siRNA Targeting Research Articles

SiRNAEfficacyDB: An experimentally supported small interfering RNA efficacy database.

Small interfering RNA (siRNA) has revolutionised biomedical research and drug development through precise post-transcriptional gene silencing technology. Despite its immense potential, siRNA therapy still faces technical challenges, such as delivery efficiency, targeting specificity, and molecular stability. To address these challenges and facilitate siRNA drug development, we have developed siRNAEfficacyDB, a comprehensive database that integrates experimentally validated siRNA efficacy data. This database contains 3544 siRNA records, covering 42 target genes and 5 cell lines. It provides detailed information on siRNA sequences, target genes, inhibition efficiencies, experimental techniques, cell lines, siRNA concentrations, and incubation times. siRNAEfficacyDB offers a user-friendly web interface that makes it easy to query, browse and analyse data, enabling efficient access to siRNA-related information. In summary, siRNAEfficacyDB provides a useful data foundation for siRNA drug design and optimisation, serving as a valuable resource for advancing computer-aided siRNA design research and nucleic acid drug development. siRNAEfficacyDB is freely available at https://cellknowledge.com.cn/siRNAEfficacy for non-commercial use.

PLAC1 augments the malignant phenotype of cervical cancer through the mTOR/HIF-1α/snail signaling pathway.

This study investigated the molecular mechanisms of placenta-specific protein 1 (PLAC1) in cervical cancer (CCa), aiming to elucidate its role in tumorigenesis through in vitro and in vivo experiments. CCa cell lines with overexpressed or silenced PLAC1 were established to evaluate its impact on cell cycle, apoptosis and the expression of key proteins in the PLAC1/mTOR/HIF-1α/Snail signaling pathways. Functional assays were conducted to assess the influence of the PLAC1/mTOR/HIF-1α/Snail regulatory pathway on cell proliferation, migration and invasion. The role of the mTOR signaling pathway in PLAC1-mediated modulation of CCa characteristics was validated using a mTOR activator (MHY1485) and a mTOR inhibitor (Rapamycin) respectively. HIF1A siRNA was introduced to confirm the role of HIF1A. Furthermore, an in vivo nude mouse model was constructed to confirm PLAC1's influence on tumorigenesis and metastasis in CCa. PLAC1 upregulated hypoxia-inducible factor (HIF)-1α and Snail, promoting CCa cell proliferation, migration, and invasion via the mTOR/HIF-1α/Snail pathway. Enrichment analysis of PLAC1-associated differentially expressed genes implicated their involvement in CCa and tumor promotion. In a xenograft mouse model, PLAC1 exhibited a pro-tumorigenic effect, which can be reversed by siRNA targeting HIF1A. This study enhances our understanding of PLAC1's role and molecular mechanisms in CCa progression, highlighting its potential as a diagnostic, prognostic, and therapeutic marker for the management of CCa.

Abstract A001: HNRNPH1 regulates the alternative splicing of transcripts expressed by cancer-associated genes

Abstract The RNA binding protein HNRNPH1 is a critical regulator of alternative splicing (AS). We have previously demonstrated that HNRNPH1 mediates an exon exclusion event required for the expression of the mRNA encoding the fusion oncoprotein expressed in a subset (∼37%) of Ewing sarcomas. Furthermore, studies by others have shown that HNRNPH1 regulates a mutually exclusive exon (MXE) AS event (exon-18a versus exon-18b) that defines the expression of TCF3 transcript variants, which, when dysregulated, promotes tumorigenesis in Burkitt’s lymphoma. In this study, we have interrogated the transcriptome-wide effects of depleting HNRNPH1 by RNAi to further explore its regulation of transcripts expressed by cancer-associated genes. We performed whole transcriptome RNA sequencing (RNAseq; short-read, Illumina; long-read, PacBio IsoSeq) of two cell lines (HEK-293T and HT-1080) post 48-hour transfection with a control siRNA (siNeg) or an siRNA targeting HNRNPH1 (siHNRNPH1). We then assembled a workflow to quantify changes in individual AS events (percent-spliced-in, ΔPSI=±0.1) using rMATS and MAJIQ, and differential transcript expression (DTE) using RSEM (pValue≤0.05) and EBSeq (FDR≤0.05). RNAseq analyses were validated using PCR assays. The rMATS analysis revealed skipped exons (SE) as the most frequent event effected in HEK-293T and HT-1080 cells following HNRNPH1 silencing, with 1069 SE or MXE events from 607 genes common to both. Gene annotation (Metascape) of these 607 genes identified 49 as cancer-associated. Consistent with previous studies, we detected a significant change in the TCF3-exon-18a/18b MXE event using rMATS (decreased PSI for 18a) and MAJIQ (negative ΔPSI for 18a and positive ΔPSI for 18b) following HNRNPH1 silencing in both cell lines. Furthermore, DTE revealed a corresponding decrease in 18a-containing TCF3-transcripts and an increase in 18b-containing TCF3-transcripts, validating our analytical pipeline. Next, we extended our analysis to uncharacterized HNRNPH1-regulated AS events, beginning with examination of transcripts encoding the mitotic kinase AURKA. Previous studies have shown that AS of AURKA transcripts localizes to the untranslated regions (UTRs), particularly the 5’UTR. However, regulators of these AS events are unknown, and it is unclear how these UTR variants affect AURKA protein expression. Long-read RNAseq of AURKA mRNAs from both cell lines revealed extensive 5’UTR AS, and the rMATS and MAJIQ analyses showed decreased PSI and negative ΔPSI for a specific 5’UTR-exon following HNRNPH1 silencing. DTE revealed corresponding decreases in abundance of AURKA transcripts containing this exon (AURKA-203, -205, -209, and -213) upon HNRNPH1 depletion, which we confirmed with variant-specific RT-PCR. Furthermore, immunoblot analysis showed a decrease in AURKA protein expression following the silencing of HNRNPH1. Our assessment of AURKA AS suggests HNRNPH1 mediates the inclusion of a specific AURKA-5’UTR-exon, providing evidence for a previously uncharacterized post-transcriptional regulatory mechanism of AURKA expression. Citation Format: Tayvia Brownmiller, Patricio Pichling, Tamara L Jones, Ioannis Grammatikakis, Soumya Sundara Rajan, Erica C Pehrsson. HNRNPH1 regulates the alternative splicing of transcripts expressed by cancer-associated genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A001.

