellulose. All these results will shed light on the regulation of NATs and nat-siRNAs in C. sativa.

Knockdown siRNA Targeting GPR55 Reveals Significant Differences Between the Anti-inflammatory Actions of KLS-13019 and Cannabidiol.

KLS-13019 was reported previously to reverse paclitaxel-induced mechanical allodynia in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). Recent studies demonstrated that paclitaxel-induced increases in inflammatory markers (GPR55, NLRP3, and IL-1ß) of dorsal root ganglion (DRG) cultures were shown to be reversed by KLS-13019 treatment. The mechanism of action for KLS-13019-mediated reversal of paclitaxel-induced neuroinflammation now has been explored using GPR55 siRNA. Pre-treatment of DRG cultures with GPR55 siRNA produced a 21% decrease of immunoreactive (IR) area for GPR55 in cell bodies and a 59% decrease in neuritic IR area, as determined by high-content imaging. Using a 24-h reversal treatment paradigm, paclitaxel-induced increases in the inflammatory markers were reversed back to control levels after KLS-3019 treatment. Decreases in these inflammatory markers produced by KLS-13019 were significantly attenuated by GPR55 siRNA co-treatment, with mean IR area responses being attenuated by 56% in neurites and 53% in cell bodies. These data indicate that the percentage decreases in siRNA-mediated attenuation of KLS-13019-related efficacy on the inflammatory markers were similar to the percentage knockdown observed for neuritic GPR55 IR area. Similar studies conducted with cannabidiol (CBD), the parent compound of KLS-13019, produced low efficacy (25%) reversal of all inflammatory markers that were poorly attenuated (29%) by GPR55 siRNA. CBD was shown previously to be ineffective in reversing paclitaxel-induced mechanical allodynia. The present studies indicated significant differences between the anti-inflammatory properties of KLS-13019 and CBD which may play a role in their observed differences in the reversibility of mechanical allodynia in a mouse model of CIPN.

[Research progress of piRNA as a biomarker for male infertility].

piRNA is a class of small non-coding RNA which specifically binds with PIWI protein. It is mainly expressed in germ cells and involved in the regulation of spermatogenesis. The role of piRNA pathway in the regulation of spermatogenesis mainly includes inhibition of transposons, induction of mRNA translation or degradation, and mediation of degradation of Miwi ubiquitination in late-stage sperm cells. With the detection of piRNA in seminal plasma, more attention has been attracted to whether piRNA can be used as a non-invasive molecular biomarker for the evaluation of spermatogenesis. This paper has reviewed recent studies on the mechanism of piRNA pathways mediating spermatogenesis and potential roles of piRNA disorders in the diagnosis and treatment of male infertility.

Cross-Reactive Polyclonal Antibodies Raised Against GalNAc-Conjugated siRNA Recognize Mostly the GalNAc Moiety.

Small interfering RNA (siRNA) is gaining momentum as a therapeutic modality with six approved products. Since siRNA has the potential to elicit undesired immune responses in patients, immunogenicity assessment is required during clinical development by regulatory authorities. In this study, anti-siRNA polyclonal antibodies were generated through animal immunization. These cross-reactive polyclonal antibodies recognized mostly the N-acetylgalactosamine (GalNAc) moiety with a small fraction against sequence-independent epitopes. We demonstrate that the polyclonal antibodies can be utilized as immunogenicity assay positive controls for the same class of GalNAc-conjugated siRNAs. In addition, anti-GalNAc mAbs showed desired sensitivity and drug tolerance, supporting their use as alternative surrogate positive controls. These findings can guide positive control selection and immunogenicity assay development for GalNAc-conjugated siRNAs and other oligonucleotide therapeutics.

[The Regulatory Effect of RNA m6 A Methylation Modification on KDM4B Gene Expression in t (8;21) AML Cells by MeRIP-qPCR].

