Knockdown Of Target Genes Research Articles

The Effects of DDI1 on Inducing Differentiation in Ovine Preadipocytes via Oar-miR-432.

Reducing fat deposition in sheep (Ovis aries) tails is one of the most important ways to combat rising costs and control consumer preference. Our previous studies have shown that oar-miR-432 is differentially expressed in the tail adipose tissue of Hu (a fat-tailed sheep breed) and Tibetan (a thin-tailed sheep breed) sheep and is a key factor in the negative regulation of fat deposition through BMP2 in ovine preadipocytes. This study investigated the effect of oar-miR-432 and its target genes in ovine preadipocytes. A dual luciferase assay revealed that DDI1 is a direct target gene of oar-miR-432. We transfected an oar-miR-432 mimic and inhibitor into preadipocytes to analyze the expression of target genes. Overexpression of oar-miR-432 inhibits DDI1 expression, whereas inhibition showed the opposite results. Compared with thin-tailed sheep, DDI1 was highly expressed in the fat-tailed sheep at the mRNA and protein levels. Furthermore, we transfected the overexpression and knockdown target genes into preadipocytes to analyze their influence after inducing differentiation. Knockdown of DDI1 induced ovine preadipocyte differentiation into adipocytes but suppressed oar-miR-432 expression. Conversely, the overexpression of DDI1 significantly inhibited differentiation but promoted oar-miR-432 expression. DDI1 overexpression also decreased the content of triglycerides. Additionally, DDI1 is a nested gene in intron 1 of PDGFD. When DDI1 was overexpressed, the PDGFD expression also increased, whereas DDI1 knockdown showed the opposite results. This is the first study to reveal the biological mechanisms by which oar-miR-432 inhibits preadipocytes through DDI1 and provides insight into the molecular regulatory mechanisms of DDI1 in ovine preadipocytes. These results have important applications in animal breeding and obesity-related human diseases.

Inefficient uptake of small interfering RNAs is responsible for their inability to trigger RNA interference in Colorado potato beetle cells.

There has been limited success in the usage of exogenous small interference RNA (siRNA) or small hairpin RNA (shRNA) to trigger RNA interference (RNAi) in insects. Instead, long double-stranded RNAs (dsRNA) are used to induce knockdown of target genes in insects. Here, we compared the potency of si/sh RNAs and dsRNA in Colorado potato beetle (CPB) cells. CPB cells showed highly efficient RNAi response to dsRNA. However, si/sh RNAs were inefficient in triggering RNAi in CPB cells. Confocal microscopy observations of Cy3 labeled-si/sh RNA cellular uptake revealed reduced si/sh RNA uptake compared to dsRNA. si/sh RNAs were stable in the conditioned media of CPB cells. Although in a small amount, when internalized by CPB cells, the si/sh RNAs were processed by the Dicer enzyme. Lipid-mediated transfection and chimeric dsRNA approaches were used to improve the delivery of si/sh RNAs. Our results suggest that the uptake of si/sh RNAs is inefficient in CPB cells, resulting in ineffective RNAi response. However, with the help of effective delivery methods, si/sh RNA could be a useful option for developing target-specific RNAi-mediated biopesticides.

A PPIX-binding probe facilitates discovery of PPIX-induced cell death modulation by peroxiredoxin

