Evaluation Of Gene Expression Research Articles

Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses

Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean–Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species (Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility.

90 Leveraging the Potential of Molecular and Genetic Markers to Improve Feed Efficiency in Beef Cattle

Abstract Genetic and other molecular markers are powerful tools with the potential to improve livestock production traits, like feed efficiency. Feed efficiency is a complex biological trait mediated by many genes. Molecular based research, including transcriptome studies to evaluate gene expression profiles to determine the genes and pathways that contribute to feed efficiency in beef cattle have been widely performed over the last decade. Despite its widespread use, challenges remain in transcriptomic data analysis, in particular non-reproducibility of results between studies. These discrepancies exist for a number of reasons including small numbers of replicates, technical differences (e.g. sample preparation, sequencing platform), and biological differences (e.g., environmental, management, and genetic effects). Recent developments in technology and analytic tools provide an opportunity to integrate and synthesize the results of existing and future data. The potential to utilize these tools to have greater impact on feed efficiency will also be discussed. USDA is an equal opportunity provider and employer.

249 The Effects of Breed and Early Supplementation on Bovine Intramuscular Fat Deposition, Adipogenic and Lipogenic Gene Expression

Abstract Wagyu cattle are known to deposit greater amounts of intramuscular fat, which can directly increase quality grade and carcass value. Supplementation can positively influence animal production and overall nutrient acquisition when used strategically. The objectives of this study were to evaluate 1) the effect of breed and early supplementation on intramuscular and subcutaneous fat deposition in Wagyu and Hereford cross calves, and 2) changes in adipogenic and lipogenic gene expression in longissimus muscle (LM) biopsies from Wagyu and Hereford cross calves during an early supplementation period. Angus cows (n = 34) were artificially inseminated with semen from a Wagyu bull. A Hereford bull was utilized as a clean-up bull. From this breeding, Wagyu cross steers (n = 8) and Hereford cross steers (n = 9) were produced and were placed on study on day 100 of age. At this time, calves were allowed access to four SuperSmart Feeder stalls and were individually supplemented with steam-rolled corn at 0.5% body weight (BWT). Calves were ultrasounded on day 0, day 43, and day 86 of the study to evaluate intramuscular fat deposition and backfat thickness. An longissimus muscle (LM) biopsy was taken on day 0 and day 86 of the study to evaluate gene expression. Supplemented steers tended (P < 0.10) to have more backfat than non-supplemented steers. Intramuscular fat amount differed (P < 0.05) by breed, while ribeye area tended (P < 0.10) to differ by breed. Wagyu exhibited a greater (P < 0.05) amount of intramuscular fat at the beginning and at the end of the study. Breed did not (P > 0.05) influence backfat thickness. Neither breed nor supplementation affected (P > 0.05) average daily gain or final body weights. Platelet-derived growth factor alpha (PDGRFA) and SREBP cleavage-activating protein (SCAP) differed (P < 0.05) between biopsy phases. Fatty acid binding protein-4 (FABP4) and stearoyl-coA desaturase-1 (SCD1) did not differ (P > 0.05) between breed or biopsy phase. In conclusion, changes in gene expression could indicate enhanced preadipocyte proliferation during this stage of calf development. Overall, supplementation altered backfat deposition but did not alter intramuscular fat levels. Opposingly, sire breed influenced intramuscular fat levels at an early age. A deeper understanding of the physical and molecular effects of supplementation and sire breed could provide additional avenues for manipulating adipose tissue.

A single-cell atlas of immunocytes in the spleen of a mouse model of Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is a disorder characterized by rare X-linked genetic immune deficiency with mutations in the Was gene, which is specifically expressed in hematopoietic cells. The spleen plays a major role in hematopoiesis and red blood cell clearance. However, to date, comprehensive analyses of the spleen in wild-type (WT) and WASp-deficient (WAS-KO) mice, especially at the transcriptome level, have not been reported. In this study, single-cell RNA sequencing (scRNA-seq) was adopted to identify various types of immune cells and investigate the mechanisms underlying immune deficiency. We identified 30 clusters and 10 major cell subtypes among 11,269 cells; these cell types included B cells, T cells, dendritic cells (DCs), natural killer (NK) cells, monocytes, macrophages, granulocytes, stem cells and erythrocytes. Moreover, we evaluated gene expression differences among cell subtypes, identified differentially expressed genes (DEGs), and performed enrichment analyses to identify the reasons for the dysfunction in these different cell populations in WAS. Furthermore, some key genes were identified based on a comparison of the DEGs in each cell type involved in specific and nonspecific immune responses, and further analysis showed that these key genes were previously undiscovered pathology-related genes in WAS-KO mice. In summary, we present a landscape of immune cells in the spleen of WAS-KO mice based on detailed data obtained at single-cell resolution. These unprecedented data revealed the transcriptional characteristics of specific and nonspecific immune cells, and the key genes were identified, laying a foundation for future studies of WAS, especially studies into novel and underexplored mechanisms that may improve gene therapies for WAS.

