Target Gene Profiles Research Articles

Immunogenic cell death mediated TLR3/4-activated MSCs in U87 GBM cell line

Background and aimsGlioblastoma (GBM) is an aggressive primary brain cancer with no promising curative therapies. It has been indicated that MSCs can interact with the tumour microenvironment (TME) through the secretion of soluble mediators regulating intercellular signalling within the TME. TLRs are a multigene family of pattern recognition receptors with evolutionarily conserved regions and are widely expressed in immune and other body cells. MSCs by TLRs can recognize conserved molecular components (DAPMPs and PAPMPs) and activate signalling pathways, which regulate immune and inflammatory responses. MSCs may exert immunomodulatory functions through interaction with their expressed toll-like receptors (TLRs) and exert a protective effect against tumour antigens. As an emerging approach, we aimed to monitor the U87 cell line growth, migration and death markers following specific TLR3/4-primed-MSCs-CMs treatment. Methods and resultsWe investigated the phenotypic and functional outcomes of primed-CMs and glioma cell line co-culture following short-term, low-dose TLR3/4 priming. The gene expression profile of target genes, including apoptotic markers and related genes, was analyzed by qRT-PCR. MicroRNA-Seq examined the miRNA expression patterns, and flow cytometry evaluated the cell viability and cycle stages. The results showed significant changes in apoptosis and likely necroptosis-related markers following TLR3/4-primed-MSCs-CMs exposure in the glioma cell line. Notably, we observed a considerable induction of selective pro-apoptotic markers and both the early and late stages of apoptosis in treated U87 cell lines. Additionally, the migration rate of glioma cells significantly decreased following MSCs-CM treatment. ConclusionOur findings confirmed that the exposure of TLR3/4-activated-MSCs-CMs with glioma tumour cells possibly changes the immunogenicity of the tumour microenvironment and induces immunogenic programmed cell death. Our results can support the idea that TLR3/4-primed-MSCs can lead to innate immune-mediated cell death and modify tumour cell biology in invasive and metastatic cancers.

400 Brain AVMs-Related MicroRNAs: Machine Learning Algorithm for Expression Profiles of Target Genes

INTRODUCTION: microRNAs (miRNAs) are a class of non-coding RNAs playing a myriad of important roles in regulating gene expression. Of note, recent work demonstrated a critical role of miRNAs in the genesis and progression of brain arteriovenous malformations (bAVMs). METHODS: A PRISMA-based literature review was performed using PubMed/Medline database with the following search terms: "brain arteriovenous malformations", "cerebral arteriovenous malformations", "microRNA", and "miRNA". All preclinical and clinical studies written in English, regardless of date, were selected. For our bioinformatic analyses, miRWalk and miRTarBase machine learning algorithms were employed; the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was quired for associated pathways/functions. RESULTS: 4 studies were ultimately included in the final analyses. Sequencing data consistently revealed the decreased expression of miR-18a in bAVM-endothelial cells, resulting in increased levels of vascular endodermal growth factor (VEGF), Id-1, matrix metalloproteinase, and growth signals. Our analyses also suggest that the downregulation of miR-137 and miR-195* within vascular smooth muscle cells (VSMCs) may foster the activation of inflammation, aberrant angiogenesis, and phenotypic switching. In the peripheral blood, the overexpression of miR-7-5p, miR-629-5p, miR-199a-5p, miR-200b-3p, and let-7b-5p may contribute to endothelial proliferation and nidus development. The machine learning algorithms employed confirmed associations between miRNA-related target networks, vascular rearrangement, and bAVM progression. CONCLUSIONS: miRNAs expression appears to be critical in managing bAVMs' post-transcriptional signals. Targets of microRNAs regulate canonical vascular proliferation and reshaping. Although additional scientific evidence is needed, the identification of bAVM miRNA signatures may facilitate the development of novel prognostic/diagnostic tools and molecular therapies for bAVMs.

