Reducing FKBP51 Expression in the Ventral Hippocampus Decreases Auditory Fear Conditioning in Male Rats.

Fear conditioning evokes a physiologic release of glucocorticoids that assists learning. As a cochaperone in the glucocorticoid receptor complex, FKBP51 modulates stress-induced glucocorticoid signaling and may influence conditioned fear responses. This study combines molecular and behavioral approaches to examine whether locally reducing FKBP51 expression in the ventral hippocampus is sufficient to affect fear-related behaviors. We hypothesized that reducing FKBP51 expression in the VH would increase glucocorticoid signaling to alter auditory fear conditioning. Adult male rats were injected with an adeno-associated virus (AAV) vector expressing short hairpin - RNAs (shRNA) targeting FKBP5 into the ventral hippocampus to reduce FKBP5 levels or a control AAV. Infusion of FKBP5-shRNA into the ventral hippocampus decreased auditory fear acquisition and recall. Although animals injected with FKBP5-shRNA showed less freezing during extinction recall, the difference was due to a reduced fear recall rather than improved extinction. Reducing ventral hippocampus FKBP51 did not affect exploratory behavior in either the open field test or the elevated zero maze test but did increase passive behavior in the forced swim test, suggesting that the reduction in auditory fear recall was not due to more active responses to acute stress. Furthermore, lower ventral hippocampus FKBP51 levels did not alter corticosterone release in response to restraint stress, suggesting that the reduced fear recall was not due to lower corticosterone release. Our findings suggest FKBP51 in the ventral hippocampus plays a selective role in modulating fear-learning processes and passive behavioral responses to acute stress rather than hypothalamic-pituitary-adrenal axis reactivity or exploratory responses.

Mesenchymal Stromal Cells Nanovesicles Carry microRNA with Nephroprotective Proprieties Regardless of Aging.

Containing information molecules from their parent cells and inclining to fuse with targeted cells, bone marrow mesenchymal stromal cells-derived extracellular vesicles (MSCs- EV) are valuable in nanomedicine. BACKGROUND: The effects of aging on the paracrine mechanism and in the production and action of MSCs-EV and their cargos of miR-26a and siRNA-26a for the treatment of tubular renal cells under nephrotoxicity injury remain unelucidated. OBJECTIVE: The purpose of this study was to evaluate MSCs-EV of different ages and their ability to deliver the cargos of miR-26a and siRNA-26a to target renal tubular cells affected by nephrotoxicity injury. METHODS: In a model of gentamicin-induced nephrotoxicity, renal tubular cells treated with MSCs-EV expressing or not expressing microRNA-26a were analyzed. Western blotting was utilized to evaluate cell cycle markers, and MTT assay was utilized to evaluate auto-renovation capacity. RESULTS: Tubular cells under nephrotoxicity injury showed decreased proliferative capacity, but the treatment in the tubular renal cells under nephrotoxicity injury with MSCs-EV expressing microRNA-26a showed nephroprotective effects, regardless of EV age. While the treatment with EV-mediated siRNA-26a failed to preserve the nephroprotective effects equally, regardless of age. CONCLUSION: Mesenchymal stromal cell nanovesicles carry microRNA with nephroprotective proprieties regardless of aging.

Absence of Heme Oxygenase-1 Affects Trophoblastic Spheroid Implantation and Provokes Dysregulation of Stress and Angiogenesis Gene Expression in the Uterus.

The enzyme heme oxygenase-1 (HO-1) is pivotal in reproductive processes, particularly in placental and vascular development. This study investigated the role of HO-1 and its byproduct, carbon monoxide (CO), in trophoblastic spheroid implantation. In order to deepen our understanding of the role of HO-1 during implantation, we conducted in vivo experiments on virgin and pregnant mice, aiming to unravel the cellular and molecular mechanisms. Using siRNA, HO-1 was knocked down in JEG-3 and BeWo cells and trophoblastic spheroids were generated with or without CO treatment. Adhesion assays were performed after transferring the spheroids to RL-95 endometrial epithelial cell layers. Additionally, angiogenesis, stress, and toxicity RT2-Profiler™ PCR SuperArray and PCR analyses were performed in uterine murine samples. HO-1 knockdown by siRNA impeded implantation in the 3D culture model, but this effect could be reversed by CO. Uteruses from virgin Hmox1-/- females exhibited altered expression of angiogenesis and stress markers. Furthermore, there was a distinct expression pattern of cytokines and chemokines in uteruses from gestation day 14 in Hmox1-/- females compared to Hmox1+/+ females. This study strongly supports the essential role of HO-1 during implantation. Moreover, CO appears to have the potential to compensate for the lack of HO-1 during the spheroid attachment process. The absence of HO-1 results in dysregulation of angiogenesis and stress-related genes in the uterus, possibly contributing to implantation failure.

