Small Interfering RNA Knockdown Research Articles

Mutant GNAS drives a pyloric metaplasia with tumor suppressive glycans in intraductal papillary mucinous neoplasia.

Intraductal Papillary Mucinous Neoplasms (IPMNs) are cystic lesions and bona fide precursors for pancreatic ductal adenocarcinoma (PDAC). Recent studies have shown that pancreatic precancer is characterized by a transcriptomic program similar to gastric metaplasia. The aims of this study were to assay IPMN for pyloric markers, to identify molecular drivers, and to determine a functional role for this program in the pancreas. Pyloric marker expression was evaluated by RNA-seq and multiplex immunostaining in patient samples. Cell lines and organoids expressing KrasG12D +/- GNASR201C underwent RNA sequencing. A PyScenic-based regulon analysis was performed to identify molecular drivers, and candidates were evaluated by RNA-seq, immunostaining, and small interfering RNA knockdown. Glycosylation profiling was performed to identify GNASR201C-driven changes. Glycan abundance was evaluated in patient samples. Pyloric markers were identified in human IPMN. GNASR201C drove expression of this program as well as an indolent phenotype characterized by distinct glycosyltransferase changes. Glycan profiling identified an increase in LacdiNAcs and loss of pro-tumorigenic Lewis antigens. Knockdown of transcription factors Spdef or Creb3l1 or chitinase treatment reduced LacdiNAc deposition and reversed the indolent phenotype. LacdiNAc and 3-sulfoLeA/C abundance discriminated low from high grade patient IPMN. GNASR201C drives an indolent phenotype in IPMN by amplifying a differentiated, pyloric phenotype through SPDEF/CREB3L1 which is characterized by distinct glycans. Acting as a glycan rheostat, mutant GNAS elevates LacdiNAcs at the expense of pro-tumorigenic acidic Lewis epitopes, inhibiting cancer cell invasion and disease progression. LacdiNAc and 3-Sulfo-LeA/C are mutually exclusive and may serve as markers of disease progression.

Inflammatory priming of human mesenchymal stem cells induces osteogenic differentiation via the early response gene IER3.

Mesenchymal stem cells (MSCs) have gained tremendous interest due to their overall potent pro-regenerative and immunomodulatory properties. In recent years, various invitro and preclinical studies have investigated different priming ("licensing") approaches to enhance MSC functions for specific therapeutic purposes. In this study, we primed bone marrow-derived human MSCs (hMSCs) with an inflammation cocktail designed to mimic the elevated levels of inflammatory mediators found in serum of patients with severe injuries, such as bone fractures. We observed a significantly enhanced osteogenic differentiation potential of primed hMSCs compared to untreated controls. By RNA-sequencing analysis, we identified the immediate early response 3 (IER3) gene as one of the top-regulated genes upon inflammatory priming. Small interfering RNA knockdown experiments established IER3 as a novel positive regulator of osteogenic differentiation. Mechanistic analysis further revealed that IER3 deletion significantly downregulated bone marrow stromal cell antigen 2 (BST2) expression and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in hMSCs, suggesting that IER3 regulates osteogenic differentiation through BST2 and ERK1/2 signaling pathway activation. On the basis of these findings, we propose IER3 as a novel therapeutic target to promote hMSC osteoblastogenesis, which might be of high clinical relevance, for example, in patients with osteoporosis or compromised fracture healing.

Melatonin inhibits voltage-gated potassium KV4.2 channels and negatively regulates melatonin secretion in rat pineal glands.

Melatonin is synthesized in and secreted from the pineal glands and regulates circadian rhythms. Although melatonin has been reported to modulate the activity of ion channels in several tissues, its effects on pineal ion channels remain unclear. In the present study, the effects of melatonin on voltage-gated K+ (KV) channels, which play a role in regulating the resting membrane potential, were examined in rat pinealocytes. The application of melatonin reduced pineal KV currents in a concentration-dependent manner (IC50 = 309 µM). An expression analysis revealed that KV4.2 channels were highly expressed in rat pineal glands. Melatonin-sensitive currents were abolished by the small interfering RNA knockdown of KV4.2 channels in rat pinealocytes. In human embryonic kidney 293 (HEK293) cells expressing KV4.2 channels, melatonin decreased outward currents (IC50 = 479 µM). Inhibitory effects were mediated by a shift in the voltage dependence of steady-state inactivation in a hyperpolarizing direction. This inhibition was observed even in the presence of 100 nM luzindole, an antagonist of melatonin receptors. Melatonin also blocked the activity of KV4.3, KV1.1, and KV1.5 channels in reconstituted HEK293 cells. The application of 1 mM melatonin caused membrane depolarization in rat pinealocytes. Furthermore, KV4.2 channel inhibition by 5 mM 4-aminopyridine attenuated melatonin secretion induced by 1 µM noradrenaline in rat pineal glands. These results strongly suggest that melatonin directly inhibited KV4.2 channels and caused membrane depolarization in pinealocytes, resulting in a decrease in melatonin secretion through parasympathetic signaling pathway. This mechanism may function as a negative-feedback mechanism of melatonin secretion in pineal glands. NEW & NOTEWORTHY Melatonin is a hormone that is synthesized in and secreted from the pineal glands, which regulates circadian rhythms. However, the effects of melatonin on pineal ion channels remain unclear. The present study demonstrated that melatonin directly inhibited voltage-gated potassium KV4.2 channels, which are highly expressed in rat pinealocytes, and induced membrane depolarization, resulting in a decrease in melatonin secretion. This mechanism may function as a negative-feedback mechanism of melatonin secretion in pineal glands.