Abstract 4139141: Predicted efficacy of inclisiran on cardiovascular outcomes in lower extremity peripheral artery disease: results of the In Silico Sirius study

Introduction: Symptomatic lower extremity peripheral artery disease (PAD) patients have the highest risk of cardiovascular (CV) outcomes among patients with established atherosclerotic cardiovascular disease (ASCVD). Inclisiran, an siRNA targeting PCSK9 mRNA, reduces LDL-C levels. In SIRIUS in silico trial (NCT05974345), inclisiran was predicted to lower CV events in ASCVD patients. Research question/Hypothesis: SIRIUS in silico study aims to predict the efficacy of inclisiran on CV outcomes in subgroups of patients with or without PAD. Methods: The SIRIUS in silico trial was conducted using a knowledge-based mechanistic computational model of ASCVD applied to a virtual ASCVD population with LDL-C ≥ 70 mg/dL. Each virtual patient is its own control. This model was previously calibrated and validated before running the trial. SIRIUS compared the efficacy of inclisiran vs placebo on top of High Intensity (HI) statins with or without ezetimibe on 3-Point-MACE defined as a composite of time to first occurrence of CV death, nonfatal myocardial infarction (MI) or nonfatal ischemic stroke (IS) over 5 years. Occurrence of major acute limb events (MALE) was also individually assessed in time-to-first-event analyses. Results: Among 204, 691 virtual SIRIUS ASCVD patients, 28,072 (13.7%) had PAD. At 5 years, the mean predicted percentage reduction in LDL-C with inclisiran as compared to placebo was -49.3% and -49.8% in patients with or without PAD, respectively. The predicted rate of 3P-MACE in the inclisiran arm was consistently lower than in the placebo arm in patients with PAD (17.62% vs 22.88%; Hazard Ratio (HR): 0.75 medium uncertainty) and in patients without PAD (10.33% vs 13.64%; HR: 0.75 low uncertainty). Compared to placebo, inclisiran was also predicted to consistently reduce MALE in patients with or without PAD (2.71% vs 4.11%; HR: 0.65 medium uncertainty and 0.19% vs 0.29%; HR: 0.66 high uncertainty respectively). Conclusion: Pending the results of ORION-4 and VICTORION-2-P, this first In Silico trial in virtual PAD patients predicted a potential effect of inclisiran on 3P-MACE and MALE reductions over 5 years follow-up.

Abstract 4140151: Network Analysis of Differentially Expressed Genes after Silencing Dynamin 2 in Pulmonary Arterial Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is a rare cardiopulmonary disease that can cause right heart failure and even death. In PAH, excessive mitochondrial fission is responsible for the hyperproliferation of pulmonary artery smooth muscle cells (PASMC), which is incompletely mediated by the large GTPase dynamin-related protein 1 (Drp1). We hypothesize that another GTPase, Dynamin 2 (DNM2), elevated in PASMC of PAH patients, is responsible for the final stage of mitochondrial fission. Objectives: 1. Identify the differentially expressed gene (DEG) signature in PAH PASMC after silencing DNM2 using small interfering RNA (siRNA) by means of RNA sequencing (RNA-seq); 2. Identify the biological processes or pathways associated with these DEGs. Methods: Human PAH PASMCs (n=5 cell line) were transfected with control siRNA or siRNA targeting DNM2. 3’RNA-seq was used to create libraries which we sequenced on the Illumina Nextseq550. Bioinformatic analysis revealed DEGs which we placed into context using the STRING: protein query database and visualized using Cytoscape 3.10.2. Network functional enrichment analysis was performed using STRING. Results: Inhibiting DNM2 significantly reduced mitochondrial fission and slowed PAH PASMC proliferation. RNA-seq identified 156 DEGs (80 down-regulated, 56 up-regulated). The top 5 down-regulated DEGs were DNM2 , RGCC , TICAM1 , RASSF3 , MRPL39 and the top 5 up-regulated DEGs were ITIF1 , ARL6IP1 , POLR3G , CBX1 , CMPK2 . We tested the biologic relevance of RGCC which encodes the Regulator of Cell Cycle using siRNA and showed inhibition of cell proliferation in human PAH PASMC. Network analysis identified 152 edges amongst all DEGs. The largest subnetwork contains 77 nodes and 151 edges. PXDN , which encodes Peroxidasin, a heme-containing peroxidase that is secreted into the extracellular matrix, has the highest degree of connectivity (15 connections), the highest betweenness centrality (0.266), and the highest closeness centrality (0.441). Enrichment analysis found that the p53 signaling pathway and the cytokine signaling in the immune system are the top KEGG and Reactome pathways affected by siDNM2, respectively. Conclusions: DNM2 appears to be an important regulator of the increased mitochondrial fission in PAH. Silencing DNM2 not only inhibits fission but also slows cell proliferation, apparently by reducing the expression of RGCC. Silencing DNM2 in human PAH PASMC results in the disruption of 136 genes.