CR5/CCL5 axis is linked to a poor outcome, and inhibition reduces metastasis in oral squamous cell carcinoma.

PURPOSE: The CCR5/CCL5 axis is essential for interactions between malignant cells and microenvironment components, promoting tumor progression in oral squamous cell carcinoma (OSCC). This study aims to evaluate the association of CCL5 and CCR5 with the behavior of oral cancer and assess the therapeutic potential of a CCR5 antagonist. METHODS: A retrospective study to analyze CCR5 and CCL5 expression on paraffin-embedded tissues was performed. In cell lines, rhCCL5 was added to induce CCR5-related pathways, and Maraviroc and shRNA against CCR5 were used to neutralize the receptor. Finally, an in vivo murine orthotopic xenograft model of tongue cancer was used to evaluate Maraviroc as an oncologic therapy. After 15 days, the mice were killed, and the primary tumors and cervical lymph nodes were analyzed. RESULTS: The expression of CCR5 was associated with clinical stage and metastasis, and CCL5 was related to overall survival. Adding rhCCL5 induced cell proliferation, while shRNA and Maraviroc reduced it in a dose-dependent manner. Maraviroc treatment also increased apoptosis and modified cytoskeletal organization. In vivo, Maraviroc reduced neck metastasis. CONCLUSIONS: The effects of CCR5 antagonists in OSCC have been poorly studied, and this study reports in vitro and in vivo evidence for the effects of Maraviroc in OSCC. Our results suggest that the CCR5/CCL5 axis plays a role in oral cancer behavior, and that its inhibition is a promising new therapy alternative.

Preclinical study of LMP1-RNAi-based anti-tumor therapy in EBV-positive nasopharyngeal carcinoma.

RNA interference (RNAi) treatment has been proven to be an important therapeutic approach in cancer based on downregulation of target-oncogenes, but its clinical efficacy still needs further investigation. LMP1 is usually presented by Epstein-Barr virus (EBV)-positive tumor cells like EBV-associated nasopharyngeal carcinoma (NPC) and acts as an oncogene in tumorigenesis. However, the mechanism of LMP1 as a proto-oncogene in nasopharyngeal carcinoma is still unclear. Two sequence-specific shRNAs 1 and 2 were designed to target the different nucleotide loci of EBV latent antigen LMP1 gene and a series of in vivo and in vitro experiments were performed to investigate the therapeutic effect of sequence-specific shRNAs targeting LMP1 and its related molecular mechanisms in EBV-positive NPC. LMP1-shRNA2 generated a truncated LMP1 mRNA and protein, whereas LMP1-shRNA1 completely blocked LMP1 mRNA and protein expression. Both LMP1-shRNAs inhibited the proliferation and migration of NPC cells overexpressing LMP1 (NPC-LMP1) as well as the NPC-associated myeloid-derived suppressor cell (MDSC) expansion in vitro. However, LMP1-shRNA2 maintained the immunogenicity of NPC-LMP1 cells, which provoked MHC-class I-dependent T cell recognition. LMP1-shRNAs inhibited tumor growth in nude mice but did not reach statistical significance compared to control groups, while the LDH nanoparticle loaded LMP1-shRNAs and the antigen-specific T cells induced by NPC-LMP1 cells treated with LMP1-shRNA2 significantly reduced tumor growth in vivo. LMP1-RNAi-based anti-tumor therapy could be a new hope for the clinical efficacy of RNAi treatment of tumors like NPC.