Selection of reliable reference genes for gene expression studies involving peripheral blood mononuclear cells in small ruminants

Quantitative PCR (qPCR) is a highly sensitive, cost effective, and routinely used molecular assay for analyzing the gene expression patterns of specific target genes across tissues, pathological conditions, treatment regimes, and physiological states. However, normalization of expression profiles of target genes using stable reference genes (RGs) is a critical step to ensure the accuracy of relative quantification using qPCR. In this study, we evaluated the stability of fourteen potential existing reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, and UXT) in the peripheral blood mononuclear cells (PBMCs) of healthy sheep and goats to determine the most stable RGs. These candidate genes belong to different functional classes and were chosen based on published literature on commonly used RGs in different livestock species. Four different analytical approaches (geNorm, NormFinder, BestKeeper, and ΔCt analysis) as well as RefFinder, an online tool which integrates the geometric means of these four prominent stability algorithms were utilized to determine a comprehensive ranking of the investigated genes. Our data indicates that PPIB, BACH1, ACTB, and PPIA are the most suitable RGs, while RPLP0, GAPDH and RPS15 are the most variable and unsuitable genes for normalization of qPCR data in the PBMCs of sheep and goats. The results of this study provide useful resource for researchers engaged in unravelling the transcriptional landscape of PBMCs of small ruminants for various scientific investigations.

Identification and validation of stable reference genes for expression profiling of target genes in diverse ovine tissues

Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1, and B2M emerged as the most stable RGs, while RPS9, RPS15, and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1, and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1, and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues.

Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable.

Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Because clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC/RNA sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2,500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NOD/SCID gamma mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Because inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL/MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL/MAPK. We report on a novel mechanism of AXL/MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN.

Formononetin attenuates high glucose-induced neurotoxicity by negatively regulating oxidative stress and mitochondrial dysfunction in Schwann cells via activation of SIRT3

High glucose induces Schwann cells death and neurotoxicity. Formononetin was originally found in Astragalus membranaceus and showed anti-tumor and anti-neuroinflammation properties. The aim of this study is to explore the molecular mechanism underlying the neuroprotective effects of formononetin and identify its direct protein target. The effects of formononetin on oxidative stress and mitochondrial dysfunction in Schwann cells induced by high glucose were investigated. High glucose treatment significantly induced oxidative stress, mitochondrial dysfunction and apoptosis in Schwann cells, while these effects were partially or completely prevented by co-treatment with formononetin. Mechanistically, we found that SIRT3/PGC-1α/SOD2 pathway was activated by formononetin under high glucose conditions as evidenced by western blotting. Knockdown of SIRT3 by siRNA delivery reversed the protective effects of formononetin on high glucose-induced Schwann cells injury and changes in expression profile of SIRT3 downstream target genes. Molecular docking, thermal shift assay and surface plasmon resonance assay revealed a direct binding between formononetin and SIRT3. Taken together, we identified a novel SIRT3 activator formononetin and revealed its beneficial effects on high glucose-induced neurotoxicity, suggesting that targeting SIRT3 in Schwann cells may be a new approach for treatment of peripheral nerve regeneration related diseases such as diabetic peripheral neuropathy.

Acquired resistance to a GPRC5D-directed T-cell engager in multiple myeloma is mediated by genetic or epigenetic target inactivation.

Bispecific antibodies targeting GPRC5D demonstrated promising efficacy in multiple myeloma, but acquired resistance usually occurs within a few months. Using a single-nucleus multi-omic strategy in three patients from the MYRACLE cohort (ClinicalTrials.gov registration: NCT03807128 ), we identified two resistance mechanisms, by bi-allelic genetic inactivation of GPRC5D or by long-range epigenetic silencing of its promoter and enhancer regions. Molecular profiling of target genes may help to guide the choice of immunotherapy and early detection of resistance in multiple myeloma.

Posttranslational splicing modifications as a key mechanism in cytarabine resistance in acute myeloid leukemia

Despite the approval of several drugs for AML, cytarabine is still widely used as a therapeutic approach. However, 85% of patients show resistance and only 10% overcome the disease. Using RNA-seq and phosphoproteomics, we show that RNA splicing and serine-arginine-rich (SR) proteins phosphorylation were altered during cytarabine resistance. Moreover, phosphorylation of SR proteins at diagnosis were significantly lower in responder than non-responder patients, pointing to their utility to predict response. These changes correlated with altered transcriptomic profiles of SR protein target genes. Notably, splicing inhibitors were therapeutically effective in treating sensitive and resistant AML cells as monotherapy or combination with other approved drugs. H3B-8800 and venetoclax combination showed the best efficacy in vitro, demonstrating synergistic effects in patient samples and no toxicity in healthy hematopoietic progenitors. Our results establish that RNA splicing inhibition, alone or combined with venetoclax, could be useful for the treatment of newly diagnosed or relapsed/refractory AML.