OBJECTIVE: To confirm the direct regulatory effect of WTAP-mediated RNA m6A modification on the KDM4B gene in t (8;21) acute myeloid leukemia (AML) cells through MeRIP combined with reverse transcription real-time quantitative PCR (RT-qPCR) technology. METHODS: The lentivirus-mediated shRNA target WTAP or KDM4B gene was used to transfect the t (8;21) AML cell lines: Kasumi-1 and SKNO-1, and cells transfected with randomly shuffled shRNA as the control. Using the Ultrapure RNA Extraction Kit (DNase I) to extract RNA. The Magna MeRIPTM m6A Kit was used to enrich methylated modified fragments, and detect the m6A methylated RNA regions by RT-qPCR, and the protein and mRNA expression levels of WTAP and KDM4B in cells were detected by Western blot and reverse transcription real-time quantitative PCR (RT-qPCR). Colony formation assays were used to detect the colony ability of cells in vitro. RESULTS: Silencing the expression of WTAP in Kasumi-1 cells, the enrichment of m6A methylation modification was significantly decreased in the 3'UTR of KDM4B mRNA(P < 0.01), and the protein(P < 0.001) and mRNA (Kasumi-1:P < 0.001; SKNO-1: P < 0.01) expression levels of KDM4B were also significantly inhibited in Kasumi-1 and SKNO-1 cells upon WTAP knockdown (all P < 0.01), accompanied by a significant decrease in the colony-forming ability of both cell lines (both P < 0.01). CONCLUSION: In t(8;21) AML cell lines, WTAP could regulate the expression of KDM4B by regulating the m6A modification of the 3'UTR of KDM4B mRNA, and silencing the expression of KDM4B could inhibit the cellular proliferation in vitro.

The curious case of the disappearing piRNAs.

Small non-coding RNAs are key regulators of gene expression across eukaryotes. Piwi-interacting small RNAs (piRNAs) are a specific type of small non-coding RNAs, conserved across animals, which are best known as regulators of genome stability through their ability to target transposable elements for silencing. Despite the near ubiquitous presence of piRNAs in animal lineages, there are some examples where the piRNA pathway has been lost completely, most dramatically in nematodes where loss has occurred in at least four independent lineages. In this perspective I will provide an evaluation of the presence of piRNAs across animals, explaining how it is known that piRNAs are missing from certain organisms. I will then consider possible explanations for why the piRNA pathway might have been lost and evaluate the evidence in favor of each possible mechanism. While it is still impossible to provide definitive answers, these theories will prompt further investigations into why such a highly conserved pathway can nevertheless become dispensable in certain lineages. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

Gene Silencing of Angiopoietin-like 3 (ANGPTL3) Induced De Novo Lipogenesis and Lipid Accumulation in Huh7 Cell Line.

Angiopoietin-like 3 (ANGPTL3) is a hepatokine acting as a negative regulator of lipoprotein lipase (LPL). Vupanorsen, an ANGPTL3 directed antisense oligonucleotide, showed an unexpected increase in liver fat content in humans. Here, we investigated the molecular mechanism linking ANGPTL3 silencing to hepatocyte fat accumulation. Human hepatocarcinoma Huh7 cells were treated with small interfering RNA (siRNA) directed to ANGPTL3, human recombinant ANGPTL3 (recANGPTL3), or their combination. Using Western blot, Oil Red-O, biochemical assays, and ELISA, we analyzed the expression of genes and proteins involved in lipid metabolism. Oil Red-O staining demonstrated that lipid content increased after 48 h of ANGPTL3 silencing (5.89 ± 0.33 fold), incubation with recANGPTL3 (4.08 ± 0.35 fold), or their combination (8.56 ± 0.18 fold), compared to untreated cells. This effect was also confirmed in Huh7-LX2 spheroids. A total of 48 h of ANGPTL3 silencing induced the expression of genes involved in the de novo lipogenesis, such as fatty acid synthase, stearoyl-CoA desaturase, ATP citrate lyase, and Acetyl-Coenzyme A Carboxylase 1 together with the proprotein convertase subtilisin/kexin 9 (PCSK9). Time-course experiments revealed that 6 h post transfection with ANGPTL3-siRNA, the cholesterol esterification by Acyl-coenzyme A cholesterol acyltransferase (ACAT) was reduced, as well as total cholesterol content, while an opposite effect was observed at 48 h. Under the same experimental conditions, no differences in secreted apoB and PCSK9 were observed. Since PCSK9 was altered by the treatment, we tested a possible co-regulation between the two genes. The effect of ANGPTL3-siRNA on the expression of genes involved in the de novo lipogenesis was not counteracted by gene silencing of PCSK9. In conclusion, our in vitro study suggests that ANGPTL3 silencing determines lipid accumulation in Huh7 cells by inducing the de novo lipogenesis independently from PCSK9.

Microdroplets Encapsulated with NFATc1-siRNA and Exosomes-Derived from MSCs Onto 3D Porous PLA Scaffold for Regulating Osteoclastogenesis and Promoting Osteogenesis.