Collagen V α1 Chain Decrease in Papillary Dermis from Early Systemic Sclerosis: A New Proposal in Cutaneous Fibrosis Molecular Structure.

Cutaneous fibrosis is one of the main features of systemic sclerosis (SSc). Recent findings correlated abnormal collagen V (Col V) deposition in dermis with skin thickening and disease activity in SSc. Considering that Col V is an important regulator of collagen fibrillogenesis, understanding the role of Col V in the first two years of the skin fibrosis in SSc (early SSc) can help to determine new targets for future treatments. In this study, we analyzed the morphological, ultrastructural and molecular features of α1(V) and α2(V) chains and the expression of their coding genes COL5A1 and COL5A2 in collagen fibrillogenesis in early-SSc. Skin biopsies were obtained from seven consecutive treatment-naïve patients with SSc-related fibrosis and four healthy controls. Our data showed increased α1(V) and α2(V) chain expression in the reticular dermis of early-SSc patients; however, immunofluorescence and ultrastructural immunogold staining determined a significant decreased expression of the α1(V) chain along the dermoepidermal junction in the papillary dermis from early-SSc-patients in relation to the control (12.77 ± 1.34 vs. 66.84 ± 3.36; p < 0.0001). The immunoblot confirmed the decreased expression of the α1(V) chain by the cutaneous fibroblasts of early-SSc, despite the increased COL5A1 and COL5A2 gene expression. In contrast, the α2(V) chain was overexpressed in the small vessels (63.18 ± 3.56 vs. 12.16 ± 0.81; p < 0.0001) and capillaries (60.88 ± 5.82 vs. 15.11 ± 3.80; p < 0.0001) in the reticular dermis of early-SSc patients. Furthermore, COLVA2 siRNA in SSc cutaneous fibroblasts resulted in a decreased α1(V) chain expression. These results highlight an intense decrease in the α1(V) chain along the dermoepidermal junction, suggesting an altered molecular histoarchitecture in the SSc papillary dermis, with a possible decrease in the expression of the α1(V)3 homotrimeric isoform, which could interfere with the thickening and cutaneous fibrosis related to SSc.

First characterization of PIWI-interacting RNA clusters in a cichlid fish with a B chromosome.

BACKGROUND: B chromosomes are extra elements found in several eukaryote species. Usually, they do not express a phenotype in the host. However, advances in bioinformatics over the last decades have allowed us to describe several genes and molecular functions related to B chromosomes. These advances enable investigations of the relationship between the B chromosome and the host to understand how this element has been preserved in genomes. However, considering that transposable elements (TEs) are highly abundant in this supernumerary chromosome, there is a lack of knowledge concerning the dynamics of TE control in B-carrying cells. Thus, the present study characterized PIWI-interacting RNA (piRNA) clusters and pathways responsible for silencing the mobilization of TEs in gonads of the cichlid fish Astatotilapia latifasciata carrying the B chromosome. RESULTS: Through small RNA-seq and genome assembly, we predicted and annotated piRNA clusters in the A. latifasciata genome for the first time. We observed that these clusters had biased expression related to sex and the presence of the B chromosome. Furthermore, three piRNA clusters, named curupira, were identified in the B chromosome. Two of them were expressed exclusively in gonads of samples with the B chromosome. The composition of these curupira sequences was derived from LTR, LINE, and DNA elements, representing old and recent transposition events in the A. latifasciata genome and the B chromosome. The presence of the B chromosome also affected the expression of piRNA pathway genes. The mitochondrial cardiolipin hydrolase-like (pld6) gene is present in the B chromosome, as previously reported, and an increase in its expression was detected in gonads with the B chromosome. CONCLUSIONS: Due to the high abundance of TEs in the B chromosome, it was possible to investigate the origin of piRNA from these jumping genes. We hypothesize that the B chromosome has evolved its own genomic guardians to prevent uncontrolled TE mobilization. Furthermore, we also detected an expression bias in the presence of the B chromosome over A. latifasciata piRNA clusters and pathway genes.

Impact of Degree of Ionization and PEGylation on the Stability of Nanoparticles of Chitosan Derivatives at Physiological Conditions.