Expression profile of plasma microRNAs and target genes in patients with complicated pregnancy

Aim: to assess features of placental transcriptome in patients with complicated pregnancy course and outcome.Materials and Methods. A prospective observational comparative study in parallel groups was carried out by examining 44 patients divided into three groups: group 1 included 13 pregnant women with moderate and severe preeclampsia (PE), group 2 consisted of 12 patients with fetal growth retardation (FGR), control group included 19 clinically healthy women with uncomplicated pregnancy and delivery. All women enrolled to the study underwent a comprehensive examination, including history collection, general and obstetric-gynecological examination, laboratory and instrumental studies. Special research methods were used during the study, which included placental tissue sampling followed by analyzed expression profile of 12 microRNAs using real-time polymerase chain reaction (PCR).Results. The PCR data indicated about lack of expression for analyzed miR-210-3p, miR-320-3p, miR-1304-5p and miR-375-5p in placental tissue samples in FGR patients. No significant differences in placental miRNA levels were observed in FGR vs. PE and control group. Analysis of placenta-specific microRNAs in women with PE vs. control group showed a significantly down modulated expression level for miR-517a-3p (p = 0.025), miR-517c-3p (p = 0.036), miR-574-5p (p = 0.015) along with upregulated expression of miR-20a-5p (p = 0.046).Conclusion. The data obtained evidence that pregnancy-related complications are characterized by specific molecular changes at placental transcriptome level.

Argon-stimulated nitric oxide production and its function in alfalfa cadmium tolerance

Recent results showed that argon may have great potential in both medicines (especially) and agriculture. However, how argon positively influences crop physiology remains elusive. Here, we observed that the stimulation of nitric oxide (NO) production upon cadmium (Cd) stress in hydroponic alfalfa root tissues was strengthened by argon-rich water and/or a NO-releasing compound. The pharmacological results further indicated that above potential source of NO stimulation achieved by argon might be attributed to NO synthase (NOS) and nitrate reductase (NR). Under hydroponic and pot conditions, the improvement of Cd tolerance elicited by argon, confirmed by the alleviation in the plant growth inhibition, oxidative damage, and Cd accumulation, was sensitive to the scavenger of NO. These results suggested a crucial role of argon-induced NO synthesis in response to Cd stress. Subsequent evidence showed that the improved iron homeostasis and increased S-nitrosylation were also dependent on argon-stimulated NO. Above results were matched with the transcriptional profiles of representative target genes involved in heavy metal detoxification, antioxidant defence, and iron homeostasis. Taken together, our results clearly indicated that argon-stimulated NO production contributes to Cd tolerance by favoring important defense strategies against heavy metal exposure.

Mutational profiling of true single circulating tumor cells (sCTC) in metastatic lung cancer to determine genomic heterogeneity and drug-resistance signatures.