Genetic risk factors for COVID-19 and influenza are largely distinct

Coronavirus disease 2019 (COVID-19) and influenza are respiratory illnesses caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses, respectively. Both diseases share symptoms and clinical risk factors1, but the extent to which these conditions have a common genetic etiology is unknown. This is partly because host genetic risk factors are well characterized for COVID-19 but not for influenza, with the largest published genome-wide association studies for these conditions including >2 million individuals2 and about 1,000 individuals3–6, respectively. Shared genetic risk factors could point to targets to prevent or treat both infections. Through a genetic study of 18,334 cases with a positive test for influenza and 276,295 controls, we show that published COVID-19 risk variants are not associated with influenza. Furthermore, we discovered and replicated an association between influenza infection and noncoding variants in B3GALT5 and ST6GAL1, neither of which was associated with COVID-19. In vitro small interfering RNA knockdown of ST6GAL1—an enzyme that adds sialic acid to the cell surface, which is used for viral entry—reduced influenza infectivity by 57%. These results mirror the observation that variants that downregulate ACE2, the SARS-CoV-2 receptor, protect against COVID-19 (ref. 7). Collectively, these findings highlight downregulation of key cell surface receptors used for viral entry as treatment opportunities to prevent COVID-19 and influenza.

Corosolic acid attenuates platelet-derived growth factor signaling in macrophages and smooth muscle cells of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease that is characterized by vascular remodeling of the pulmonary artery. Pulmonary vascular remodeling is primarily caused by the excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), which are facilitated by perivascular inflammatory cells including macrophages. Corosolic acid (CRA) is a natural pentacyclic triterpenoid that exerts anti-inflammatory effects. In the present study, the effects of CRA on the viability of macrophages were examined using monocrotaline (MCT)-induced PAH rats and human monocyte-derived macrophages. Although we previously reported that CRA inhibited signal transducer and activator of transcription 3 (STAT3) signaling and ameliorated pulmonary vascular remodeling in PAH, the inhibitory mechanism remains unclear. Therefore, the underlying mechanisms were investigated using PASMCs from idiopathic PAH (IPAH) patients. In MCT-PAH rats, CRA inhibited the accumulation of macrophages around remodeled pulmonary arteries. CRA reduced the viability of human monocyte-derived macrophages. In IPAH-PASMCs, CRA attenuated cell proliferation and migration facilitated by platelet-derived growth factor (PDGF)-BB released from macrophages and PASMCs. CRA also downregulated the expression of PDGF receptor β and its signaling pathways, STAT3 and nuclear factor-κB (NF-κB). In addition, CRA attenuated the phosphorylation of PDGF receptor β and STAT3 following the PDGF-BB simulation. The expression and phosphorylation levels of PDGF receptor β after the PDGF-BB stimulation were reduced by the small interfering RNA knockdown of NF-κB, but not STAT3, in IPAH-PASMCs. In conclusion, CRA attenuated the PDGF-PDGF receptor β-STAT3 and PDGF-PDGF receptor β-NF-κB signaling axis in macrophages and PASMCs, and thus, ameliorated pulmonary vascular remodeling in PAH.

Protective roles of peroxiporins AQP0 and AQP11 in human astrocyte and neuronal cell lines in response to oxidative and inflammatory stressors.