Introduction: Osteoporotic-related fractures remains a significant public health concern, thus imposing substantial burdens on our society. Excessive activation of osteoclastic activity is one of the main contributing factors for osteoporosis-related fractures. While polylactic acid (PLA) is frequently employed as a biodegradable scaffold in tissue engineering, it lacks sufficient biological activity. Microdroplets (MDs) have been explored as an ultrasound-responsive drug delivery method, and mesenchymal stem cell (MSC)-derived exosomes have shown therapeutic effects in diverse preclinical investigations. Thus, this study aimed to develop a novel bioactive hybrid PLA scaffold by integrating MDs-NFATc1-silencing siRNA to target osteoclast formation and MSCs-exosomes (MSC-Exo) to influence osteogenic differentiation (MDs-NFATc1/PLA-Exo). Methods: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) were used for exosome isolation. Transmission electron microscopy (TEM) and confocal laser scanning microscopy were used for exosome and MDs morphological characterization, respectively. The MDs-NFATc1/PLA-Exo scaffold was fabricated through poly(dopamine) and fibrin gel coating. Biocompatibility was assessed using RAW 264.7 macrophages and hBMSCs. Osteoclast formations were examined via TRAP staining. Osteogenic differentiation of hBMSCs and cytokine expression modulation were also investigated. Results: MSC-Exo exhibited a cup-shaped structure and effective internalization into cells, while MDs displayed a spherical morphology with a well-defined core-shell structure. Following ultrasound stimulation, the internalization study demonstrated efficient delivery of bioactive MDs into recipient cells. Biocompatibility studies indicated no cytotoxicity of MDs-NFATc1/PLA-Exo scaffolds in RAW 264.7 macrophages and hBMSCs. Both MDs-NFATc1/PLA and MDs-NFATc1/PLA-Exo treatments significantly reduced osteoclast differentiation and formation. In addition, our results further indicated MDs-NFATc1/PLA-Exo scaffold significantly enhanced osteogenic differentiation of hBMSCs and modulated cytokine expression. Discussion: These findings suggest that the bioactive MDs-NFATc1/PLA-Exo scaffold holds promise as an innovative structure for bone tissue regeneration. By specifically targeting osteoclast formation and promoting osteogenic differentiation, this hybrid scaffold may address key challenges in osteoporosis-related fractures.

The role of piRNAs in predicting and prognosing in cancer: a focus on piRNA-823 (a systematic review and meta-analysis).

INTRODUCTION: This article examines the potential of using liquid biopsy with piRNAs to study cancer survival outcomes. While previous studies have explored the relationship between piRNA expression and cancer patient outcomes, a comprehensive investigation is still lacking. To address this gap, we conducted a systematic review and meta-analysis of existing literature. METHODS: We searched major online databases up to February 2024 to identify articles reporting on the role of piRNA in cancer patient survival outcomes. Our meta-analysis used a random-effects model to pool hazard ratios with 95% confidence intervals (CI) and assess the prognostic value of deregulated piRNA-823. For survival analysis, the Kaplan-Meier method and COX analysis were used. RESULTS: Out of 6104 articles screened, 20 met our inclusion criteria. Our analysis revealed that dysregulated piRNA expression is associated with cancer patient survival outcomes. Specifically, our meta-analysis found that overexpression of piR-823 is significantly linked with poorer overall survival in patients with colorectal cancer and renal cell cancer (HR: 3.82, 95% CI = [1.81, 8.04], I2 = 70%). CONCLUSION: Our findings suggest that various piRNAs may play a role in cancer survival outcomes and that piRNA-823 in particular holds promise as a prognostic biomarker for multiple human cancers. IMPLICATIONS FOR CANCER SURVIVORS: Our systematic review and meta-analysis of piRNA-823 has important implications for cancer survivors. Our findings suggest that piRNA-823 can be used as a prognostic biomarker for predicting cancer recurrence and survival rates. This information can help clinicians develop personalized treatment plans for cancer survivors, which can improve their quality of life and reduce the risk of recurrence.

Identification and expression patterns of somatic piRNAs and PIWI genes in Riptortus pedestris (Hemiptera: Alydidae).

RNA interference (RNAi)-based gene silencing is a feasible and sustainable technology for the management of hemipteran pests by double-stranded RNA involvement, including small-interfering RNA, microRNA, and Piwi-interacting RNA (piRNA) pathways, that may help to decrease the usage of chemical insecticides. However, only a few data are available on the somatic piRNAs and their biogenesis genes in Riptortus pedestris, which serves as a significant pest of soybean (Glycine max). In this study, two family members of the PIWI gene were identified and characterized in R. pedestris, containing Argonaute3 (RpAgo3) and Aubergine (RpAub) genes with conserved protein domains, and their clusters were validated by phylogenetic analysis. In addition, they were widely expressed in all developmental stages of the whole body of R. pedestris and had lower expression levels in R. pedestris guts under different rearing conditions based on previous transcriptome sequencing. Furthermore, abundant clean reads were filtered to a total number of 45,998 piRNAs with uridine bias at the first nucleotide (nt) position and 26-32 nt in length by mapping onto the reference genome of R. pedestris according to our previous whole-transcriptome sequencing. Finally, our data revealed that gut bacterial changes were significantly positively or negatively associated with differentially expressed piRNAs among the five comparison groups with Pearson correlation analysis. In conclusion, these findings paved new avenues for the application of RNAi-based biopesticides for broad-spectrum hemipteran pest control.