METTL3-Mediated STING Upregulation and Activation in Kupffer Cells Contribute to Radiation-Induced Liver Disease via Pyroptosis

Purpose: Radiation therapy is a vital adjuvant treatment for liver cancer, although the challenge of radiation-induced liver diseases (RILDs) limits its implementation. Kupffer cells (KCs) are a crucial cell population of the hepatic immune system and their biological function can be modulated by multiple epigenetic RNA modifications, including N6-methyladenosine (m6A) methylation. However, the mechanism for m6A methylation in KC-induced inflammatory response in RILD remains unclear. The present study investigated the function of m6A modification in KCs contributing to RILD. Methods and Materials: Methylated RNA-immunoprecipitation sequencing (MeRIP-seq) and RNA transcriptome sequencing were used to explore the m6A methylation profile of primary KCs isolated from mice after irradiation with 3 × 8 Gy. Western blotting and quantitative real-time polymerase chain reaction were used to evaluate gene expression. DNA pull-down and CHIP assays were performed to verify target gene binding and identify binding site. Results: MeRIP-seq revealed a significantly increased m6A modification level in human KCs after irradiation, suggesting the potential role of upregulated m6A in RILD. In addition, the study results corroborated that methyltransferase-like 3 (METTL3) acts as a main modulator to promote the methylation and gene expression of TEAD1, leading to STING-NLRP3 signaling activation. Importantly, it was shown that IGF2BP2 functions as an m6A “reader” to recognize methylated TEAD1 mRNA and promote its stability. METTL3/TEAD1 knockdown abolished the activation of STING-NLRP3 signaling and protected against RILD in addition to suppressing inflammatory cytokines and hepatocyte apoptosis. Moreover, clinically collected human normal liver tissue samples post-irradiation showed increased expression of STING and IL-1β in KCs compared to the non-irradiation group. Notably, STING pharmacological inhibition alleviated irradiation-induced liver injury in mice, indicating its potential therapeutic role in RILD. Conclusions: The results of our study reveal that TEAD1-STING-NLRP3 signaling activation contributes to RILD via METTL3-dependent m6A modification.

Cell-type-specific gene expression and regulation in the cerebral cortex and kidney of atypical Setbp1S858R Schinzel Giedion Syndrome mice.

Schinzel Giedion Syndrome (SGS) is an ultra-rare autosomal dominant Mendelian disease presenting with abnormalities spanning multiple organ systems. The most notable phenotypes involve severe developmental delay, progressive brain atrophy, and drug-resistant seizures. SGS is caused by spontaneous variants in SETBP1, which encodes for the epigenetic hub SETBP1 transcription factor (TF). SETBP1 variants causing classical SGS cluster at the degron, disrupting SETBP1 protein degradation and resulting in toxic accumulation, while those located outside cause milder atypical SGS. Due to the multisystem phenotype, we evaluated gene expression and regulatory programs altered in atypical SGS by snRNA-seq of the cerebral cortex and kidney of Setbp1S858R heterozygous mice (corresponds to the human likely pathogenic SETBP1S867R variant) compared to matched wild-type mice by constructing cell-type-specific regulatory networks. Setbp1 was differentially expressed in excitatory neurons, but known SETBP1 targets were differentially expressed and regulated in many cell types. Our findings suggest molecular drivers underlying neurodevelopmental phenotypes in classical SGS also drive atypical SGS, persist after birth, and are present in the kidney. Our results indicate SETBP1's role as an epigenetic hub leads to cell-type-specific differences in TF activity, gene targeting, and regulatory rewiring. This research provides a framework for investigating cell-type-specific variant impact on gene expression and regulation.