In addition to aquaporin (AQP) classes AQP1, AQP4 and AQP9 known to be expressed in mammalian brain, our recent transcriptomic analyses identified AQP0 and AQP11 in human cortex and hippocampus at levels correlated with age and Alzheimer's disease (AD) status; however, protein localization remained unknown. Roles of AQP0 and AQP11 in transporting hydrogen peroxide (H2O2) in lens and kidney prompted our hypothesis that up-regulation in brain might similarly be protective. Established cell lines for astroglia (1321N1) and neurons (SHSY5Y, differentiated with retinoic acid) were used to monitor changes in transcript levels for human AQPs (AQP0 to AQP12) in response to inflammation (simulated with 10-100 ng/ml lipopolysaccharide [LPS], 24 h), and hypoxia (5 min N2, followed by 0 to 24 h normoxia). AQP transcripts up-regulated in both 1321N1 and SHSY5Y included AQP0, AQP1 and AQP11. Immunocytochemistry in 1321N1 cells confirmed protein expression for AQP0 and AQP11 in plasma membrane and endoplasmic reticulum; AQP11 increased 10-fold after LPS and AQP0 increased 0.3-fold. In SHSY5Y cells, AQP0 expression increased 0.2-fold after 24 h LPS; AQP11 showed no appreciable change. Proposed peroxiporin roles were tested using melondialdehyde (MDA) assays to quantify lipid peroxidation levels after brief H2O2. Boosting peroxiporin expression by LPS pretreatment lowered subsequent H2O2-induced MDA responses (∼50%) compared with controls; conversely small interfering RNA knockdown of AQP0 in 1321N1 increased lipid peroxidation (∼17%) after H2O2, with a similar trend for AQP11 siRNA. Interventions that increase native brain peroxiporin activity are promising as new approaches to mitigate damage caused by aging and neurodegeneration.

Abstract 6873: Prognostic impact of squamous cell carcinoma antigen as a mediator of anti-inflammatory macrophage polarization in tumor microenvironment

Abstract Squamous cell carcinoma antigen 1 (SCCA1) is a potential biomarker and prognostic indicator for many epithelial cancers including cervix, head and neck, breast, and lung. Cancer patients with high serum levels of SCCA commonly experience treatment resistance and recurrence. We analyze SCCA expression in correlation with tumor microenvironment from 66 human cervical tumor biopsies and find that high serum SCCA is associated with increased macrophage infiltration in tumors and poor overall survival. In a single cell RNAseq analysis of 8 cervical tumors, patients with high SCCA1 show increased immunosuppressive genes (C1QA, C1QC, APOE) and reduced MHC molecules (HLA-DR, HLA-DQA1) in their tumor associated macrophages (TAMs), corresponding to decreased expression of T-cell cytotoxic-associated genes (IFNG, GZMB, PRF1). Thus, we aim to understand the impact of elevated SCCA1 on macrophages and its role in shaping the immune response. Co-culture of peritoneal macrophages isolated from SCCA1-treated mice with T cells from syngeneic wild-type mice, showed significant suppression in T cell proliferation. Downregulation of antigen presentation molecules was also observed SCCA1-treated macrophages and resulted in impaired ability to induce T cell activation by peptide-pulsed macrophages. Compared with treatment-naïve mice, SCCA1-treated macrophages showed increased anti-inflammatory cytokine CCL22, IL10 and immunosuppression regulators ARG1, PD-L1 expression. Moreover, human monocyte-derived M2 macrophages treated with SCCA1 during the differentiation process demonstrated inhibition toward CD3/28-induced T cell proliferation, accompanied by enhanced expression of cytokines, IL10 and CCL18. We further found that targeting SCCA1 by small interfering RNA knockdown significantly reduced tumor growth and macrophage infiltration, resulting in improved T-cell antitumor immunity. Lastly, analysis of cervical cancer patients with chemoradiation therapy showed that patients with high SCCA are associated with increased exhaustion (LAG3, CTLA4) and decreased antitumor gene signature (GZMB, IFNG) during treatment. In summary, SCCA expression promotes immunosuppressive macrophage polarization and function, contributing to T cell exhaustion phenotype and poor treatment response. Citation Format: Liyun Chen, Victoria Shi, Eric Liu, Jasmine Yang, Abhay Singh, Matthew Inkman, Jin Zhang, Julie Schwarz, Stephanie Markovina. Prognostic impact of squamous cell carcinoma antigen as a mediator of anti-inflammatory macrophage polarization in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6873.

Anti-Inflammatory Effect of High-Density Lipoprotein Blunted by Delivery of Altered MicroRNA Cargo in Patients With Rheumatoid Arthritis.