While heme synthesis requires the formation of a potentially lethal intermediate, protoporphyrin IX (PPIX), surprisingly little is known about the mechanism of its toxicity, aside from its phototoxicity. The cellular protein interactions of PPIX might provide insight into modulators of PPIX-induced cell death. Here we report the development of PPB, a biotin-conjugated, PPIX-probe that captures proteins capable of interacting with PPIX. Quantitative proteomics in a diverse panel of mammalian cell lines reveal a high degree of concordance for PPB-interacting proteins identified for each cell line. Most differences are quantitative, despite marked differences in PPIX formation and sensitivity. Pathway and quantitative difference analysis indicate that iron and heme metabolism proteins are prominent among PPB-bound proteins in fibroblasts, which undergo PPIX-mediated death determined to occur through ferroptosis. PPB proteomic data (available at PRIDE ProteomeXchange # PXD042631) reveal that redox proteins from PRDX family of glutathione peroxidases interact with PPIX. Targeted gene knockdown of the mitochondrial PRDX3, but not PRDX1 or 2, enhance PPIX-induced death in fibroblasts, an effect blocked by the radical-trapping antioxidant, ferrostatin-1. Increased PPIX formation and death was also observed in a T-lymphoblastoid ferrochelatase-deficient leukemia cell line, suggesting that PPIX elevation might serve as a potential strategy for killing certain leukemias.

A Comparative Analysis of RNAi Trigger Uptake and Distribution in Mosquito Vectors of Disease.

In mosquitoes, the utilization of RNAi for functional genetics is widespread, usually mediated through introduced double-stranded RNAs (dsRNAs) with sequence identity to a gene of interest. However, RNAi in mosquitoes is often hampered by inconsistencies in target gene knockdown between experimental setups. While the core RNAi pathway is known to function in most mosquito strains, the uptake and biodistribution of dsRNAs across different mosquito species and life stages have yet to be extensively explored as a source of variation in RNAi experiments. To better understand mosquito-RNAi dynamics, the biodistribution of a dsRNA to a heterologous gene, LacZ (iLacZ), was tracked following various routes of exposure in the larval and adult stages of Aedes aegypti, Anopheles gambiae, and Culex pipiens. iLacZ was largely limited to the gut lumen when exposed per os, or to the cuticle when topically applied, but spread through the hemocoel when injected. Uptake of dsRNA was noted in a subset of cells including: hemocytes, pericardial cells of the dorsal vessel, ovarian follicles, and ganglia of the ventral nerve cord. These cell types are all known to undergo phagocytosis, pinocytosis, or both, and as such may actively take up RNAi triggers. In Ae. aegypti, iLacZ was detected for up to one week post exposure by Northern blotting, but uptake and degradation drastically differed across tissues. The results presented here reveal that the uptake of RNAi triggers is distinct and specific to the cell type in vivo.

Identification and validation of G protein-coupled receptors modulating flow-dependent signaling pathways in vascular endothelial cells

Vascular endothelial cells are exposed to mechanical forces due to their presence at the interface between the vessel wall and flowing blood. The patterns of these mechanical forces (laminar vs. turbulent) regulate endothelial cell function and play an important role in determining endothelial phenotype and ultimately cardiovascular health. One of the key transcriptional mediators of the positive effects of laminar flow patterns on endothelial cell phenotype is the zinc-finger transcription factor, krüppel-like factor 2 (KLF2). Given its importance in maintaining a healthy endothelium, we sought to identify endothelial regulators of the KLF2 transcriptional program as potential new therapeutic approaches to treating cardiovascular disease. Using an approach that utilized both bioinformatics and targeted gene knockdown, we identified endothelial GPCRs capable of modulating KLF2 expression. Genetic screening using siRNAs directed to these GPCRs identified 12 potential GPCR targets that could modulate the KLF2 program, including a subset capable of regulating flow-induced KLF2 expression in primary endothelial cells. Among these targets, we describe the ability of several GPCRs (GPR116, SSTR3, GPR101, LGR4) to affect KLF2 transcriptional activation. We also identify these targets as potential validated targets for the development of novel treatments targeting the endothelium. Finally, we highlight the initiation of drug discovery efforts for LGR4 and report the identification of the first known synthetic ligands to this receptor as a proof-of-concept for pathway-directed phenotypic screening to identify novel drug targets.

WED-514 - Knockdown of a novel target gene enhances liver regeneration and attenuates chronic liver damage related liver fibrosis

WED-514 - Knockdown of a novel target gene enhances liver regeneration and attenuates chronic liver damage related liver fibrosis

Molecular Characterization of U6 Promoters from Orange-Spotted Grouper (Epinephelus coioides) and Its Application in DNA Vector-Based RNAi Technology.