e21024 Background: Omics of single Circulating tumor cells (CTCs) is highly envisioned though challenging. . Genotyping of TRUE sCTC capture and omics data can determine the molecular heterogeneity of a single cell without the stringent need for deep sequencing compared to tissue or blood (1/100th fold compared to ctDNA). Here, we show the utility of sCTC genome profiling to determine inter and intra-patient mutational heterogeneity and compare the outcome with matched ensembled ctDNA mutational profiles. Methods: OncoRADAR an antibody-affinity-based platform was used to capture live sCTCs without cell fixing. 50 sCTCs (90 %) and clusters (10 %) were isolated from 20 metastatic lung cancer pts for mutational genotyping. Amplified whole genomes were subjected to targeted hybrid-capture comprehensive genome profiling. Libraries prepared using a custom-designed comprehensive gene panel OncoIndx, targeting 600 cancer-relevant exons including MSI and HRR pathway genes, were sequenced in paired-end mode on the Illumina platform. Variant calling was performed using an in-house developed bioinformatics pipeline. Pathway enrichment analysis was performed manually using data enriched from different gene pathway databases. Results: 81 mutations were detected in 56 genes across all sCTCs with pathogenic, likely pathogenic, and unknown significance. The mutational landscape included SNVs (38.6%), splice variants (10%), frameshift mutations (0.8%), and truncations (50.4%). From a subset of patients (10 %), multiple sCTCs showed PTEN variant cooccurring with ESR1 and MET. This suggest a clonal selection and emergence of subclones during progression. Pathways related to drug resistance, migration, deregulated cell cycle, and signaling were significantly enriched in multiple sCTCs when compared to plasma genotyping. Low average TMB (1.89), LOH (3.9) and HRD (14.67%), and high MSI (28.68%) scores were observed. In contrast, matching ctDNA had higher average LOH (18.67), HRD (45.08%), and lower MSI (0.99) scores. Multiple sCTCs from a small fraction of patients had comparable values for genome stability indicators, suggesting maintained genome stability. Genotyping of sCTC among patients showed a high variability compared to comprehensive analysis at the ctDNA level. Conclusions: Targeted gene profiling of true sCTCs revealed multiple variants that remained undetected in ctDNA, enabling a better understanding of tumor heterogeneity. Analysis of multiple sCTCs from the same patient underscores the importance of clonal evolution and plasticity during metastasis. Longitudinal monitoring of sCTCs may reveal personalized markers for disease prevalence, metastasis and relapse. sCTC genotyping may help design personalized therapy with better precision.

Comprehensive plasma genotyping reveals mutational signatures that confer resistance to tyrosine kinase inhibitors (TKI) in patients with advanced lung cancer.

e20505 Background: Targeting actionable mutations with TKI has altered the treatment paradigm for progressive lung cancers. Significant fraction of patients remain unresponsive to TKI due to cooccurrence of activating EGFR mutations (mEGFR) with other driver mutations. Biopsy of the advanced patients is challenging and is often inconclusive due to tumor heterogeneity. Plasma genotyping using a comprehensive gene panel poses a viable alternative to obtain a broad mutational landscape for Tx decisions. Here we demonstrate the clinical utility of our comprehensive gene panel for detecting actionable mutations and novel alterations from advanced lung cancer patients. Methods: Mutational landscape of 82 advanced-stage lung cancer patients was monitored longitudinally using target-hybridization enrichment of ctDNA and FFPE derived genomic DNA (tDNA). Libraries prepared using a custom designed comprehensive gene panel OncoIndx, targeting 600 cancer relevant exons including MSI and HRR pathway genes, sequenced on Illumina platform. Variant calling was performed using an in-house developed bioinformatics pipeline. Results: Cumulatively, 551 mutations from ctDNA (n = 70) and tDNA (n = 12) samples with pathogenic, likely pathogenic and unknown significance were detected. This included SNVs (31.64 %), truncations (44.73%), splice variants (8.73%), frameshift (6 %), CNVs (4.18 %) and indels (2.9 %). Genome-wide alterations conceded low TMB (10-18) and high average HRD (43-45%) scores in ctDNA and tDNA respectively. mEGFR was the most frequently detected alteration (45.12 %), and had comparable occurrence in ctDNA and tDNA. 28 % ctDNA samples with mEGFR co-occurred with targetable ATM, RAD54 or TP53 mutations and had high HRD scores. Other actionable variants of RAS, ALK, ROS, BRAF and MET were primarily detected in ctDNA samples, albeit at low frequency. Surprisingly, 10 % patients with MET alterations that potentiate TKI resistance were concomitant with mEGFR, and showed high genome instability. Single or multiple mutations in HRR pathway genes were detected in 45 % ctDNA samples. Alterations likely to lead therapy resistance were principally observed in ctDNA samples suggesting that liquid biopsy provides elaborate cancer mutation landscape. Conclusions: Monitoring patients longitudinally for the Tx response is highly implicated. Targeted gene profiling of ctDNA detected multiple variants that were inconspicuous in tDNA. We observed a significant enrichment of variants co-occurring with mEGFR, a new finding on ctDNA mutational analysis. Further investigation into this may suggest better treatment strategies to benefit from TKI resistance. Comprehensive plasma genotyping suggests that patients positive for TKI blunting MET, RAS, BRAF mutations with or without mEGFR co-occurrence may benefit from HRD inhibitors in combination with TKI. Clinical trial information: CTRI/2016/08/007149 .