High-density lipoprotein (HDL) has well-characterized anti-atherogenic cholesterol efflux and antioxidant functions. Another function of HDL uncharacterized in rheumatoid arthritis (RA) is its ability to transport microRNAs (miRNAs) between cells and thus alter cellular function. The study's purpose was to determine if HDL-miRNA cargo is altered and affects inflammation in RA. HDL-microRNAs were characterized in 30 RA and 30 control participants by next generation sequencing and quantitative polymerase chain reaction. The most abundant differentially expressed miRNA was evaluated further. The function of miR-1246 was assessed by miRNA mimics, antagomiRs, small interfering RNA knockdown, and luciferase assays. Monocyte-derived macrophages were treated with miR-1246-loaded HDL and unmodified HDL from RA and control participants to measure delivery of miR-1246 and its effect on interleukin-6 (IL-6). The most abundant miRNA on HDL was miR-1246; it was significantly enriched two-fold on HDL from RA versus control participants. HDL-mediated miR-1246 delivery to macrophages significantly increased IL6 expression 43-fold. miR-1246 delivery significantly decreased DUSP3 1.5-fold and DUSP3 small interfering RNA knockdown increased macrophage IL6 expression. Luciferase assay indicated DUSP3 is a direct target of miR-1246. Unmodified HDL from RA delivered 1.6-fold more miR-1246 versus control participant HDL. Unmodified HDL from both RA and control participants attenuated activated macrophage IL6 expression, but this effect was significantly blunted in RA so that IL6 expression was 3.4-fold higher after RA versus control HDL treatment. HDL-miR-1246 was increased in RA versus control participants and delivery of miR-1246 to macrophages increased IL-6 expression by targeting DUSP3. The altered HDL-miRNA cargo in RA blunted HDL's anti-inflammatory effect.

Differential Effects of PTH (1-34), PTHrP (1-36), and Abaloparatide on the Murine Osteoblast Transcriptome.

Teriparatide (PTH (1-34)), PTHrP (1-36), and abaloparatide (ABL) have been used for the treatment of osteoporosis, but their efficacy long term is significantly limited. The 3 peptides exert time- and dose-dependent differential responses in osteoblasts, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. Treatment of mouse calvarial osteoblasts with 1 nM of the peptides for 4 hours results in RNA sequencing data with PTH (1-34) regulating 367 genes, including 194 unique genes; PTHrP (1-36) regulating 117 genes, including 15 unique genes; and ABL regulating 179 genes, including 20 unique genes. There were 83 genes shared among all 3 peptides. Gene ontology analyses showed similarities in Wnt signaling, cAMP-mediated signaling, ossification, but differences in morphogenesis of a branching structure in biological processes; receptor ligand activity, transcription factor activity, and cytokine receptor/binding activity in molecular functions. The peptides increased Vdr, Cited1, and Pde10a messenger RNAs (mRNAs) in a pattern similar to Rankl, that is, PTH (1-34) greater than ABL greater than PTHrP (1-36). mRNA abundance of other genes, including Wnt4, Wnt7, Wnt11, Sfrp4, Dkk1, Kcnk10, Hdac4, Epn3, Tcf7, Crem, Fzd5, Ppp2r2a, and Dvl3, showed that some genes were regulated similarly by all 3 peptides; others were not. Finally, small interfering RNA knockdowns of SIK1/2/3 and CRTC1/2/3 in PTH (1-34)-treated cells revealed that Vdr and Wnt4 genes are regulated by salt-inducible kinases (SIKs) and CREB-regulated transcriptional coactivators (CRTCs), while others are not. Although many studies have examined PTH signaling in the osteoblast/osteocyte, ours is the first to compare the global effects of these peptides on the osteoblast transcriptome or to analyze the roles of the SIKs and CRTCs.