The U6 promoter, a typical RNA polymerase III promoter, is widely used to transcribe small RNAs in vector-based siRNA systems. The RNAi efficiency is mainly dependent on the transcriptional activity of the U6 promoter. However, studies have found that U6 promoters isolated from some fishes do not work well in distantly related species. To isolate a U6 promoter with high transcriptional efficiency from fish, in this study, we cloned five U6 promoters in orange-spotted grouper, of which only the grouper U6-1 (GU6-1) promoter contains the OCT element in the distant region. Functional studies revealed that the GU6-1 promoter has high transcriptional ability, which could efficiently transcribe shRNA and result in target gene knockdown in vitro and in vivo. Subsequently, the deletion or mutation of the OCT motif resulted in a significant decrease in promoter transcriptional activity, demonstrating that the OCT element plays an important role in enhancing the grouper U6 promoter transcription. Moreover, the transcriptional activity of the GU6-1 promoter showed little species specificity. It not only works in the grouper but also possesses high transcriptional activity in the zebrafish. Knockdown of the mstn gene in zebrafish and grouper through shRNA driven by the GU6-1 promoter could promote fish growth, suggesting that the GU6-1 promoter can be used as a potential molecular tool in aquaculture practice.

FLI1 Regulates Histamine Decarboxylase Expression to Control Inflammation Signaling and Leukemia Progression.

Histamine decarboxylase (HDC) catalyzes decarboxylation of histidine to generate histamine. This enzyme affects several biological processes including inflammation, allergy, asthma, and cancer, although the underlying mechanism is not fully understood. The present study provides a novel insight into the relationship between the transcription factor FLI1 and its downstream target HDC, and their effects on inflammation and leukemia progression. Promoter analysis combined with chromatin immunoprecipitation (ChIp) was used to demonstrate binding of FLI1 to the promoter of HDC in leukemic cells. Western blotting and RT-qPCR were used to determine expression of HDC and allergy response genes, and lentivirus shRNA was used to knock-down target genes. Proliferation, cell cycle, apoptosis assays and molecular docking were used to determine the effect of HDC inhibitors in culture. An animal model of leukemia was employed to test the effect of HDC inhibitory compounds in vivo. Results presented herein demonstrate that FLI1 transcriptionally regulates HDC by direct binding to its promoter. Using genetic and pharmacological inhibition of HDC, or the addition of histamine, the enzymatic product of HDC, we show neither have a discernable effect on leukemic cell proliferation in culture. However, HDC controls several inflammatory genes including IL1B and CXCR2 that may influence leukemia progression in vivo through the tumor microenvironment. Indeed, diacerein, an IL1B inhibitor, strongly blocked Fli-1-induced leukemia in mice. In addition to allergy, FLI1 is shown to regulate genes associated with asthma such as IL1B, CPA3 and CXCR2. Toward treatment of these inflammatory conditions, epigallocatechin (EGC), a tea polyphenolic compound, is found strongly inhibit HDC independently of FLI1 and its downstream effector GATA2. Moreover, the HDC inhibitor, tetrandrine, suppressed HDC transcription by directly binding to and inhibiting the FLI1 DNA binding domain, and like other FLI1 inhibitors, tetrandrine strongly suppressed cell proliferation in culture and leukemia progression in vivo. These results suggest a role for the transcription factor FLI1 in inflammation signaling and leukemia progression through HDC and point to the HDC pathway as potential therapeutics for FLI1-driven leukemia.

Engineering Female Germline Stem Cells with Exocytotic Polymer Dots.