Dietary medium-chain triglycerides upregulate metabolic gene expression in the gastrocnemius and downregulate expression of muscle-degradation gene in the soleus in aged bromodomain-containing 4+/- heterozygous mice

PurposeBromodomain containing 4 (BRD4) positively regulates metabolic gene expression, and Brd4+/- heterozygous mice exhibit leanness and malnutrition-like features. We evaluated gene expression changes, particularly of metabolism and muscle synthesis/degradation, in the skeletal muscles of aged Brd4+/- mice fed a high medium-chain triglyceride (MCT) diet. MethodsEach organ weight of C57BL/6J male wild-type and Brd4+/-/C57BL/6J mice were assessed at approximately 100 weeks old. Male Brd4+/-/C57BL/6J mice (aged 54–84 weeks) were fed a control or MCT diet for approximately 5 months and mRNA expression changes in the gastrocnemius and soleus were quantified using mRNA microarray analysis and qRT-PCR. ResultsGastrocnemius and soleus weights decreased markedly in aged Brd4+/-, compared to wild-type mice; MCT-fed Brd4+/- mice had decreased mesenteric adipose tissue weight but no decrease in gastrocnemius and soleus weights. Metabolic gene expression was downregulated more in the gastrocnemius than in the soleus. Metabolic genes, including glycolytic and citric acid cycle, and Myh3, mediating regeneration of the skeletal muscle, in gastrocnemius, were upregulated in MCT-diet-fed Brd4+/-, whereas Murf1, which promotes degradation, was downregulated in soleus. ConclusionsAn MCT-rich diet altered mRNA expression of metabolic and muscle-regeneration and -degradation genes in the gastrocnemius and soleus of Brd4+/- mice.

Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.

Crocin Suppresses Colorectal Cancer Cell Proliferation by Regulating miR-143/145 and KRAS/RREB1 Pathways.

As a chemoprevention agent, crocin effectively decreases the risk of human cancers, including colorectal cancer (CRC). However, the mechanism underlying the anti-cancer effects of crocin is not entirely explained. Considering that in this study, we investigated the crocin effect on miR-143/145 and related signaling pathways in CRC cells. HCT-116 and HT-29 CRC cells were treated with different concentrations of crocin and then were subjected to MTT and qRT-PCR assays to investigate cell viability and miR-143/miR-145, KRAS, and RREB1 expression, respectively. Also, western blotting was performed to evaluate gene expression at protein levels. Our results showed that treating CRC cells with crocin decreases cell viability by upregulating miR-143/145 expression and reducing KRAS and RREB1 expression dose-dependently. These effects on gene expression in CRC cells were reversed by removing crocin from the media after 48 h. Furthermore, western blotting results exhibited that crocin significantly reduced the protein expression of KRAS and RREB1. Also, it was found that treatment of CRC cells by crocin led to the inactivation of AKT by decreasing its phosphorylation. This study suggests that crocin may inhibit CRC cell proliferation by modulating KRAS, REEB1, and AKT signaling pathways mediated through miR-143/145 upregulation.

3D culture induction of adipogenic differentiation in 3T3-L1 preadipocytes exhibits adipocyte-specific molecular expression patterns and metabolic functions

Adipose tissues are closely related to physiological functions and pathological conditions in most organs. Although differentiated 3T3-L1 preadipocytes have been used for in vitro adipose studies, the difference in cellular characteristics of adipogenic differentiation in two-dimensional (2D) culture and three-dimensional (3D) culture remain unclear. In this study, we evaluated gene expression patterns using RNA sequencing and metabolic functions using an extracellular flux analyzer in 3T3-L1 preadipocytes with and without adipogenic induction in 2D culture and 3D culture. In 2D culture, 565 up-regulated genes and 391 down-regulated genes were identified as differentially expressed genes (DEGs) by adipogenic induction of 3T3-L1 preadipocytes, whereas only 69 up-regulated genes and 59 down-regulated genes were identified as DEGs in 3D culture. Ingenuity Pathway Analysis (IPA) revealed that genes associated with lipid metabolism were identified as 2 out of the top 3 causal networks related to diseases and function in 3D spheroids, whereas only one network related to lipid metabolism was identified within the top 9 of these causal networks in the 2D planar cells, suggesting that adipogenic induction in the 3D culture condition exhibits a more adipocyte-specific gene expression pattern in 3T3-L1 preadipocytes. Real-time metabolic analysis revealed that the metabolic capacity shifted from glycolysis to mitochondrial respiration in differentiated 3T3-L1 cells in the 3D culture condition but not in those in the 2D cultured condition, suggesting that adipogenic differentiation in 3D culture induces a metabolic phenotype of well-differentiated adipocytes. Consistently, expression levels of mitochondria-encoded genes including mt-Nd6, mt-Cytb, and mt-Co1 were significantly increased by adipogenic induction of 3T3-L1 preadipocytes in 3D culture compared with those in 2D culture. Taken together, the findings suggest that induction of adipogenesis in 3D culture provides a more adipocyte-specific gene expression pattern and enhances mitochondrial respiration, resulting in more adipocyte-like cellular properties.