MicroRNA-322 overexpression reduces neural tube defects in diabetic pregnancies

Hyperglycemia from pregestational diabetes mellitus induces neural tube defects in the developing fetus. Folate supplementation is the only effective way to prevent neural tube defects; however, some cases of neural tube defects are resistant to folate. Excess folate has been linked to higher maternal cancer risk and infant allergy. Therefore, additional interventions are needed. Understanding the mechanisms underlying maternal diabetes mellitus-induced neural tube defects can identify potential targets for preventing such defects. Despite not yet being in clinical use, growing evidence suggests that microRNAs are important intermediates in embryonic development and can serve as both biomarkers and drug targets for disease intervention. Our previous studies showed that maternal diabetes mellitus invivo activates the inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) in the developing embryo and that a high glucose condition invitro reduces microRNA-322 (miR-322) levels. IRE1α is an RNA endonuclease; however, it is unknown whether IRE1α targets and degrades miR-322 specifically or whether miR-322 degradation leads to neural tube defects via apoptosis. We hypothesize that IRE1α can inhibit miR-322 in maternal diabetes mellitus-induced neural tube defects and that restoring miR-322 expression in developing neuroepithelium ameliorates neural tube defects. This study aimed to identify potential targets for preventing maternal diabetes mellitus-induced neural tube defects and to investigate the roles and relationship of a microRNA and an RNA endonuclease in mouse embryos exposed to maternal diabetes mellitus. To determine whether miR-322 reduction is necessary for neural tube defect formation in pregnancies complicated by diabetes mellitus, male mice carrying a transgene expressing miR-322 were mated with nondiabetic or diabetic wide-type female mice to generate embryos with or without miR-322 overexpression. At embryonic day 8.5 when the neural tube is not yet closed, embryos were harvested for the assessment of 3 miR-322 transcripts (primary, precursor, and mature miR-322), tumor necrosis factor receptor-associated factor 3 (TRAF3), and neuroepithelium cell survival. Neural tube defect incidences were determined in embryonic day 10.5 embryos when the neural tube should be closed if there is no neural tube defect formation. To identify which miR-322 transcript is affected by maternal diabetes mellitus and high glucose conditions, 3 miR-322 transcripts were assessed in embryos from dams with or without diabetes mellitus and in C17.2 mouse neural stem cells treated with different concentrations of glucose and at different time points. To determine whether the endonuclease IRE1α targets miR-322, small interfering RNA knockdown of IRE1α or overexpression of inositol-requiring transmembrane kinase/endoribonuclease 1α by DNA plasmid transfection was used to determine the effect of IRE1α deficiency or overexpression on miR-322 expression. RNA immunoprecipitation was performed to reveal the direct targets of inositol-requiring transmembrane kinase/endoribonuclease 1α. Maternal diabetes mellitus suppressed miR-322 expression in the developing neuroepithelium. Restoring miR-322 expression in the neuroepithelium blocked maternal diabetes mellitus-induced caspase-3 and caspase-8 cleavage and cell apoptosis, leading to a neural tube defect reduction. Reversal of maternal diabetes mellitus-inhibited miR-322 via transgenic overexpression prevented TRAF3 up-regulation in embryos exposed to maternal diabetes mellitus. Activated IRE1α acted as an endonuclease and degraded precursor miR-322, resulting in mature miR-322 reduction. This study supports the crucial role of the IRE1α-microRNA-TRAF3 circuit in the induction of neuroepithelial cell apoptosis and neural tube defect formation in pregnancies complicated by diabetes mellitus and identifies IRE1α and miR-322 as potential targets for preventing maternal diabetes mellitus-induced neural tube defects.

Interleukin-4 Promotes Human Metapneumovirus Replication Through the JAK/STAT6 Pathway.

Respiratory virus infections are the main causes of pediatric diseases. Human metapneumovirus (hMPV) is an enveloped RNA virus similar to severe acute respiratory syndrome coronavirus type 2, both of which have emerged as important new respiratory viruses. Recent studies have found that interleukin-4 (IL-4) is involved in the replication of a variety of viruses, and its role differs in different viruses. The purpose of this study was to investigate the effect of IL-4 on hMPV and to elucidate its mechanism of action. We found that hMPV infection promoted the expression of IL-4 in human bronchial epithelial cells. The replication of the virus was reduced using small interfering RNA knockdown of IL-4 expression, while the addition of exogenous recombinant human IL-4 to IL-4 knockdown cells restored viral replication ability. These results demonstrate that the expression of IL-4 is closely related to the replication of hMPV; moreover, further experiments revealed that IL-4 promotes the replication of hMPV through a mechanism dependent on the Janus kinase/signal transductor and transcription activator 6 signaling pathway. Therefore, anti-IL-4 strategies may be a promising avenue for the treatment of hMPV infection, representing an important breakthrough for children at risk from hMPV infection.

C1 inhibitor and prolylcarboxypeptidase modulate prekallikrein activation on endothelial cells

We examined how prekallikrein (PK) activation on human microvascular endothelial cells (HMVECs) is regulated by the ambient concentration of C1 inhibitor (C1INH) and prolylcarboxypeptidase (PRCP). We sought to examine the specificity of PK activation on HMVECs by PRCP and the role of C1INH to regulate it, high-molecular-weight kininogen (HK) cleavage, and bradykinin (BK) liberation. Investigations were performed on cultured HMVECs. Immunofluorescence, enzymatic activity assays, immunoblots, small interfering RNA knockdowns, and cell transfections were used to perform these studies. Cultured HMVECs constitutively coexpressed PK, HK, C1INH, and PRCP. PK activation on HMVECs was modulated by the ambient C1INH concentration. In the absence of C1INH, forming PKa on HMVECs cleaved 120-kDa HK completely to a 65-kDa H-chain and a 46-kDa L-chain in 60 minutes. In the presence of 2 μM C1INH, only 50% of the HK became cleaved. C1INH concentrations (0.0-2.5 μM) decreased but did not abolish BK liberated from HK by activated PK. Factor XII did not activate when incubated with HMVECs alone for 1 hour. However, if incubated in the presence of HK and PK, factor XII became activated. The specificity of PK activation on HMVECs by PRCP was shown by several inhibitors to each enzyme. Furthermore, PRCP small interfering RNA knockdowns magnified C1INH inhibitory activity on PK activation, and PRCP transfections reduced C1INH inhibition at any given concentration. These combined studies indicated that on HMVECs, PK activation and HK cleavage to liberate BK weremodulated by the local concentrations of C1INH and PRCP.