Germline stem cells (GSCs) are the only cell population capable of passing genetic information to offspring, making them attractive targets in reproductive biology and fertility research. However, it is generally more difficult to introduce exogenous biomolecules into GSCs than other cell types, impeding the exploration and manipulation of these cells for biomedical purposes. Herein, semiconductor polymer dots (Pdots)-based nanocomplex Pdot-siRNA is developed and achieves effective knockdown of target genes in female germline stem cells (FGSCs). Advantage of high fluorescence brightness of Pdots is taken for comprehensive investigation of their cellular uptake, intracellular trafficking, and exocytosis in FGSCs. Importantly, Pdots show excellent biocompatibility and minimally disturb the differentiation of FGSCs. Intracellular Pdots escape from the lysosomes and undergo active exocytosis, which makes them ideal nanocarriers for bioactive cargos. Moreover, Pdot-siRNA can penetrate into 3D ovarian organoids derived from FGSCs and down-regulate the expression levels of target genes. This study investigates the interface between a type of theranostic nanoparticles and FGSCs for the first time and sheds light on the manipulation and medical application of FGSCs.

Targeted and high-throughput gene knockdown in diverse bacteria using synthetic sRNAs

Synthetic sRNAs allow knockdown of target genes at translational level, but have been restricted to a limited number of bacteria. Here, we report the development of a broad-host-range synthetic sRNA (BHR-sRNA) platform employing the RoxS scaffold and the Hfq chaperone from Bacillus subtilis. BHR-sRNA is tested in 16 bacterial species including commensal, probiotic, pathogenic, and industrial bacteria, with >50% of target gene knockdown achieved in 12 bacterial species. For medical applications, virulence factors in Staphylococcus epidermidis and Klebsiella pneumoniae are knocked down to mitigate their virulence-associated phenotypes. For metabolic engineering applications, high performance Corynebacterium glutamicum strains capable of producing valerolactam (bulk chemical) and methyl anthranilate (fine chemical) are developed by combinatorial knockdown of target genes. A genome-scale sRNA library covering 2959 C. glutamicum genes is constructed for high-throughput colorimetric screening of indigoidine (natural colorant) overproducers. The BHR-sRNA platform will expedite engineering of diverse bacteria of both industrial and medical interest.

Abstract 3973: Precision RNAi using synthetic shRNAmir target sites

Abstract Genetic loss-of-function methods including short-hairpin RNAs (shRNAs) and CRISPR are key methods for target gene validation across a variety of different disease areas. While these methods have revolutionized target gene discovery and characterization, both methods suffer from limitations in the context of in vivo target validation due to off-target effects and insufficient knock-down for shRNAs and resistant clones for CRISPR. In this study, we describe a method, artificial RNA interference (ARTi) that overcomes these limitations by fundamentally changing the basic experimental strategy of RNAi-based loss-of-function studies. Instead of newly designing gene-specific shRNA for individual target genes, ARTi utilizes ultra-effective and selective artificial miRNA-based shRNAs that do not match any transcribed gene in the target genome. In addition, these sequences are optimized for ultra-efficient miRNA processing and knockdown of synthetic target genes, and are deeply characterized to not trigger major off-target effects, enabling highly stringent target validation in vivo with unprecedented temporal control, selectivity and potency. We validate the approach by studying the in vitro and in vivo phenotypes of EGFR, KRAS and STAG1, genes relevant to cancer biology. This loss-of-function strategy will enable novel experimental strategies in therapeutic target validation and will be instrumental in guiding the lead optimization process by establishing genetic benchmark phenotypes. Citation Format: Ralph Neumüller, Thomas Hoffmann, Alexandra Hörmann, Maja Corcokovic, Johannes Zuber. Precision RNAi using synthetic shRNAmir target sites. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3973.