TMIC-79. IDH1-R132H REWIRES AUTOPHAGY IN THE GLIOMA MICROENVIRONMENT TO FACILITATE TUMOR PROGRESSION AND RESPONSE TO RADIATION

Abstract Gliomas are primary brain tumors with a poor prognosis. IDH1R132H exhibits a gain of function mutation leading to 2-hydroxyglutarate (2HG) production. We previously demonstrated, 2-HG mediated epigenetic rewiring is associated with better prognosis and its presence impacts several cellular functions including immune responses and enhanced DNA-damage response in IDH1R132H gliomas harboring p53 and ATRX loss-of-function mutations. Also, IDH1R132H elicits metabolic reprogramming which implies that IDH1R132H could modify the energetic state of gliomas with consequences in tumor biology and therapeutic responses. In this study, RNA-seq and ChIP-seq data revealed human and mouse IDH1R132H gliomas have downmodulated gene ontologies related to mitochondrial metabolism. This was paralleled by a decrease in glycolysis, rendering autophagy as a source of energy for IDH1R132H glioma cells. We also found that IDH1R132H-neurospheres have decreased levels of oxygen consumption and extracellular acidification rates, characteristic of an autophagic/quiescent state when compared to IDH1WT-neurospheres. Analysis of the autophagy pathway revealed that both human and mouse IDH1R132H gliomas have increased expression of pULK1-S555, pATG4b-S383, enhanced conversion of LC3I to LC3II, and decreased expression of pULK1-S757, p62 indicating that IDH1R132H cells have augmented autophagy activity compared to IDH1WT cells. Blocking autophagy selectively impairs the growth of IDH1R132H glioma cells in vitro. Targeting autophagy by systemic administration of synthetic protein nanoparticles packaged with siRNA targeting Atg7 sensitized IDH1R132H glioma cells to radiation-induced cell death, resulting in tumor regression, long-term survival, and immunological memory when used in combination with irradiation. Our results thus indicate that the metabolic changes in IDH1R132H glioma cells lead to compromised mitochondrial functions and increased autophagic activity which contributes to their radioresistance, representing a novel therapeutic target for IDH1R132H cells. *Corresponding author: Maria G. Castro: mariacas@med.umich.edu Equal contributions: KB-FN-AM-AM-CT Funding: This work was supported by National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) Grants: [R37-NS094804, R01-NS122165, R21-NS123879-01 awarded to MGC]; [R01-NS122378, R01-NS122234 awarded to PRL], Ian’s Friends & Chad Tough Foundation.

Clinical Significance of HHLA2 as a Novel Therapeutic Target for Colorectal Cancer.

Colorectal cancer (CRC) is a high-indence malignance of the digestive system with a high mortality rate in the world. The results are desired to provide an important theoretical basis for discovering new therapeutic targets for CRC. The expression of human endogenous retrovirus-H-long terminal repeat association protein 2 (HHLA2) in human CRC was detected to explore its correlationship with clinicopathological features and prognosis of patients and its potential in treating CRC. Western blot was employed to detect HHLA2 expression in fresh tissues obtained from 6 CRC patients' excisions, including cancer, paracancer, and normal issues. Immunohistochemical staining was employed to determine HHLA2 expression in paraffin-embedded specimens of 139 patients with colorectal cancer, and its relationship with the clinicopathological profiles and survival was analyzed. Small interfering RNA (siRNA) targeting HHLA2 was used to transfect CRC cells to silent HHLA2. MTT, plate colony formation, cell scratch, and Transwell assay were conducted to observe the proliferation, migration, and invasion of CRC cells. HHLA2 protein was expressed in human colorectal cancer tissues, paracancer tissues and normal tissues, which was significantly upregulated in cancer tissues (P<0.01). HHLA2 expression level in CRC tissues showed a close correlationship with the invasion depth of the tumor (P=0.000), metastasis of regional lymph nodes (P=0.018), clinical stage (P=0.010), and patient survival (P=0.011). Correlation with gender (P=0.873), age (P=0.864), location of the tumor (P=0.768), degree of tumor differentiation (P=0.569) and distant metastasis (P=0.494) exhibited no significance. The survival time of CRC patients with high and low HHLA2 expression groups was 43.231 months and 55.649 months, respectively, with a statistical difference between the two groups (P=0.001). Silencing HHLA2 inhibited proliferation, migration and invasion of CRC cells significantly. HHLA2 is overexpressed in CRC tissues which is associated with poor prognosis of patients. HHLA2 might be recognized as a new candidate for adjuvant diagnosis and prognosis of CRC, as well as a promised new target for immunotherapy of CRC.