Novel genetic regulators of fibrinogen synthesis identified by an in vitro experimental platform

BackgroundFibrinogen has an established, essential role in both coagulation and inflammatory pathways, and these processes are deeply intertwined in the development of thrombotic and atherosclerotic diseases. Previous studies aimed to better understand the (patho) physiological actions of fibrinogen by characterizing the genomic contribution to circulating fibrinogen levels. ObjectivesEstablish an in vitro approach to define functional roles between genes within these loci and fibrinogen synthesis. MethodsCandidate genes were selected on the basis of their proximity to genetic variants associated with fibrinogen levels and expression in hepatocytes and HepG2 cells. HepG2 cells were transfected with small interfering RNAs targeting candidate genes and cultured in the absence or presence of the proinflammatory cytokine interleukin-6. Effects on fibrinogen protein production, gene expression, and cell growth were assessed by immunoblotting, real-time polymerase chain reaction, and cell counts, respectively. ResultsHepG2 cells secreted fibrinogen, and stimulation with interleukin-6 increased fibrinogen production by 3.4 ± 1.2 fold. In the absence of interleukin-6, small interfering RNA knockdown of FGA, IL6R, or EEPD1 decreased fibrinogen production, and knockdown of LEPR, PDIA5, PLEC, SHANK3, or CPS1 increased production. In the presence of interleukin-6, knockdown of FGA, IL6R, or ATXN2L decreased fibrinogen production. Knockdown of FGA, IL6R, EEPD1, LEPR, PDIA5, PLEC, or CPS1 altered transcription of one or more fibrinogen genes. Knocking down ATXN2L suppressed inducible but not basal fibrinogen production via a post-transcriptional mechanism. ConclusionsWe established an in vitro platform to define the impact of select gene products on fibrinogen production. Genes identified in our screen may reveal cellular mechanisms that drive fibrinogen production as well as fibrin(ogen)-mediated (patho)physiological mechanisms.

Cancer-Associated Fibroblasts Induce Proliferation and Therapeutic Resistance to Everolimus in Neuroendocrine Tumors through STAT3 Activation

Introduction: Cancer-associated fibroblasts (CAF) have been identified as relevant contributors to cancer progression and drug resistance in many tumors. Although neuroendocrine tumors (NET) are often associated with a strong stromal reaction, no study has addressed whether CAF are involved in progression and therapeutic resistance in NET. The aim of this study was to characterize the role of CAF in NET. Methods: We established primary CAF cultures derived from NET liver metastases to study the effect on NET cell lines NT-3 and BON. Immunohistochemistry was performed on tissue sections of primary and metastatic NET tissue. Results: Immunohistochemistry identified CAF dispersed in between tumor cells and within fibrotic bands separating tumor cell clusters in NET. Stimulating NET cells with CAF decreased expression of SSTR2 and chromogranin A and induced expression of CXCR4. CAF induced a 2.3-fold increase in proliferation and completely reversed the response to everolimus in NT-3 cells. We identified STAT3 as the main signaling pathway induced by CAF. STAT3 targeting by small interfering RNA knockdown and inhibitors prevented CAF-induced proliferation and restored everolimus responsiveness. STAT3 activation in NET tissue was associated with decreased chromogranin A expression, increased Ki-67 index, and decreased 5-year overall and progression-free survival. CAF directly influence proliferation and therapeutic response in NET cells. Conclusion: Identifying STAT3 as the contributing pathway of this so far neglected tumor-stroma interaction has the potential to become a new therapeutic target to halt tumor growth and to restore therapeutic responsiveness in NET.

Seneca Valley Virus Enters PK-15 Cells via Caveolae-Mediated Endocytosis and Macropinocytosis Dependent on Low-pH, Dynamin, Rab5, and Rab7.