MP14-17 SMARCD1 REGULATED BY MICRORNA-99A-5P CONTRIBUTES TO GEMCITABINE RESISTANCE IN BLADDER CANCER VIA SUPPRESSION OF CELLULAR SENESCENCE

You have accessJournal of UrologyCME1 Apr 2023MP14-17 SMARCD1 REGULATED BY MICRORNA-99A-5P CONTRIBUTES TO GEMCITABINE RESISTANCE IN BLADDER CANCER VIA SUPPRESSION OF CELLULAR SENESCENCE Junya Arima, Hirofumi Yoshino, Ichiro Kawahara, Wataru Fukumoto, Issei Kawakami, Motoki Tamai, Shuichi Tatarano, and Hideki Enokida Junya ArimaJunya Arima More articles by this author , Hirofumi YoshinoHirofumi Yoshino More articles by this author , Ichiro KawaharaIchiro Kawahara More articles by this author , Wataru FukumotoWataru Fukumoto More articles by this author , Issei KawakamiIssei Kawakami More articles by this author , Motoki TamaiMotoki Tamai More articles by this author , Shuichi TataranoShuichi Tatarano More articles by this author , and Hideki EnokidaHideki Enokida More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with advanced bladder cancer are generally treated with a combination of chemotherapeutics, including gemcitabine, but the effect is limited due to acquisition of drug resistance. Several microRNAs (miRNAs) have been proven to be associated with drug resistance. Thus, in this study, we focused on miRNAs and investigated the mechanism of gemcitabine resistance. METHODS: First, human BC cell lines (BOY, T24) were cultured in gemcitabine supplemented culture medium for 12 months to establish gemcitabine-resistant cells (GEM-R-BOY, GEM-R-T24). Small RNA sequencing analyses were performed to search for miRNAs involved in gemcitabine resistance. The candidate miRNA was transduced into gemcitabine-resistant cells for functional analysis. Target genes of the candidate miRNA were subjected to RNA sequencing analysis, and functional analyses were performed by knockdown of target genes. RESULTS: Established gemcitabine-resistant cells showed more than 3-fold resistance to gemcitabine in IC50 compared to the parental cells in vitro, and also showed significant gemcitabine resistance in vivo. miR-99a-5p, selected as a candidate miRNA for gemcitabine resistance, was down-regulated compared to its parental cells. In gain-of-function studies, miR-99a-5p inhibited proliferation, migration and invasion both in parent and gemcitabine-resistant cells. In addition, miR-99a-5p restored the susceptibility to gemcitabine in gemcitabine-resistant cells. As a target of miR-99a-5p, we focused on SMARCD1 based on RNA sequence analysis. Dual luciferase reporter assays showed that miR-99a-5p directly regulated SMARCD1. Loss-of-function studies with si-RNAs revealed suppression of proliferative, migratory, and invasive abilities of cancer cells both in parent and gemcitabine-resistant cells, and restored their sensitivity to gemcitabine in gemcitabine-resistant cells. Furthermore, β-galactosidase staining showed that miR-99a-5p induction and SMARCD1 suppression contributed to cellular senescence through p21 induction. CONCLUSIONS: We found that miR-99a-5p overexpression or SMARCD1 down regulation induced cellular senescence with recovery of gemcitabine sensitivity. The identification of miR-99a-5p and SMARCD1 as key molecules to overcome gemcitabine resistance may lead to a better understanding of BC and the development of new therapeutic strategies to treat this disease. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e189 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Junya Arima More articles by this author Hirofumi Yoshino More articles by this author Ichiro Kawahara More articles by this author Wataru Fukumoto More articles by this author Issei Kawakami More articles by this author Motoki Tamai More articles by this author Shuichi Tatarano More articles by this author Hideki Enokida More articles by this author Expand All Advertisement PDF downloadLoading ...