Berberine safeguards sepsis-triggered acute gastric damage and inhibits pyroptosis in gastric epithelial cells via suppressing the ubiquitination and degradation of Nrf2.

Berberine (BBR), a widely recognized traditional Chinese medicine, has attracted considerable attention for its promising anti-inflammatory effects. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) effectively safeguards against organ damage stemming from sepsis-induced oxidative stress and inflammatory responses. This study examined the potential of BBR in alleviating sepsis-induced acute gastric injury, with a particular focus on elucidating whether its mechanism of action involves the activation of the Nrf2 signaling pathway. Following intraperitoneal injection of BBR, mice were subjected to the cecal ligation and puncture (CLP) method to induce sepsis. In vitro experiments involved pre-treating the normal gastric epithelial cells (GES-1) with BBR, followed by treatment with lipopolysaccharide (LPS). Functional assays were then performed to assess cell proliferation and apoptosis. To validate the role of Nrf2 in pyroptosis and inflammation, siRNA targeting Nrf2 (si-Nrf2) was transfected into LPS-treated GES-1 cells. Additionally, mice were administered the Nrf2 inhibitor ML385 to confirm the protective effects of BBR in vivo. BBR displayed a dose-dependent effect in mitigating gastric tissue damage, suppressing the release of inflammatory cytokines, and reducing the expression of NLRP3, ASC, and GSDMD-N. In vitro, BBR fostered GES-1 cell proliferation, hindered apoptosis, and suppressed the levels of TNF-α, IL-18, IL-1β, NLRP3, ASC, and GSDMD-N. Further analysis revealed that knocking down Nrf2 reversed BBR's inhibitory effect on pyroptosis in LPS-treated GES-1 cells. Through binding to Keap1, BBR efficiently prevented the ubiquitination and degradation of Nrf2, ultimately promoting its nuclear translocation. In vivo experiments confirmed that ML385 reversed the protective effect of BBR on pyroptosis and inflammation. Our research reveals that BBR interacts with Keap1 to activate the Keap1/Nrf2 signaling pathway in gastric epithelial cells, thereby suppressing pyroptosis and inflammation in sepsis-induced acute gastric injury.

Prolonged glutamine starvation reactivates mTOR to inhibit autophagy and initiate autophagic lysosome reformation to maintain cell viability.

Autophagy, a cellular recycling mechanism, utilizes lysosomes for cellular degradation. Prolonged autophagy reduces the pool of functional lysosomes in the cell. However, lysosomal homeostasis is maintained through the regeneration of functional lysosomes during the terminal stage of autophagy, i.e. Autophagic lysosome reformation (ALR). Through confocal microscopy during glutamine starvation, we unravel the regeneration of tubules from autolysosomes by undertaking significant membrane remodeling, which majorly depends on mTOR reactivation, RAB7 dissociation, phosphatidyl inositol 3 phosphate (PI3P) dependent dynamin 2 and clathrin recruitment. In glutamine-starved cells, we found mTOR is the central modulator in regulating ALR initiation, and its pharmacological inhibition with rapamycin leads to a decrease in lysosomal tubulation. Moreover, RAB7 and Clathrin are essential for tubule elongation and it showed that siRNA targeting RAB7 and Clathrin restricts tubule initiation under glutamine starvation. In this setting, we examined the physiological relevance of ALR during prolonged glutamine deprivation and found that genetic and pharmacological inhibition of critical proteins involved in ALR promotes cell death in oral cancer cells, establishing ALR is essential for maintaining cell survival during stress.

Double Braking Effects of Nanomedicine on Mitochondrial Permeability Transition Pore for Treating Idiopathic Pulmonary Fibrosis.

Mitochondrial permeability transition pore (mPTP) opening is a key hallmark of injured type II alveolar epithelial cells (AECIIs) in idiopathic pulmonary fibrosis (IPF). Inhibiting mPTP opening in AECIIs is considered a potential IPF treatment. Herein, a "double braking" strategy on mPTP by cyclosporin A (CsA) derived ionizable lipid with 3D structure (3D-lipid) binding cyclophilin D (CypD) and siRNA downregulating mitochondrial calcium uniporter (MCU) expression is proposed for treating IPF. 3D-lipid and MCU targeting siRNA (siMCU) are co-assembled to form stable 3D-LNP/siMCU nanoparticles (NPs), along with helper lipids. In vitro results demonstrated that these NPs effectively inhibit mPTP opening by 3D-lipid binding with CypD and siRNA downregulating MCU expression, thereby decreasing damage-associated molecular patterns (DAMPs) release and suppressing epithelial-to-mesenchymal transition (EMT) process in bleomycin-induced A549 cells. In vivo results revealed that 3D-LNP/siMCU NPs effectively ameliorated collagen deposition, pro-fibrotic factors secretion, and fibroblast activation in bleomycin-induced pulmonary fibrosis (PF) mouse models. Moreover, compared to the commercial MC3-based formulation, optimized Opt-MC3/siRNA NPs with incorporating 3D-lipid as the fifth component, showed superior therapeutic efficacy against PF due to their enhanced stability and higher gene silencing efficiency. Overall, the nanomedicine containing 3D-lipid and siMCU will be a promising and potential approach for IPF treatment.