Adipose-derived mesenchymal stem cells -conditioned medium effects on Glioma U87 cell line migration, apoptosis, and gene expression

The conditioned medium of mesenchymal stem cells (MSCs) has controversial roles in cancer, either promoting or suppressing tumor growth. Our research on the results of adipose tissue-derived MSC (AD-MSC)-conditioned media on U87 glioma cells was motivated by the disputed role of mesenchymal stem cells (MSCs) in cancer, which may either promote or inhibit tumor growth. Using flow cytometry, AD-MSCs were identified, verified, and their conditioned media was used to treat U87 cells. Through RT-qPCR, scratch assay, and apoptosis analysis, we evaluated gene expression (SOX4, H19, and CCAT1), cell migration, and apoptosis in U87 cells.The conditioned media greatly increased the expression of SOX4 and H19, but not CCAT1. Although there were few differences in migration and apoptosis, both were slightly increased in the treated group.These outcomes have drawn attention to the complexity of the interactions between MSCs and glioma cells. This complexity requires further research to identify the specific mechanisms governing MSC-mediated impacts on the development of glioblastoma multiforme (GBM).

High-throughput sequencing reveals Jatrorrhizine inhibits colorectal cancer growth by ferroptosis-related genes

BackgroundColorectal cancer is a malignant tumor that poses a serious threat to human health. The main objective of this study is to investigate the mechanism by which Jatrorrhizine (JAT), a root extract from Stephania Epigaea Lo, exerts its anticancer effects in colorectal cancer.MethodsWe initially assessed the inhibitory properties of JAT on SW480 cells using MTT and cell scratch assays. Flow cytometry was employed to detect cell apoptosis. Differentially expressed genes were identified through high-throughput sequencing, and they were subjected to functional enrichment and signaling pathway analysis and PPI network construction. RT-qPCR was used to evaluate gene expression and identify critical differentially expressed genes. Finally, the function and role of differentially expressed genes produced by JAT-treated SW480 cells in colorectal cancer will be further analyzed using the TCGA database.ResultsOur study demonstrated that JAT exhibits inhibitory effects on SW480 cells at concentrations of 12.5µM, 25µM, 50µM, and 75µM without inducing cell apoptosis. Through high-throughput sequencing, we identified 244 differentially expressed genes. KEGG and GO analysis of high-throughput sequencing results showed that differentially expressed genes were significantly enriched in MAPK, Wnt, and P53 signaling pathways. Notably, JAT significantly altered the expression of genes associated with ferroptosis. Subsequent RT-qPCR showed that the expression of ferroptosis genes SLC2A3 and ASNS was significantly lower in JAT-treated SW480 cells than in the control group. Analysis by TCGA data also showed that ferroptosis genes SLC2A3 and ASNS were significantly highly expressed in COAD. The prognosis of SLC2A3 was significantly worse in COAD compared to the normal group. SLC2A3 may be a core target of JAT for the treatment of COAD.ConclusionsJAT can inhibit COAD growth by ferroptosis-related genes. And it is a potential natural substance for the treatment of COAD.

Evaluating Gene Expression and Methylation Profiles of TCF4, MBP, and EGR1 in Peripheral Blood of Drug-Free Patients with Schizophrenia: Correlations with Psychopathology, Intelligence, and Cognitive Impairment.

Discovery and validation of new, reliable diagnostic and predictive biomarkers for schizophrenia (SCZ) are an ongoing effort. Here, we assessed the mRNA expression and DNA methylation of the TCF4, MBP, and EGR1 genes in the blood of patients with SCZ and evaluated their relationships to psychopathology and cognitive impairments. Quantitative real-time PCR and quantitative methylation-specific PCR methods were used to assess the expression level and promoter DNA methylation status of these genes in 70 drug-free SCZ patients and 72 healthy controls. The correlation of molecular changes with psychopathology and cognitive performance of participants was evaluated. We observed downregulation of TCF4 and upregulation of MBP mRNA levels in SCZ cases, relative to controls in our study. DNA methylation status at the promoter region of TCF4 demonstrated an altered pattern in SCZ as well. Additionally, TCF4 mRNA levels were inversely correlated with PANSS and Stroop total errors and positively correlated with WAIS total score and working memory, consistent with previous studies by our group. In contrast, MBP mRNA level was significantly positively correlated with PANSS and Stroop total errors and inversely correlated with WAIS total score and working memory. These epigenetic and expression signatures can help to assemble a peripheral biomarker-based diagnostic panel for SCZ.