Silencing of heat shock protein 90 (hsp90): Effect on development and infectivity of Ichthyophthirius multifiliis

BackgroundRecently, an increasing number of ichthyophthiriasis outbreaks has been reported, leading to high economic losses in fisheries and aquaculture. Although several strategies, including chemotherapeutics and immunoprophylaxis, have been implemented to control the parasite, no effective method is available. Hence, it is crucial to discover novel drug targets and vaccine candidates against Ichthyophthirius multifiliis. For this reason, understanding the parasite stage biology, host–pathogen interactions, molecular factors, regulation of major aspects during the invasion, and signaling pathways of the parasite can promote further prospects for disease management. Unfortunately, functional studies have been hampered in this ciliate due to the lack of robust methods for efficient nucleic acid delivery and genetic manipulation. In the current study, we used antisense technology to investigate the effects of targeted gene knockdown on the development and infectivity of I. multifiliis. Antisense oligonucleotides (ASOs) and their gold nanoconjugates were used to silence the heat shock protein 90 (hsp90) of I. multifiliis. Parasite stages were monitored for motility and development. In addition, the ability of the treated parasites to infect fish and cause disease was evaluated.ResultsWe demonstrated that ASOs were rapidly internalized by I. multifiliis and distributed diffusely throughout the cytosol. Knocking down of I. multifiliis hsp90 dramatically limited the growth and development of the parasite. In vivo exposure of common carp (Cyprinus carpio) showed reduced infectivity of ASO-treated theronts compared with the control group. No mortalities were recorded in the fish groups exposed to theronts pre-treated with ASOs compared with the 100% mortality observed in the non-treated control fish.ConclusionThis study presents a gene regulation approach for investigating gene function in I. multifiliis in vitro. In addition, we provide genetic evidence for the crucial role of hsp90 in the growth and development of the parasite, suggesting hsp90 as a novel therapeutic target for successful disease management. Further, this study introduces a useful tool and provides a significant contribution to the assessing and understanding of gene function in I. multifiliis.

Bacterial cellulose: A highly versatile nanomaterial.

Bacterial cellulose: A highly versatile nanomaterial.

An evaluation of the role of miR-361-5p in senescence and systemic ageing

Senescent cells are key regulators of ageing and age-associated disease. MicroRNAs (miRs) are a key component of the molecular machinery governing cellular senescence, with several known to regulate important genes associated with this process.We sought to identify miRs associated with both senescence and reversal by pinpointing those showing opposing directionality of effect in senescence and in response to senotherapy. Cellular senescence phenotypes were assessed in primary human endothelial cells following targeted manipulation of emergent miRNAs. Finally, the effect of conserved target gene knockdown on lifespan and healthspan was assessed in a C. elegans system in vivo.Three miRNAs (miR-5787, miR-3665 and miR-361-5p) demonstrated associations with both senescence and rejuvenation, but miR-361-5p alone demonstrated opposing effects in senescence and rescue. Treatment of late passage human endothelial cells with a miR-361-5p mimic caused a 14 % decrease in the senescent load of the culture. RNAi gene knockdown of conserved miR-361-5p target genes in a C. elegans model however resulted in adverse effects on healthspan and/or lifespan.Although miR-361-5p may attenuate aspects of the senescence phenotype in human primary endothelial cells, many of its validated target genes also play essential roles in the regulation or formation of the cytoskeletal network, or its interaction with the extracellular matrix. These processes are essential for cell survival and cell function. Targeting miR-361-5p alone may not represent a promising target for future senotherapy; more sophisticated approaches to attenuate its interaction with specific targets without roles in essential cell processes would be required.

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.

A robust, flow-based, microfluidic device for siRNA-mediated gene knockdown in glioblastoma spheroids

Glioblastoma (GBM) is a deadly disease with a poor prognosis, there is therefore a crucial need for novel therapeutic targets. Current preclinical models of GBM fail to predict clinical outcomes, thus, new translationally relevant models are urgently needed for reliable therapeutic target validation. 3D spheroid culture of cancer cells has been shown to better reflect tumour biology than 2D monolayer culture, as has culturing cells in flow-based microfluidic devices, which mimic key aspects of the tumour microenvironment. Gene knockdown by siRNA is a key preclinical target validation tool, however, siRNA-mediated knockdown of cancer spheroids in microfluidic culture has not yet been demonstrated. Here we describe a simple and robust microfluidic device that can maintain GBM spheroids (U87 cells) for at least 7 days. Via RNA sequencing analysis, we demonstrate that spheroids grown in microfluidic culture are more proliferative than spheroids grown in static plate culture and downregulate genes associated with cell adhesion, potentially offering insights into the metastatic process. Comparison of target gene (PRMT2 and RAB21) knockdown using siRNA between 2D monolayer cultured cells, static spheroid culture and spheroids maintained in the microfluidic device showed that gene expression (as measured by quantitative-PCR) was significantly reduced in all culture systems. Knockdown was most efficient in cells grown in 2D monolayer culture followed by static spheroid culture, but we also demonstrate [Formula: see text] knockdown efficiency using the microfluidic device. In summary, this study describes an easy-to-use microfluidic culture platform and provides evidence that pre-clinical siRNA-mediated target validation studies will be possible in flow systems that mimic tumour physiology.