Nanocarriers Targeting Circular RNA ADARB1 Boost Radiosensitivity of Nasopharyngeal Carcinoma through Synergically Promoting Ferroptosis.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor of the head and neck, prevalent in regions such as Southern China and Southeast Asia. Radiotherapy serves as the primary clinical treatment for this carcinoma. However, resistance to radiotherapy is a fundamental cause of treatment failure and patient mortality, with the underlying mechanisms yet to be fully elucidated. We identified a recently characterized circular RNA, circADARB1, which is markedly upregulated in NPC tissues and closely associated with poor prognosis and radiotherapy resistance. Both in vitro and in vivo experiments demonstrated that circADARB1 inhibited ferroptosis, thereby inducing radiotherapy resistance in NPC cells. Building on these findings, we synthesized a biomimetic nanomaterial consisting of semiconducting polymer nanoparticles wrapped in cell membranes, designed to deliver both siRNA targeting circADARB1 and iron ions. The application of this nanomaterial not only efficiently suppressed the expression of circADARB1 and boosted intracellular iron concentrations, but also enhanced ferroptosis induced by radiotherapy, improving the radiosensitivity of NPC cells. Furthermore, our study revealed that circADARB1 upregulated the expression of heat shock protein HSP90B1, which repaired misfolded SLC7A11 and GPX4 proteins triggered by radiotherapy, thereby preserving their stability and biological functions. Mechanistically, SLC7A11 facilitated cysteine transportation into cells and glutathione synthesis, while GPX4 employed glutathione to mitigate intracellular lipid peroxidation induced by radiotherapy, shielding cells from oxidative damage and inhibiting ferroptosis, and ultimately leading to radiotherapy resistance in NPC cells. Our investigation elucidates molecular mechanisms with substantial clinical relevance, highlights the promising application prospects of nanotechnology in precision cancer therapy.

The development of small-molecule ERBB4 agonists as a treatment for heart failure

Abstract The neuregulin-1 (NRG1)/ERBB4 signaling pathway has emerged as a cardioprotective pathway and is a promising target for the treatment of chronic heart failure. Activation of ERBB4 signaling is known to decrease cardiomyocyte cell death and hypertrophy, and fibroblast collagen synthesis. Recombinant NRG1 (rNRG1) is currently tested in phase III clinical trials for heart failure, but its need for intravenous administration is a disadvantage. In an attempt to circumvent this, we hypothesized that small-molecule-induced activation of ERBB4 is feasible and would recapitulate the effects of its natural ligand on myocytes and fibroblasts. To this end, we screened 10,240 compounds for their ability to induce homodimerization of ERBB4. We identified a series of 8 similar compounds (named EF-1 – EF-8) that concentration-dependently induced ERBB4 dimerization, with EF-1 being the most potent and effective compound (n = 4-5 independent repeats in each group; Emax = 27.9 ± 4.8% relative to NRG1, EC50 = 10.5 ± 4.5 x 10-6). EF-1 showed neither cytotoxicity nor increased cell proliferation of tumor cell lines. In vitro, EF-1 significantly decreased in a concentration-dependent manner hydrogen peroxide–induced cardiomyocyte cell death (n = 4 independent repeats in each group; P&amp;lt;0.0001 compared to siERBB4, Fig. 1A), angiotensin-II (AngII)-induced cardiomyocyte hypertrophy (n = 20 individual cardiomyocytes in each group; P&amp;lt;0.0001 compared to AngII/vehicle, Fig. 1B), and collagen expression in cultured human fibroblasts (n = 3 independent repeats in each group; P=0.03 compared to siERBB4, Fig. 1C). The observed effects in cultured cardiomyocytes and fibroblasts could be abrogated by siRNA targeting ERBB4, indicating that they were mediated by ERBB4. Moreover, when used in vivo, EF-1 (2 mg/kg/day) significantly decreased AngII-induced left ventricular Col1a1 and Col3a1 mRNA expression (n = 4-5 mice in each group; P=0.02 and P=0.004 compared to AngII/vehicle, respectively) and inhibited AngII-induced myocardial total and interstitial fibrosis in wild-type mice (n = 4-5 mice in each group, Fig. 2), but not in Erbb4-null mice. Moreover, EF-1 decreased acute cardiotoxicity (assessed by troponin release) in wild-type mice treated with doxorubicin (DOX; n = 8-9 mice in each group; P&amp;lt;0.0001 compared to DOX/vehicle), but not in Erbb4-null mice. In conclusion, we show that small-molecule-induced ERBB4 dimerization and activation is feasible, and recapitulates anti-fibrotic and cardiomyocyte protective effects in the heart in an ERBB4-dependent manner, both in vitro and in vivo. This could be the start for the further development of small-molecule ERBB4 agonists as a novel class of drugs to treat heart failure. Cardiomyocyte-protective effect in vitroAnti-fibrotic effect in vivo