Expression profile of plasma microRNAs and target genes in patients with complicated pregnancy

Aim: comparative analysis of the expression profile of plasma microRNAs and target genes in patients with complicated pregnancy.Materials and Methods. A prospective observational comparative study in parallel groups was carried out. The study included 73 women divided into three groups: the main group – 42 patients with preeclampsia (PE), the comparison group – 12 pregnant women with fetal growth retardation (FGR), the control group – 19 clinically healthy women with uncomplicated pregnancy. An examination was performed, which included the analysis of clinical characteristics and the study of microRNA expression in blood plasma using the real-time polymerase chain reaction.Results. MicroRNA hsa-miR-210-5p and hsa-miR-1972 were not identified in any plasma sample. Analyzing plasma microRNAs in group of women with PE showed significant changes in the expression levels of hsa-miR-517a-3p (p = 0.025), hsa-miR-517c-3p (p = 0.036), hsa-miR-574-5p (p = 0.015), hsa-miR-517a-3p (p < 0.001) and an increase in miR-20a-5p (p = 0.046) compared to control group. No significant differences were found in the miRNA expression profile in group of women with FGR compared to control group. Assessing an influence of the studied microRNAs on regulatory signaling pathways allowed to establish that hsa-miR-miR-146a-5p, -181a-5p, -210-3p, -517a-3p, -517c-3p, -574-3p, -574-5p, -1304-5p are potential regulators of the reaction cascades involved in the PE pathogenesis.Conclusion. The changes revealed in the circulating blood plasma miRNA level indicate the presence of specific transcriptomic alterations during complicated course of pregnancy.

Distinct gene expression patterns of SOX2 and SOX2OT variants in different types of brain tumours

Numerous investigations have been recently published on the dysregulated expression of long-noncoding RNAs (lncRNAs) in various cancer types, emphasizing that abnormal lncRNA expression is a major contributor to tumourigenesis. A broad spectrum of lncRNAs is expressed in the central nervous system, where these RNAs seem to play key roles in brain development and function. In addition to expressing SOX2, a master regulator of pluripotency that lies within its third intron, lncRNA SOX2OT has a proposed role in regulating neural development. Based on our previous studies, alternative splicing of SOX2OT generates two alternatively spliced variants (SOX2OT-S1 and SOX2OT-S2). The present study investigated the expression patterns of SOX2OT variants and SOX2 in three principal types of brain tumours (gliomas, meningiomas and pituitary adenomas) and in four brain tumour cell lines (U87-MG, 1321N1, A172 and DAOY). Total RNAwas extracted from 34 human brain tumour specimens, and the expression profile of target genes was measured using a real-time reverse transcription PCR approach. Our data revealed distinct expression patterns for SOX2OT variants and SOX2 in the brain tumour samples, indicating their potential involvement in brain tumourigenesis. Moreover, our results highlighted the potential usefulness of SOX2OT-S1, SOX2OT-S2, and SOX2 in molecular diagnosis and brain tumour classification.

WpLogicNet: logic gate and structure inference in gene regulatory networks.