The advent of RNA-based therapeutics for metabolic syndrome and associated conditions: a comprehensive review of the literature.

Metabolic syndrome (MetS) is a prevalent and intricate health condition affecting a significant global population, characterized by a cluster of metabolic and hormonal disorders disrupting lipid and glucose metabolism pathways. Clinical manifestations encompass obesity, dyslipidemia, insulin resistance, and hypertension, contributing to heightened risks of diabetes and cardiovascular diseases. Existing medications often fall short in addressing the syndrome's multifaceted nature, leading to suboptimal treatment outcomes and potential long-term health risks. This scenario underscores the pressing need for innovative therapeutic approaches in MetS management. RNA-based treatments, employing small interfering RNAs (siRNAs), microRNAs (miRNAs), and antisense oligonucleotides (ASOs), emerge as promising strategies to target underlying biological abnormalities. However, a summary of research available on the role of RNA-based therapeutics in MetS and related co-morbidities is limited. Murine models and human studies have been separately interrogated to determine whether there have been recent advancements in RNA-based therapeutics to offer a comprehensive understanding of treatment available for MetS. In a narrative fashion, we searched for relevant articles pertaining to MetS co-morbidities such as cardiovascular disease, fatty liver disease, dementia, colorectal cancer, and endocrine abnormalities. We emphasize the urgency of exploring novel therapeutic avenues to address the intricate pathophysiology of MetS and underscore the potential of RNA-based treatments, coupled with advanced delivery systems, as a transformative approach for achieving more comprehensive and efficacious outcomes in MetS patients.

Ultrasound enhanced siRNA delivery using cationic liposome-microbubble complexes for the treatment of squamous cell carcinoma.

Rationale: Microbubble (MB) contrast agents combined with ultrasound targeted microbubble cavitation (UTMC) are a promising platform for site-specific therapeutic oligonucleotide delivery. We investigated UTMC-mediated delivery of siRNA directed against epidermal growth factor receptor (EGFR), to squamous cell carcinoma (SCC) via a novel MB-liposome complex (LPX). Methods: LPXs were constructed by conjugation of cationic liposomes to the surface of C4F10 gas-filled lipid MBs using biotin/avidin chemistry, then loaded with siRNA via electrostatic interaction. Luciferase-expressing SCC-VII cells (SCC-VII-Luc) were cultured in Petri dishes. The Petri dishes were filled with media in which LPXs loaded with siRNA against firefly luciferase (Luc siRNA) were suspended. Ultrasound (US) (1 MHz, 100-µs pulse, 10% duty cycle) was delivered to the dishes for 10 sec at varying acoustic pressures and luciferase assay was performed 24 hr later. In vivo siRNA delivery was studied in SCC-VII tumor-bearing mice intravenously infused with a 0.5 mL saline suspension of EGFR siRNA LPX (7×108 LPX, ~30 µg siRNA) for 20 min during concurrent US (1 MHz, 0.5 MPa spatial peak temporal peak negative pressure, five 100-µs pulses every 1 ms; each pulse train repeated every 2 sec to allow reperfusion of LPX into the tumor). Mice were sacrificed 2 days post treatment and tumor EGFR expression was measured (Western blot). Other mice (n=23) received either EGFR siRNA-loaded LPX + UTMC or negative control (NC) siRNA-loaded LPX + UTMC on days 0 and 3, or no treatment ("sham"). Tumor volume was serially measured by high-resolution 3D US imaging. Results: Luc siRNA LPX + UTMC caused significant luciferase knockdown vs. no treatment control, p<0.05) in SCC-VII-Luc cells at acoustic pressures 0.25 MPa to 0.9 MPa, while no significant silencing effect was seen at lower pressure (0.125 MPa). In vivo, EGFR siRNA LPX + UTMC reduced tumor EGFR expression by ~30% and significantly inhibited tumor growth by day 9 (~40% decrease in tumor volume vs. NC siRNA LPX + UTMC, p<0.05). Conclusions: Luc siRNA LPXs + UTMC achieved functional delivery of Luc siRNA to SCC-VII-Luc cells in vitro. EGFR siRNA LPX + UTMC inhibited tumor growth and suppressed EGFR expression in vivo, suggesting that this platform holds promise for non-invasive, image-guided targeted delivery of therapeutic siRNA for cancer treatment.