SMAD3 silencing induces Epithelial-to-Mesenchymal Transition (EMT) towards cardiac pericyte progenitors with proangiogenic potential in hiPSC-derived epicardial cells

Abstract Background The epicardium is only a single mesothelial layer and "quiescent" in the adult heart. However, once the heart is injured, the epicardium reactivates and acts as a key for cardiac regeneration through epithelium-to-mesenchymal transition (EMT). While the canonical TGF-β-SMAD pathway plays a central role in regulating EMT, SMAD3 functions independent of this pathway in epicardium remain unknown. Purpose The purpose of this study is to investigate the effects of SMAD3 downregulation on EMT in epicardial cells. Methods First, we differentiated human iPSC-derived epicardial cells (EPI cells) by mimicking the process of cardiac mesoderm development. Next, siRNA targeting SMAD3 was transfected into EPI cells to silence SMAD3. We performed qRT-PCR, western blotting, immunocytochemistry, flow cytometry, transcriptional analysis, endothelial tube formation assay, and paracrine experiments using FUCCI (fluorescent ubiquitination-based cell cycle indicator) system to evaluate the phenotype of EPI cells with silenced SMAD3. Results We found that SMAD3 silencing in EPI cells resulted in loss of epicardial identity and upregulation of multiple EMT markers. Additionally, NG2 and ENG (CD105), both cardiac pericyte markers, were highly expressed in SMAD3-silenced EPI cells. RNA-sequence also revealed that SMAD3 silencing in EPI cells induced EMT facilitated towards cardiac pericyte progenitors with proangiogenic potential. In endothelial tube formation assay, SMAD3-silenced EPI cells demonstrated enhanced tube formation compared to the control (1.48-fold increase in the number of junctions, n=3; p&amp;lt;0.0001), which indicated their angiogenic potential. At last, we revealed the paracrine effect of SMAD3-silenced EPI cells to enhance the proliferative reactivation in iPSC-derived cardiomyocytes by showing upregulation in CDK6/CCND1 axis and FUCCI-green. Conclusion Our findings demonstrate a new role of SMAD3 biology in the human epicardium, specifically in the generation of cardiac pericyte progenitor cells that contribute to better responses in angiogenesis of the heart microvasculature enabling the possibility to continue exploiting epicardial biology for effective cardiac regeneration.

Decoding Cancer through Silencing the Mitochondrial Gatekeeper VDAC1.

Mitochondria serve as central hubs for regulating numerous cellular processes that include metabolism, apoptosis, cell cycle progression, proliferation, differentiation, epigenetics, immune signaling, and aging. The voltage-dependent anion channel 1 (VDAC1) functions as a crucial mitochondrial gatekeeper, controlling the flow of ions, such as Ca2+, nucleotides, and metabolites across the outer mitochondrial membrane, and is also integral to mitochondria-mediated apoptosis. VDAC1 functions in regulating ATP production, Ca2+ homeostasis, and apoptosis, which are essential for maintaining mitochondrial function and overall cellular health. Most cancer cells undergo metabolic reprogramming, often referred to as the "Warburg effect", supplying tumors with energy and precursors for the biosynthesis of nucleic acids, phospholipids, fatty acids, cholesterol, and porphyrins. Given its multifunctional nature and overexpression in many cancers, VDAC1 presents an attractive target for therapeutic intervention. Our research has demonstrated that silencing VDAC1 expression using specific siRNA in various tumor types leads to a metabolic rewiring of the malignant cancer phenotype. This results in a reversal of oncogenic properties that include reduced tumor growth, invasiveness, stemness, epithelial-mesenchymal transition. Additionally, VDAC1 depletion alters the tumor microenvironment by reducing angiogenesis and modifying the expression of extracellular matrix- and structure-related genes, such as collagens and glycoproteins. Furthermore, VDAC1 depletion affects several epigenetic-related enzymes and substrates, including the acetylation-related enzymes SIRT1, SIRT6, and HDAC2, which in turn modify the acetylation and methylation profiles of histone 3 and histone 4. These epigenetic changes can explain the altered expression levels of approximately 4000 genes that are associated with reversing cancer cells oncogenic properties. Given VDAC1's critical role in regulating metabolic and energy processes, targeting it offers a promising strategy for anti-cancer therapy. We also highlight the role of VDAC1 expression in various disease pathologies, including cardiovascular, neurodegenerative, and viral and bacterial infections, as explored through siRNA targeting VDAC1. Thus, this review underscores the potential of targeting VDAC1 as a strategy for addressing high-energy-demand cancers. By thoroughly understanding VDAC1's diverse roles in metabolism, energy regulation, mitochondrial functions, and other cellular processes, silencing VDAC1 emerges as a novel and strategic approach to combat cancer.