The gene regulatory process resembles a logic system in which a target gene is regulated by a logic gate among its regulators. While various computational techniques are developed for a gene regulatory network (GRN) reconstruction, the study of logical relationships has received little attention. Here, we propose a novel tool called wpLogicNet that simultaneously infers both the directed GRN structures and logic gates among genes or transcription factors (TFs) that regulate their target genes, based on continuous steady-state gene expressions. wpLogicNet proposes a framework to infer the logic gates among any number of regulators, with a low time-complexity. This distinguishes wpLogicNet from the existing logic-based models that are limited to inferring the gate between two genes or TFs. Our method applies a Bayesian mixture model to estimate the likelihood of the target gene profile and to infer the logic gate a posteriori. Furthermore, in structure-aware mode, wpLogicNet reconstructs the logic gates in TF-gene or gene-gene interaction networks with known structures. The predicted logic gates are validated on simulated datasets of TF-gene interaction networks from Escherichia coli. For the directed-edge inference, the method is validated on datasets from E.coli and DREAM project. The results show that compared to other well-known methods, wpLogicNet is more precise in reconstructing the network and logical relationships among genes. The datasets and R package of wpLogicNet are available in the github repository, https://github.com/CompBioIPM/wpLogicNet. Supplementary data are available at Bioinformatics online.

Selection of reference genes for RT-qPCR analysis in developing chicken embryonic ovary.

Normalization of the expression profiling of target genes, in a tissue-specific manner and under different experimental conditions, requires stably expressed gene(s) to be used as internal reference(s). However, to study the molecular regulation of oocyte meiosis initiation during ovary development in chicken embryos, stable reference gene(s) still need to be compared and confirmed. Six candidate genes previously used as internal references for the chicken embryo (Actb, Cvh, Dazl, Eef1a, Gapdh and Rpl15) were chosen, and their expression profiles in left ovaries dissected at five chicken embryonic days (E12.5, E15.5, E17.5, E18.5 and E20.5) were evaluated, respectively. Separately, GeNorm, NormFinder, BestKeeper and Comparative ΔCt methods were used to assess the stability of candidate reference genes, and all results were combined to give the final rank by RefFinder. All methods identified that Eef1a and Rpl15 were the two most stable internal reference genes, whereas Cvh is the most unstable one. Moreover, expression levels of three marker genes for chicken oocyte meiosis entry (Stra8, Scp3 and Dmc1) were normalized, based on Eef1a, Rpl15, or their combinations, respectively. Our findings provide the most suitable internal reference genes (Eef1a and Rpl15), to investigate further molecular regulation of ovary development and oocyte meiosis initiation in chicken embryos.

DNA methylation of GITR, OX40, 4-1BB, CD27 , and CD40 correlates with BAP1 aberrancy and prognosis in uveal melanoma.

Uveal melanoma represents an aggressive tumor that responds mostly poorly to established melanoma treatments. Comprehensive methylation profiling of the next-generation immunotherapeutic target genes, for example, members of the tumor necrosis factor receptor superfamily, might allow for the development of companion predictive biomarkers. We have analyzed CpG sites within the immune checkpoint genes GITR, OX40, 4-1BB, CD 27, and CD40 probed by the Illumina Infinium HumanMethylation450 BeadChip in N = 80 uveal melanomas included in The Cancer Genome Atlas with regard to BAP1 aberrancy, mRNA expression, and overall survival. In all analyzed immune checkpoint genes, BAP1 aberrancy was associated with decreased CpG methylation levels. We identified specific CpG sites that significantly correlated with BAP1 aberrancy, mRNA expression levels, and overall survival. Our results suggest epigenetic regulation of the analyzed immune checkpoint genes via DNA methylation in uveal melanoma and provide rationale for methylation testing in biomarker programs in clinical trials.

Brain AVMs-Related microRNAs: Machine Learning Algorithm for Expression Profiles of Target Genes.