Seneca Valley virus (SVV), a new pathogen resulting in porcine vesicular disease, is prevalent in pig herds worldwide. Although an understanding of SVV biology pathogenesis is crucial for preventing and controlling this disease, the molecular mechanisms for the entry and post-internalization of SVV, which represent crucial steps in viral infection, are not well characterized. In this study, specific inhibitors, Western blotting, and immunofluorescence detection revealed that SVV entry into PK-15 cells depends on low-pH conditions and dynamin. Furthermore, results showed that caveolae-mediated endocytosis (CavME) contributes crucially to the internalization of SVV, as evidenced by cholesterol depletion, downregulation of caveolin-1 expression by small interfering RNA knockdown, and overexpression of a caveolin-1 dominant negative (caveolin-1-DN) in SVV-infected PK-15 cells. However, SVV entry into PK-15 cells did not depend on clathrin-mediated endocytosis (CME). Furthermore, treatment with specific inhibitors demonstrated that SVV entry into PK-15 cells via macropinocytosis depended on the Na+/H+ exchanger (NHE), p21-activated kinase 1 (Pak1), and actin rearrangement, but not phosphatidylinositol 3-kinase (PI3K). Electron microscopy showed that SVV particles or proteins were localized in CavME and macropinocytosis. Finally, knockdown of GTPase Rab5 and Rab7 by siRNA significantly inhibited SVV replication, as determined by measuring viral genome copy numbers, viral protein expression, and viral titers. In this study, our results demonstrated that SVV utilizes caveolae-mediated endocytosis and macropinocytosis to enter PK-15 cells, dependent on low pH, dynamin, Rab5, and Rab7. IMPORTANCE Entry of virus into cells represents the initiation of a successful infection. As an emerging pathogen of porcine vesicular disease, clarification of the process of SVV entry into cells enables us to better understand the viral life cycle and pathogenesis. In this study, patterns of SVV internalization and key factors required were explored. We demonstrated for the first time that SVV entry into PK-15 cells via caveolae-mediated endocytosis and macropinocytosis requires Rab5 and Rab7 and is independent of clathrin-mediated endocytosis, and that low-pH conditions and dynamin are involved in the process of SVV internalization. This information increases our understanding of the patterns in which all members of the family Picornaviridae enter host cells, and provides new insights for preventing and controlling SVV infection.

Receptor for Advanced Glycation End-Products Promotes Activation of Alveolar Macrophages through the NLRP3 Inflammasome/TXNIP Axis in Acute Lung Injury.

The roles of thioredoxin-interacting protein (TXNIP) and receptor for advanced glycation end-products (RAGE)-dependent mechanisms of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-driven macrophage activation during acute lung injury are underinvestigated. Cultured THP-1 macrophages were treated with a RAGE agonist (S100A12), with or without a RAGE antagonist; cytokine release and intracytoplasmic production of reactive oxygen species (ROS) were assessed in response to small interfering RNA knockdowns of TXNIP and NLRP3. Lung expressions of TXNIP and NLRP3 and alveolar levels of IL-1β and S100A12 were measured in mice after acid-induced lung injury, with or without administration of RAGE inhibitors. Alveolar macrophages from patients with acute respiratory distress syndrome and from mechanically ventilated controls were analyzed using fluorescence-activated cell sorting. In vitro, RAGE promoted cytokine release and ROS production in macrophages and upregulated NLRP3 and TXNIP mRNA expression in response to S100A12. TXNIP inhibition downregulated NLRP3 gene expression and RAGE-mediated release of IL-1β by macrophages in vitro. In vivo, RAGE, NLRP3 and TXNIP lung expressions were upregulated during experimental acute lung injury, a phenomenon being reversed by RAGE inhibition. The numbers of cells expressing RAGE, NLRP3 and TXNIP among a specific subpopulation of CD16+CD14+CD206- (“pro-inflammatory”) alveolar macrophages were higher in patients with lung injury. This study provides a novel proof-of-concept of complex RAGE–TXNIP–NLRP3 interactions during macrophage activation in acute lung injury.

Multiomics characterization of mouse hepatoblastoma identifies yes-associated protein 1 target genes.

Hepatoblastoma (HB) is the most common primary liver malignancy in childhood and lacks targeted therapeutic options. We previously engineered, to our knowledge, the first yes-associated protein 1 (YAP1) S127A -inducible mouse model of HB, demonstrating tumor regression and redifferentiation after YAP1 withdrawal through genome-wide enhancer modulation. Probing accessibility, transcription, and YAP1 binding at regulatory elements in HB tumors may provide more insight into YAP1-driven tumorigenesis and expose exploitable vulnerabilities in HB. Using a multiomics approach, we integrated high-throughput transcriptome and chromatin profiling of our murine HB model to identify dynamic activity at candidate cis -regulatory elements (cCREs). We observed that 1301 of 305,596 cCREs exhibit "tumor-modified" (TM) accessibility in HB. We mapped 241 TM enhancers to corresponding genes using accessibility and histone H3K27Ac profiles. Anti-YAP1 cleavage under targets and tagmentation in tumors revealed 66 YAP1-bound TM cCRE/gene pairs, 31 of which decrease expression after YAP1 withdrawal. We validated the YAP1-dependent expression of a putative YAP1 target, Jun dimerization protein 2 (JDP2), in human HB cell lines using YAP1 and LATS1/2 small interfering RNA knockdown. We also confirmed YAP1-induced activity of the Jdp2 TM enhancer in vitro and discovered an analogous human enhancer in silico. Finally, we used transcription factor (TF) footprinting to identify putative YAP1 cofactors and characterize HB-specific TF activity genome wide. Our chromatin-profiling techniques define the regulatory frameworks underlying HB and identify YAP1-regulated gene/enhancer pairs. JDP2 is an extensively validated target with YAP1-dependent expression in human HB cell lines and hepatic malignancies.