Nowadays, the therapeutic efficiency of small interfering RNAs (siRNA) is still limited by the efficiency of gene therapy vectors capable of carrying them inside the target cells. In this study, siRNA nanocarriers based on low molecular weight chitosan grafted with increasing proportions (5 to 55%) of diisopropylethylamine (DIPEA) groups were developed, which allowed precise control of the degree of ionization of the polycations at pH 7.4. This approach made obtaining siRNA nanocarriers with small sizes (100-200 nm), positive surface charge and enhanced colloidal stability (up to 24 h) at physiological conditions of pH (7.4) and ionic strength (150 mmol L-1) possible. Moreover, the PEGylation improved the stability of the nanoparticles, which maintained their colloidal stability and nanometric sizes even in an albumin-containing medium. The chitosan-derivatives displayed non-cytotoxic effects in both fibroblasts (NIH/3T3) and macrophages (RAW 264.7) at high N/P ratios and polymer concentrations (up to 0.5 g L-1). Confocal microscopy showed a successful uptake of nanocarriers by RAW 264.7 macrophages and a promising ability to silence green fluorescent protein (GFP) in HeLa cells. These results were confirmed by a high level of tumor necrosis factor-α (TNFα) knockdown (higher than 60%) in LPS-stimulated macrophages treated with the siRNA-loaded nanoparticles even in the FBS-containing medium, findings that reveal a good correlation between the degree of ionization of the polycations and the physicochemical properties of nanocarriers. Overall, this study provides an approach to enhance siRNA condensation by chitosan-based carriers and highlights the potential of these nanocarriers for in vivo studies.

Melatonin Reverses the Warburg-Type Metabolism and Reduces Mitochondrial Membrane Potential of Ovarian Cancer Cells Independent of MT1 Receptor Activation.

Ovarian cancer (OC) is the most lethal gynecologic malignancy, and melatonin has shown various antitumor properties. Herein, we investigated the influence of melatonin therapy on energy metabolism and mitochondrial integrity in SKOV-3 cells and tested whether its effects depended on MT1 receptor activation. SKOV-3 cells were exposed to different melatonin concentrations, and experimental groups were divided as to the presence of MT1 receptors (melatonin groups) or receptor absence by RNAi silencing (siRNA MT1+melatonin). Intracellular melatonin levels increased after treatment with melatonin independent of the MT1. The mitochondrial membrane potential of SKOV-3 cells decreased in the group treated with the highest melatonin concentration. Melatonin reduced cellular glucose consumption, while MT1 knockdown increased its consumption. Interconversion of lactate to pyruvate increased after treatment with melatonin and was remarkable in siRNA MT1 groups. Moreover, lactate dehydrogenase activity decreased with melatonin and increased after MT1 silencing at all concentrations. The UCSC XenaBrowser tool showed a positive correlation between the human ASMTL gene and the ATP synthase genes, succinate dehydrogenase gene (SDHD), and pyruvate dehydrogenase genes (PDHA and PDHB). We conclude that melatonin changes the glycolytic phenotype and mitochondrial integrity of SKOV-3 cells independent of the MT1 receptor, thus decreasing the survival advantage of OC cells.

ANKHD1 contributes to the malignant phenotype of triple-negative breast cancer cells.

Ankyrin repeat and KH domain-containing protein 1, ANKHD1, has been identified as a regulator of signaling pathways and cellular processes of relevance in carcinogenesis. However, the role of ANKHD1 in breast cancer remains unclear. The aim of the present study was to characterize the expression pattern and involvement of ANKHD1 in the malignant phenotype of breast cancer cell lines and to investigate the clinical relevance of ANKHD1 in a breast cancer context. Gene and protein expressions were assessed in the cell lines by quantitative reverse transcription PCR and Western blot analysis, respectively, and ANKHD1 silencing through siRNA transfection was conducted for further in vitro functional assays. The expression of ANKHD1 was identified in non-tumorigenic breast epithelium and breast cancer cell lines, but differences in cellular localization were found among the neoplasia subtypes. ANKHD1 silencing reduced the viability, clonogenicity, and migration of triple-negative breast cancer (TNBC) cells. Bioinformatics analyses demonstrated that patients with triple-negative basal-like 2 and mesenchymal breast cancer subtypes had high ANKHD1 expression associated with poor recurrence-free survival. Therefore, these data indicate that ANKHD1 relevance in breast cancer varies among its subtypes, indicating the importance of ANKHD1 in TNBC.

Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus.

Post-transcriptional gene silencing (PTGS) is an evolutionarily conserved plant defense mechanism against viruses. This paper aimed to evaluate a dsDNA construct (77 bp) as a template for in vitro production of virus-derived artificial small hairpin RNAs (shRNAs) and test for their potential to trigger the RNAi mechanism in Nicotiana benthamiana plants against CMV after their foliar infiltration. This approach allowed for the production of significant amounts of shRNAs (60-mers) quickly and easily. The gene silencing was confirmed using polymerase chain reaction (PCR), immunological-based assays, and real-time PCR (qPCR). The highest levels of gene silencing were recorded for mRNAs coding for replication protein (ORF1a), the viral suppressor of RNA silencing (ORF2b), and the capsid protein (ORF3b), with 98, 94, and 70% of total transcript silencing, respectively. This protocol provides an alternative to producing significant shRNAs that can effectively trigger the RNAi mechanism against CMV.

Gene knockdown of HCN2 ion channels in the ventral tegmental area reduces ethanol consumption in alcohol preferring rats.

Background: Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) ionic channels are known to play a key role in the control of neuron excitability and have been proposed as a molecular target of ethanol. Previous studies in rats have shown that gene-induced overexpression of the HCN2 channel in the ventral tegmental area (VTA) increases the rewarding effects of ethanol and its intake by the animals.Objective: The aim of this work was to study the effects of VTA HCN2 gene knockdown in the voluntary ethanol consumption of alcohol-preferring UChB rats.Methods: Two lentiviral vectors were generated; LV-siRNA-HCN2, coding for a siRNA that elicited >95% reduction of HCN2 protein levels in vitro, and a control vector coding for a scrambled siRNA sequence. Female UChB naïve rats (n = 14) were microinjected into the VTA with LV-siRNA-HCN2 or the scrambled control vector (n = 11). Four days after, animals were given a daily free access to 10% ethanol and water for 10 days.Results: Rats treated with the LV-siRNA-HCN2 vector showed a ~ 70% reduction (p < .001) in their ethanol preference and ethanol intake compared to control animals. No changes were observed in the total fluid intake of both groups. HCN2 levels in the VTA were measured by Western blot showing a reduction of 40% (p < .05) in the rats injected with LV-siRNA-HCN2, compared to control animals.Conclusion: These results show that knockdown of HCN2 ionic channels in the VTA of UChB rats markedly reduces their voluntary ethanol intake, supporting the idea that HCN2 channels may constitute a therapeutic target for alcohol use disorders.

Single-cell transcriptional analysis of human endothelial colony-forming cells from patients with low VWF levels.

von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.

Pan-cancer analysis of non-oncogene addiction to DNA repair.

Cancer cells usually depend on the aberrant function of one or few driver genes to initiate and promote their malignancy, an attribute known as oncogene addiction. However, cancer cells might become dependent on the normal cellular functions of certain genes that are not oncogenes but ensure cell survival (non-oncogene addiction). The downregulation or silencing of DNA repair genes and the consequent genetic and epigenetic instability is key to promote malignancy, but the activation of the DNA-damage response (DDR) has been shown to become a type of non-oncogene addiction that critically supports tumour survival. In the present study, a systematic evaluation of DNA repair addiction at the pan-cancer level was performed using data derived from The Cancer Dependency Map and The Cancer Genome Atlas (TCGA). From 241 DDR genes, 59 were identified as commonly essential in cancer cell lines. However, large differences were observed in terms of dependency scores in 423 cell lines and transcriptomic alterations across 18 cancer types. Among these 59 commonly essential genes, 14 genes were exclusively associated with better overall patient survival and 19 with worse overall survival. Notably, a specific molecular signature among the latter, characterized by DDR genes like UBE2T, RFC4, POLQ, BRIP1, and H2AFX showing the weakest dependency scores, but significant upregulation was strongly associated with worse survival. The present study supports the existence and importance of non-oncogenic addiction to DNA repair in cancer and may facilitate the identification of prognostic biomarkers and therapeutic opportunities.