Comprehensive Analyses of the Intracellular and in Vivo Disposition of Fab- Small Interfering RNA Conjugate to Identify Key Issues to Improve Its in Vivo Activity.

Comprehensive analyses of intracellular disposition and in vivo pharmacokinetics were performed for small interfering RNA (siRNA) conjugated with the Fab fragment of panitumumab, a fully humanized monoclonal antibody against epidermal growth factor receptor (EGFR). The Fab-siRNA conjugate was internalized into EGFR-expressing cancer cells in an antigen-dependent manner. Intracellular disposition was quantitatively evaluated using fluorescent-labeled panitumumab and confocal microscopy. The majority of internalized panitumumab was suggested to be transferred into lysosomes. In vivo pharmacokinetics were evaluated in EGFR-expressing tumor-bearing mice. Intact Fab-siRNA was measured by immunoprecipitation using anti-Fab antibody followed by quantitative polymerase chain reaction. The Fab portion was measured by a ligand binding assay. Intact Fab-siRNA concentrations rapidly decreased in the plasma and tumor, although the Fab portion concentration remained high, suggesting extensive degradation in the linker-siRNA portion. After incubation of Fab-siRNA in mouse plasma, samples were digested with proteinase K, and extracted siRNA tagged with Fab-derived peptide was subjected to an ion-pair reversed-phase liquid chromatography with mass spectrometry analysis. Results suggested that hydrolysis from the 3' end of the antisense strand of siRNA is the major metabolizing pathway. Based on these findings, endosomal escape and stability in lysosomes, blood, and tumor are key factors to improve to achieve efficient target gene knockdown in tumors, and stabilizing the 3' end of the antisense strand was suggested to be most efficient. Our approaches clearly identified the key issues of Fab-siRNA from a pharmacokinetics aspect, which will be useful for improving the in vivo activity of siRNA conjugated with not only Fab but also other immunoproteins. SIGNIFICANCE STATEMENT: The intracellular and in vivo disposition of Fab-small interfering RNA (siRNA) conjugate was comprehensively investigated using various approaches, including newly developed analytical methods. This study clearly shows that improvements in siRNA stability in lysosomes, blood, and tumor are needed for target gene knockdown in tumors. The major metabolic pathway of Fab-siRNA is 3' exonuclease degradation, suggesting that optimization of the conjugation site to Fab might help improve stability.

Comparison of tyrosine-modified low molecular weight branched and linear polyethylenimines for siRNA delivery

Polyethylenimines (PEIs) have been previously introduced for siRNA delivery. In particular, in the case of higher molecular weight PEIs, this is associated with toxicity, while low molecular weight PEIs are often insufficient for siRNA complexation. The tyrosine-modification of PEIs has been shown to enhance PEI efficacy and biocompatibility. This paper evaluates a set of tyrosine-modified low molecular weight linear or branched polyethylenimines as efficient carriers of siRNA. Complexation efficacies and biophysical complex properties were analyzed by zeta potential, dynamic light scattering and circular dichroism measurements as well as gel electrophoresis. Biological knockdown was studied in 2 D cell culture and 3 D ex vivo tissue slice air-liquid interface culture. The results demonstrate that siRNAs were able to form stable complexes with all tested polymers. Complexation was able to protect siRNA from degradation by RNase and to mediate target gene knockdown, as determined on the mRNA level and in PC3-Luc3/EGFP and HCT116-Luc3/EGFP expressing reporter cells on the protein level, using flow cytometry and confocal microscopy. The direct comparison of the studied polymers revealed differences in biological efficacies. Moreover, the tyrosine-modified PEIs showed high biocompatibility, as determined by LDH release and mitochondria integrity (J-aggregate assay) as well as caspase 3/7 (apoptosis) and H2O2 levels (ROS). In 3 D tissue slices, complexes based on LP10Y proved to be most efficient, by combining tissue penetration with efficient gene expression knockdown.