FOXM1 Inhibition Enhances the Therapeutic Outcome of Lung Cancer Immunotherapy by Modulating PD-L1 Expression and Cell Proliferation.

Programmed death‐ligand 1 (PD‐L1) is a major target to cancer immunotherapy, and anti‐PD‐L1 and anti‐PD‐1 antibody‐mediated immunotherapy are being increasingly used. However, immune checkpoint inhibitors (ICIs) are ineffective in treating large tumors and cause various immune‐related adverse events in nontarget organs, including life‐threatening cardiotoxicity. Therefore, the development of new therapeutic strategies to overcome these limitations is crucial. The focus of this study is the forkhead box protein M1 (FOXM1), which is identified as a potential therapeutic target for cancer immunotherapy and is associated with the modulation of PD‐L1 expression. Selective small interfering RNA knockdown of FOXM1 or treatment with thiostrepton (TST) significantly reduces PD‐L1 expression in non‐small‐cell lung cancer (NSCLC) cells and inhibits proliferation. Chromatin immunoprecipitation‐PCR reveals that FOXM1 selectively upregulates PD‐L1 expression by binding directly to the PD‐L1 promoter. In vivo animal studies have shown that TST treatment significantly downregulates PD‐L1 expression in human NSCLC tumors, while greatly reducing tumor size without side effects on normal tissues. Combined treatment with TST and anti‐4‐1BB antibody in the LLC‐1 syngeneic tumor model induces synergistic therapeutic outcomes against immune resistant lung tumors as well as 2.72‐folds higher CD3+ T cells in tumor tissues compared to that in the anti‐4‐1BB antibody treatment group.

Synthesis and anti-melanoma effect of 3-O-prenyl glycyrrhetinic acid against B16F10 cells via induction of endoplasmic reticulum stress-mediated autophagy through ERK/AKT signaling pathway.

Melanoma is an aggressive form of cancer with poor prognosis and survival rates and limited therapeutic options. Here, we report the anti-melanoma effect of 3-O-prenyl glycyrrhetinic acid (NPC-402), a derivative of glycyrrhtinic acid, from a reputed medicinal plant Glycyrrhiza glabra against B16F10 cells. We studied the cytotoxic effect of NPC-402 on melanoma cells and investigated the role of mitogen-activated protein (MAP) kinase, AKT axis, and endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated autophagy as the involved signaling cascade by studying specific marker proteins. In this study, 4-phenylbutyric acid (4PBA, a chemical chaperone) and small interference RNA (siRNA) knockdown of C/EBP Homologous Protein (CHOP)/growth arrest- and DNA damage-inducible gene 153(GAD153) blocked NPC-402-mediated autophagy induction, thus confirming the role of ER stress and autophagy in melanoma cell death. NPC-402 induced oxidative stress and apoptosis in melanoma cells, which were effectively mitigated by treatment with N-acetylcysteine (NAC). In vivo studies showed that intraperitoneal (i.p.) injection of NPC-402 at 10 mg/kg (5 days in 1 week) significantly retarded angiogenesis in the Matrigel plug assay and reduced the tumor size and tumor weight without causing any significant toxic manifestation in C57BL/6J mice. We conclude that NPC-402 has a high potential to be developed as a chemotherapeutic drug against melanoma.

Safrole oxide induced 5-HT neuron-like cell differentiation of bone marrow mesenchymal stem cells by elevating G9a.

In our previous study, we found that safrole oxide (SFO) could induce bone marrow mesenchymal stem cell differentiation into neuron-like cells. However, which kind of neuron cells was induced by SFO was unknown. Here, we found that SFO could induce BMSC differentiation into 5-hydroxytryptamine (5-HT) neuron-like cells. Microarray analysis of BMSCs treated with SFO for 6h revealed a total of 35 genes changed more than twice. We selected G9a, a histone methyltransferase for further study. The upregulation of G9a was confirmed by RT-PCR and Western blot analysis. Small interfering RNA knockdown of G9a blocked SFO-induced BMSC differentiation. These results demonstrated that G9a was the pivotal factor in SFO-medicated 5-HT neuronal differentiation of BMSCs. Our findings provide a new clue for further investigating the gene control of BMSC differentiation into 5-HT neuron-like cells and provide a putative strategy for depression diseases therapies.