Transposable Element Expression and Regulation Profile in Gonads of Interspecific Hybrids of Drosophila arizonae and Drosophila mojavensis wrigleyi.

Interspecific hybridization may lead to sterility and/or inviability through differential expression of genes and transposable elements (TEs). In Drosophila, studies have reported massive TE mobilization in hybrids from interspecific crosses of species presenting high divergence times. However, few studies have examined the consequences of TE mobilization upon hybridization in recently diverged species, such as Drosophila arizonae and D. mojavensis. We have sequenced transcriptomes of D. arizonae and the subspecies D. m. wrigleyi and their reciprocal hybrids, as well as piRNAs, to analyze the impact of genomic stress on TE regulation. Our results revealed that the differential expression in both gonadal tissues of parental species was similar. Globally, ovaries and testes showed few deregulated TEs compared with both parental lines. Analyses of small RNA data showed that in ovaries, the TE upregulation is likely due to divergence of copies inherited from parental genomes and lack of piRNAs mapping to them. Nevertheless, in testes, the divergent expression of genes associated with chromatin state and piRNA pathway potentially indicates that TE differential expression is related to the divergence of regulatory genes that play a role in modulating transcriptional and post-transcriptional mechanisms.

Atypical DNA methylation, sRNA-size distribution, and female gametogenesis in Utricularia gibba.

The most studied DNA methylation pathway in plants is the RNA Directed DNA Methylation (RdDM), a conserved mechanism that involves the role of noncoding RNAs to control the expansion of the noncoding genome. Genome-wide DNA methylation levels have been reported to correlate with genome size. However, little is known about the catalog of noncoding RNAs and the impact on DNA methylation in small plant genomes with reduced noncoding regions. Because of the small length of intergenic regions in the compact genome of the carnivorous plant Utricularia gibba, we investigated its repertoire of noncoding RNA and DNA methylation landscape. Here, we report that, compared to other angiosperms, U. gibba has an unusual distribution of small RNAs and reduced global DNA methylation levels. DNA methylation was determined using a novel strategy based on long-read DNA sequencing with the Pacific Bioscience platform and confirmed by whole-genome bisulfite sequencing. Moreover, some key genes involved in the RdDM pathway may not represented by compensatory paralogs or comprise truncated proteins, for example, U. gibba DICER-LIKE 3 (DCL3), encoding a DICER endonuclease that produces 24-nt small-interfering RNAs, has lost key domains required for complete function. Our results unveil that a truncated DCL3 correlates with a decreased proportion of 24-nt small-interfering RNAs, low DNA methylation levels, and developmental abnormalities during female gametogenesis in U. gibba. Alterations in female gametogenesis are reminiscent of RdDM mutant phenotypes in Arabidopsis thaliana. It would be interesting to further study the biological implications of the DCL3 truncation in U. gibba, as it could represent an initial step in the evolution of RdDM pathway in compact genomes.

Syndecan-1 Depletion Has a Differential Impact on Hyaluronic Acid Metabolism and Tumor Cell Behavior in Luminal and Triple-Negative Breast Cancer Cells.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are major components of the glycocalyx. The secreted GAG and CD44 ligand hyaluronic acid (HA), and the cell surface PG syndecan-1 (Sdc-1) modulate the expression and activity of cytokines, chemokines, growth factors, and adhesion molecules, acting as critical regulators of tumor cell behavior. Here, we studied the effect of Sdc-1 siRNA depletion and HA treatment on hallmark processes of cancer in breast cancer cell lines of different levels of aggressiveness. We analyzed HA synthesis, and parameters relevant to tumor progression, including the stem cell phenotype, Wnt signaling constituents, cell cycle progression and apoptosis, and angiogenic markers in luminal MCF-7 and triple-negative MDA-MB-231 cells. Sdc-1 knockdown enhanced HAS-2 synthesis and HA binding in MCF-7, but not in MDA-MB-231 cells. Sdc-1-depleted MDA-MB-231 cells showed a reduced CD24-/CD44+ population. Furthermore, Sdc-1 depletion was associated with survival signals in both cell lines, affecting cell cycle progression and apoptosis evasion. These changes were linked to the altered expression of KLF4, MSI2, and miR-10b and differential changes in Erk, Akt, and PTEN signaling. We conclude that Sdc-1 knockdown differentially affects HA metabolism in luminal and triple-negative breast cancer model cell lines and impacts the stem phenotype, cell survival, and angiogenic factors.