GR113808, a serotonin receptor 4 antagonist, prevents high-fat-diet-induced obesity, fatty liver formation, and insulin resistance in C57BL/6J mice

BackgroundThe burden of nonalcoholic fatty liver disease is increasing, and limited therapeutic drugs are available for its treatment. Serotonin binds to approximately 14 serotonin receptors (HTR) and plays diverse roles in obesity and metabolic complications. In this study, we focused on the function of HTR4 on nonalcoholic fatty liver disease using GR113808, a selective HTR4 antagonist.MethodsMale C57BL/6J mice were fed high-fat diet for 12 weeks with intraperitoneal GR113808 injection, and HTR expression, weight changes, glucose and lipid metabolism, hepatic fat accumulation, changes in adipose tissue, the changes in transcriptional factors of signaling pathways, and inflammations were assessed. Hep3B cells and 3T3-L1 cells were treated with siRNA targeting HTR4 to downregulate its expression and then cultured with palmitate to mimic a high-fat diet. The changes in transcriptional factors of signaling pathways, and inflammations were assessed in those cells.ResultsAfter feeding a high-fat diet to male C57BL/6J mice, HTR4 expression in the liver and adipose tissues decreased. GR113808 suppressed body weight gain and improved glucose intolerance. Furthermore, GR113808 not only decreased fatty liver formation but also reduced adipose tissue size. Additionally, GR113808 reduced inflammatory cytokine serum levels and inflammasome complex formation in both tissues. Palmitate treatment in HTR4-downregulated Hep3B cells, also reduced peroxisome proliferator-activated receptor γ and sterol regulatory element-binding protein-1 pathway induction as well as inflammasome complex formation, thus decreasing inflammatory cytokine levels. HTR4 downregulation in 3T3-L1 cells also reduced palmitate-induced inflammasome complex formation and inflammatory cytokine production. Palmitate-induced insulin resistance in Hep3B cells, but not in 3T3-L1 cells, was improved by HTR4 downregulation.ConclusionsIn summary, GR113808 protected against fatty liver formation and improved inflammation in the liver and adipose tissue. Downregulation of HTR4 ameliorated insulin resistance in the liver. These results suggest that HTR4 could serve as a promising therapeutic target for metabolic diseases.

The effects of an aggressive breast tumor on thrombosis after antithrombin downregulation in a hypercoagulable mouse model

BackgroundDespite improvements in therapy, breast cancer still contributes to high mortality rates. Survival of these patients becomes progressively worse upon diagnosis with cancer-associated thrombosis (CAT). Unfortunately, the mechanism causing CAT has remained unclear. ObjectiveSet up an acute and non-invasive hypercoagulable mouse model with an aggressive breast cancer and study the mechanism of cancer-associated thrombosis. MethodsMice were grafted with the aggressive breast cancer cell line MDA-MB-231 or sham-treated. Subsequently, an acute imbalance in coagulation was introduced by injecting a synthetic small interfering (si) RNA targeting hepatic Serpinc1 to knockdown antithrombin – a condition known to predispose to cause a hypercoagulant state in vivo. ResultsSilencing Serpinc1 with siRNA decreased plasma antithrombin levels. siRNA treatment had no short-term effects on tumor characteristics, but increased distant metastasis within the timeframe of this study. The systemic pro-inflammatory status, with elevated platelet counts and fibrinogen levels in tumor-bearing mice, was also not affected by antithrombin silencing. While elevated fibrin deposition in the liver upon Serpinc1 targeting was not significantly affected by the presence of breast cancer, knockdown of antithrombin did significantly increase intratumoral fibrin deposition and inflammation. Surprisingly, in the presence of an aggressive tumor, a protective outcome with less clinical features coinciding with venous thrombosis were observed in mice with antithrombin knockdown. ConclusionWe conclude that the presence of a breast tumor protects hypercoagulant mice from severe consumption of coagulation factors after lowering hepatic antithrombin levels, possibly due to elevated platelet counts. However, the consequences on cancer-associated thrombosis remained inconclusive.

Ionizable Drugs Enable Intracellular Delivery of Co-Formulated siRNA.

Targeting complementary pathways in diseases such as cancer can be achieved with co-delivery of small interfering ribonucleic acid (siRNA) and small molecule drugs; however, current formulation strategies are typically limited to one, but not both. Here, ionizable small molecule drugs and siRNA are co-formulatedin drug-rich nanoparticles. Ionizable analogs of the selective estrogen receptor degrader fulvestrant self-assemble into colloidal drug aggregates and cause endosomal disruption, allowing co-delivery of siRNA against a non-druggable target. siRNA is encapsulated in lipid-stabilized, drug-rich colloidal nanoparticles where the ionizable lipid used in conventional lipid nanoparticles is replaced with an ionizable fulvestrant analog. The selection of an appropriate phospholipid and formulation buffer enables endocytosis and potent reporter gene knockdown in cancer cells. Importantly, siRNA targeting cyclin E1 is effectively delivered to drug-resistant breast cancer cells, demonstrating the utility of this approach. This strategy opens the possibility of using ionizable drugs to co-deliver RNA and ultimately improve therapeutic outcomes.