Discovery of a Novel Driver of Radioresistance in Pancreatic Cancer (PC) Using Genome-Wide RNA-Seq

<h3>Purpose/Objective(s)</h3> Discovery of genetic drivers of radioresistance in PC is critical for developing novel therapeutic strategies to combine with radiation in this radioresistant disease. In this study, we used genome-wide RNA-seq to identify genes upregulated in radioresistant (RR) generated PC cell lines and discovered the Inhibitor of DNA Binding 1 (ID1) gene as a promising radiosensitizing target. <h3>Materials/Methods</h3> RR PC cell lines MIA PaCa-2 and PANC-1 were generated by delivering daily ionizing irradiation (IR) (2 Gy/day) <i>in vitro</i> over two weeks (total 20 Gy) followed by standard clonogenic assays to calculate the dose enhancement ratio (DER) following one week from the end of IR. Parental and RR cell lines were submitted for RNA-seq analysis to identify differentially expressed genes. The Limma R package was used to calculate differential expression among genes. Log2 fold change values were calculated for each sample compared to control. Genes with an absolute fold change > 1 were considered significant. Knockdown of target genes via small interfering RNA (siRNA) was confirmed through immunoblotting. Luciferase expressing RR PANC-1 and MIA PaCa-2 with or without shRNA stable knockdown of ID1 were implanted orthotopically in the pancreas of athymic nude mice, and tumors were treated with external beam radiation using a Small Animal Radiation Research Platform to a dose of 20 Gy in 5 daily fractions. Tumor growth was measured weekly via bioluminescence. <h3>Results</h3> Following exposure to two weeks of 2 Gy daily IR <i>in vitro</i>, the two PC cell lines showed significantly greater clonogenic cell survival than parental cell lines (DER 0.4 and 0.5), indicating enhanced RR in these cells. RNA-seq analysis comparing parental and RR cell lines found upregulated seven genes (TNS4, ZDHHC8P1, APLNR, AQP3, SPP1, ID1, ID2) and seven genes downregulated (PTX3, ITGB2, EPS8L1, ALDH1L2, KCNT2, ARHGAP9, IFI16) in both RR cell lines. Western blotting confirmed either increased or decreased expression of these 14 genes in the RR cell lines compared to their parental. siRNA knockdown of each of the seven most significant upregulated genes in the RR MIA PaCa-2 and PANC-1 cells revealed ID1 as the most potent radiosensitizer in the RR cell lines (DER 1.5 and 1.6, respectively). ID1 protein expression was higher in 6 different human PC cell lines than both non-malignant pancreatic and intestinal cell lines. Orthotopic RR tumors with stable knockdown of ID1 showed significantly decreased tumor growth following <i>in vivo IR</i> than the wild-type ID1 tumors (2.5-fold decrease, p<0.001). After the last dose of IR, expression of the DNA damage marker yH2aX in tumors was significantly higher in the ID1 knockdown tumors compared to controls indicating reduced DNA repair in tumors deficient in ID1. <h3>Conclusion</h3> Our analysis indicates a novel role of ID1 in PC radioresistance. Targeting ID1 prior to IR is a promising strategy to overcome the RR of PC, and pre-clinical studies using ID1 inhibitors are ongoing.