The pluripotency transcription factor OCT4 represses heme oxygenase-1 gene expression.

In embryonic stem (ES) cells, oxidative stress control is crucial for genomic stability, self-renewal, and cell differentiation. Heme oxygenase-1 (HO-1) is a key player of the antioxidant system and is also involved in stem cell differentiation and pluripotency acquisition. We found that the HO-1 gene is expressed in ES cells and induced after promoting differentiation. Moreover, downregulation of the pluripotency transcription factor (TF) OCT4 increased HO-1 mRNA levels in ES cells, and analysis of ChIP-seq public data revealed that this TF binds to the HO-1 gene locus in pluripotent cells. Finally, ectopic expression of OCT4 in heterologous systems repressed a reporter carrying the HO-1 gene promoter and the endogenous gene. Hence, this work highlights the connection between pluripotency and redox homeostasis.

Morphological and biological properties of silica nanoparticles for CRTC3-siRNA delivery and downregulation of the RGS2 expression in preadipocytes.

The aim of this study was to characterize the morphological properties of amorphous silica nanoparticles (SiO2 NPs), their cytotoxicity and intracellular location within Human Osteoblasts (HOB). Additionally, SiO2 NPs were explored for their effectivity as carriers of CRTC3-siRNA on Human Preadipocytes (HPAd), and thus downregulate RGS2 gene expression. SiO2 NPs were synthesized using the method of Stöber at 45 °C, 56 °C, and 62 °C. These were characterized via TEM with EDS, Zeta Potential and FT-IR. Cytotoxicity was evaluated by XTT at three concentrations 50, 100 and 500 µg/mL; SiO2 NPs intracellular localization was observed through Confocal Laser Scanning Microscope. Delivering siRNA effectivity was measured by RT-qPCR. Morphology of SiO2 NPs was spherical with a range size from 64 to 119 nm; their surface charge was negative. Confocal images demonstrated that SiO2 NPs were located within cellular cytoplasm. At a SiO2 NPs concentration of 500 µg/mL HOB viability decreased, while at 50 µg/mL and 100 µg/mL cell viability was not affected regardless SiO2 NPs size. SiO2 NPs-CRTC3-siRNA are effective to down-regulate RGS2 gene expression in HPAd without cytotoxic effects. The developed SiO2 NPs-CRTC3-siRNA are a promising tool as a delivery vehicle to control obesity.

Wnt5a promotes hippocampal postsynaptic development and GluN2B-induced expression via the eIF2α HRI kinase.

Wnt signaling plays a key role in neurodevelopment and neuronal maturation. Specifically, Wnt5a stimulates postsynaptic assemblies, increases glutamatergic neurotransmission and, through calcium signaling, generates nitric oxide (NO). Trying to unveil the molecular pathway triggering these postsynaptic effects, we found that Wnt5a treatment induces a time-dependent increases in the length of the postsynaptic density (PSD), elicits novel synaptic contacts and facilitates F-actin flow both in in vitro and ex vivo models. These effects were partially abolished by the inhibition of the Heme-regulated eukaryotic initiation factor 2α (HRI) kinase, a kinase which phosphorylates the initiation translational factor eIF2α. When phosphorylated, eIF2α normally avoids the translation of proteins not needed during stress conditions, in order to avoid unnecessary energetic expenses. However, phosphorylated eIF2α promotes the translation of some proteins with more than one open reading frame in its 5' untranslated region. One of these proteins targeted by Wnt-HRI-eIF2α mediated translation is the GluN2B subunit of the NMDA receptor. The identified increase in GluN2B expression correlated with increased NMDA receptor function. Considering that NMDA receptors are crucial for excitatory synaptic transmission, the molecular pathway described here contributes to the understanding of the fast and plastic translational mechanisms activated during learning and memory processes.