Efficient Method for Liposome-Based Gene Transfer in Cattle Embryos.

In vitro embryo production (IVP) in cattle is crucial for advancing genetic enhancement and preserving valuable genetic lineages, enabling precise genetic modifications and gene studies through modern techniques. Successful genetic manipulation in cattle embryos requires efficient delivery of exogenous DNA/RNA molecules. This research investigates the efficacy of a single embryo culture system for developing genetically modified zona-free (ZF) embryos and examines the use of liposome-based SAMTOR target siRNA transfer in these individually cultured ZF embryos. The findings indicated that the individual culture system resulted in increased cleavage rates, and blastocyst rates were minimally impacted. The new culture system effectively achieved SAMTOR silencing, with 8-16 cell embryos exhibiting reduction in transcript levels compared to control. Measurement of total protein content in the spent culture media was performed to validate the single-culture approach for further analytical applications. Total protein content analysis demonstrated the system's suitability for comprehensive evaluation of the embryo-media interaction, enhancing the scope for in-depth genetic research and applications. This research sheds light into an innovative method to improve genetic editing techniques in reproduction research.

The Nociceptor Primary Cilium Contributes to Mechanical Nociceptive Threshold and Inflammatory and Neuropathic Pain.

The primary cilium, a single microtubule-based organelle protruding from the cell surface and critical for neural development, also functions in adult neurons. While some dorsal root ganglion neurons elaborate a primary cilium, whether it is expressed by and functional in nociceptors is unknown. Recent studies have shown a role of Hedgehog, whose canonical signaling is primary cilium dependent, in nociceptor sensitization. We establish the presence of primary cilia in soma of rat nociceptors, where they contribute to mechanical threshold, prostaglandin E2 (PGE2)-induced hyperalgesia, and chemotherapy-induced neuropathic pain (CIPN). Intrathecal administration of siRNA targeting Ift88, a primary cilium-specific intra-flagellar transport (IFT) protein required for ciliary integrity, resulted in attenuation of Ift88 mRNA and nociceptor primary cilia. Attenuation of primary cilia was associated with an increase in mechanical nociceptive threshold in vivo, and decrease in nociceptor excitability in vitro, abrogation of hyperalgesia, and nociceptor sensitization induced by both a prototypical pronociceptive inflammatory mediator PGE2 and paclitaxel CIPN, in a sex-specific fashion. siRNA targeting Ift52, another IFT protein, and knockdown of NompB, the Drosophila Ift88 ortholog, also abrogated CIPN and reduced baseline mechanosensitivity, respectively, providing independent confirmation for primary cilia control of nociceptor function. Hedgehog-induced hyperalgesia is attenuated by Ift88 siRNA, supporting a role for primary cilia in Hedgehog-induced hyperalgesia. Attenuation of CIPN by cyclopamine (intradermal and intra-ganglion), which inhibits Hedgehog signaling, supports a role of Hedgehog in CIPN. Our findings support a role of the nociceptor primary cilium in control of mechanical nociceptive threshold and inflammatory and neuropathic pain, the latter Hedgehog-dependent.Significance statement Many neurons have a tiny antenna-like structure, the primary cilium, protruding from their cell body, which processes information about the extracellular environment as well as regulates intracellular signaling. We report experiments aimed at understanding the role of the primary cilium in pain sensory neurons (nociceptors), including in the setting of inflammatory and neuropathic pain. We establish that nociceptors bear a primary cilium and show that this organelle regulates detection of noxious stimuli and contributes to nociceptor sensitization, using both the rat and the fruit fly as model organisms to manipulate primary cilia in pain states. We also identify primary cilium dependence of the contribution of Hedgehog to pain states.

Increased viperin expression induced by avian infectious bronchitis virus inhibits viral replication by restricting cholesterol synthesis: an in vitro study

With the emergence of new variant strains resulting from high mutation rates and genome recombination, avian infectious bronchitis virus (IBV) has caused significant economic losses to the poultry industry worldwide. Little is known about the underlying mechanisms of IBV-host interactions, particularly how IBV utilizes host metabolic pathways for efficient viral replication and transmission. In the present study, the effects of the cell membrane, viral envelope membrane, and viperin-mediated cholesterol synthesis on IBV replication were explored. Our results revealed significant increase in cholesterol levels and the expression of viperin after IBV infection. Acute cholesterol depletion in the cell membrane and viral envelope membrane by treating cells with methyl-β-cyclodextrin (MβCD) obviously inhibited IBV replication; thereafter, replenishment of the cell membrane with cholesterol successfully restored viral replication, and direct addition of exogenous cholesterol to the cell membrane significantly promoted IBV infection during the early stages of infection. In addition, overexpression of viperin effectively suppressed cholesterol synthesis, as well as IBV replication, whereas knockdown of viperin (gene silencing with siRNA targeting viperin, siViperin) significantly increased IBV replication and cholesterol levels, whereas supplementation with exogenous cholesterol to viperin-transfected cells markedly restored viral replication. In conclusion, the increase in viperin induced by IBV infection plays an important role in IBV replication by affecting cholesterol production, providing a theoretical basis for understanding the pathogenesis of IBV and discovering new potential antiviral targets.