microRNAs (miRNAs) are a class of non-coding RNAs playing a myriad of important roles in regulating gene expression. Of note, recent work demonstrated a critical role of miRNAs in the genesis and progression of brain arteriovenous malformations (bAVMs). Accordingly, here we examine miRNA signatures related to bAVMs and associated gene expression. In so doing we expound on the potential prognostic, diagnostic, and therapeutic significance of miRNAs in the clinical management of bAVMs. A PRISMA-based literature review was performed using PubMed/Medline database with the following search terms: "brain arteriovenous malformations", "cerebral arteriovenous malformations", "microRNA", and "miRNA". All preclinical and clinical studies written in English, regardless of date, were selected. For our bioinformatic analyses, miRWalk and miRTarBase machine learning algorithms were employed; the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was quired for associated pathways/functions. four studies were ultimately included in the final analyses. Sequencing data consistently revealed the decreased expression of miR-18a in bAVM-endothelial cells, resulting in increased levels of vascular endodermal growth factor (VEGF), Id-1, matrix metalloproteinase, and growth signals. Our analyses also suggest that the downregulation of miR-137 and miR-195* within vascular smooth muscle cells (VSMCs) may foster the activation of inflammation, aberrant angiogenesis, and phenotypic switching. In the peripheral blood, the overexpression of miR-7-5p, miR-629-5p, miR-199a-5p, miR-200b-3p, and let-7b-5p may contribute to endothelial proliferation and nidus development. The machine learning algorithms employed confirmed associations between miRNA-related target networks, vascular rearrangement, and bAVM progression. miRNAs expression appears to be critical in managing bAVMs' post-transcriptional signals. Targets of microRNAs regulate canonical vascular proliferation and reshaping. Although additional scientific evidence is needed, the identification of bAVM miRNA signatures may facilitate the development of novel prognostic/diagnostic tools and molecular therapies for bAVMs.

Allele-Specific Disruption of a Common STAT3 Autosomal Dominant Allele Is Not Sufficient to Restore Downstream Signaling in Patient-Derived T Cells.

Dominant negative mutations in the STAT3 gene account for autosomal dominant hyper-IgE syndrome (AD-HIES). Patients typically present high IgE serum levels, recurrent infections, and soft tissue abnormalities. While current therapies focus on alleviating the symptoms, hematopoietic stem cell transplantation (HSCT) has recently been proposed as a strategy to treat the immunological defect and stabilize the disease, especially in cases with severe lung infections. However, because of the potentially severe side effects associated with allogeneic HSCT, this has been considered only for a few patients. Autologous HSCT represents a safer alternative but it requires the removal of the dominant negative mutation in the patients’ cells prior to transplantation. Here, we developed allele-specific CRISPR-Cas9 nucleases to selectively disrupt five of the most common STAT3 dominant negative alleles. When tested ex vivo in patient-derived hematopoietic cells, allele-specific disruption frequencies varied in an allele-dependent fashion and reached up to 62% of alleles harboring the V637M mutation without detectable alterations in the healthy STAT3 allele. However, assessment of the gene expression profiles of the STAT3 downstream target genes revealed that, upon activation of those edited patient cells, mono-allelic STAT3 expression (functional haploinsufficiency) is not able to sufficiently restore STAT3-dependent signaling in edited T cells cultured in vitro. Moreover, the stochastic mutagenesis induced by the repair of the nuclease-induced DNA break could further contribute to dominant negative effects. In summary, our results advocate for precise genome editing strategies rather than allele-specific gene disruption to correct the underlying mutations in AD-HIES.

Study on the Potential Mechanism of miR-22-5p in Non-Small-Cell Lung Cancer.

Objective Non-small-cell lung cancer (NSCLC) ranks among one of the most lethal malignancies worldwide. A better and comprehensive understanding of the mechanism of its malignant progression will be helpful for clinical treating NSCLC. Methods The miRNA expression profiles and target gene profiles downloaded from the Gene Expression Omnibus and TargetScan databases were used to identify the key regulatory pattern in NSCLC by bioinformatic analysis. The regulation of miRNA to target mRNA was verified by luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. A series of the in vitro and in vivo experiments were conducted to examine the mechanism of the overexpression or knockdown of the miRNA and/or target gene. Results In this study, miR-22-5p was remarkably downregulated in NSCLC than in normal lung cells. The in vitro experiments showed that it could substantially inhibit NSCLC cell proliferation, invasion, migration, and epithelial–mesenchymal transition (EMT) progression. The results of luciferase reporter assay, qRT-PCR, and Western blot revealed that TWIST2 was a direct target gene of miR-22-5p. The results of in vitro and in vivo feedback experiments further demonstrated that miR-22-5p relied on TWIST2-induced malignant progression to regulate NSCLC proliferation, metastasis, and EMT progression. Conclusions This study revealed that miR-22-5p downregulation contributed to the malignant progression of NSCLC by targeting TWIST2. The findings highlight a potential novel pathway that could be therapeutically targeted in treating NSCLC.