Interplay between BMP2 and Notch signaling in endothelial-mesenchymal transition: implications for cardiac fibrosis.

The endothelial-to-mesenchymal transition (EndoMT) is a crucial process in cardiovascular development and disorders. Cardiac fibrosis, characterized by excessive collagen deposition, occurs in heart failure, leading to the organ remodeling. Embryonic signaling pathways such as bone morphogenetic protein 2 (BMP2) and Notch are involved in its regulation. However, the interplay between these pathways in EndoMT remains unclear. This study investigates the downstream targets of Notch and BMP2 and their effect on EndoMT markers in cardiac mesenchymal cells (CMCs) and human umbilical vein endothelial cells (HUVECs). We transduced cell cultures with vectors carrying intracellular domain of NOTCH1 (NICD) and/or BMP2 and evaluated gene expression and activation of EndoMT markers. The results suggest that the Notch and BMP2 signaling pathways have common downstream targets that regulate EndoMT. The activation of BMP2 and Notch is highly dependent on cell type, and co-cultivation of CMCs and HUVECs produced opposing cellular responses to target gene expression and α-smooth muscle actin (α-SMA) synthesis. The balance between Notch and BMP2 signaling determines the outcome of EndoMT and fibrosis in the heart. The study's findings highlight the need for further research to understand the interaction between Notch and BMP2 in the heart and develop new therapeutic strategies for treating cardiac fibrosis.

Caffeic Acid Phenethyl Ester Reduces the Adverse Effects of Nicotine on the Endometrium.

Tobacco smoke contains various toxins that negatively affect the human reproductive system. Caffeic acid phenethyl ester (CAPE), a potent antioxidant, has protective effects on the reproductive system against oxygen-free radicals, methotrexate, and pesticides. Herein, the effect of CAPE on some key markers of endometrial receptivity has been evaluated. A cross-sectional study was conducted during 2018-2019 in the Department of Clinical Biochemistry, School of Medicine, Fasa University of Medical Sciences (Fasa, Iran). Primary endometrial cells were divided into five groups, namely control, nicotine, CAPE, vehicle, and nicotine+CAPE. Real-time polymerase chain reaction (PCR) and methylation-specific PCR were performed to evaluate gene expressions and methylation, respectively. Appropriate doses of CAPE and nicotine were determined using the MTT assay. Data were analyzed using SPSS software (version 16.0) with a one-way analysis of variance. P<0.01 was considered statistically significant. The fold change was calculated using the 2-∆ΔCT method. Treatment of cells with nicotine significantly reduced the expression of C-X-C motif chemokine ligand 12 (CXCL12), fibroblast growth factor 2 (FGF2), and vascular endothelial growth factor A (VEGF-A) genes (P<0.0001). However, the expression levels increased significantly when treated with nicotine+CAPE (P<0.0001). Despite the reduced CXCL12 gene expression in cells treated with nicotine, CXCL12 was unmethylated in all study groups, indicating that the methylation status of the CXCL12 gene was not affected by nicotine or CAPE. CAPE can be a suitable agent to protect female smokers from the harmful effects of nicotine. This manuscript is available as a preprint on the Research Gate website.

Comprehensive Investigation of Cucumber Heat Shock Proteins under Abiotic Stress Conditions: A Multi-Omics Survey.

Heat-shock proteins (Hsps) are a family of proteins essential in preserving the vitality and functionality of proteins under stress conditions. Cucumber (Cucumis sativus) is a widely grown plant with high nutritional value and is used as a model organism in many studies. This study employed a genomics, transcriptomics, and metabolomics approach to investigate cucumbers' Hsps against abiotic stress conditions. Bioinformatics methods were used to identify six Hsp families in the cucumber genome and to characterize family members. Transcriptomics data from the Sequence Read Archive (SRA) database was also conducted to select CsHsp genes for further study. Real-time PCR was used to evaluate gene expression levels under different stress conditions, revealing that CssHsp-08 was a vital gene for resistance to stress conditions; including drought, salinity, cold, heat stresses, and ABA application. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of plant extracts revealed that amino acids accumulate in leaves under high temperatures and roots under drought, while sucrose accumulates in both tissues under applied most stress factors. The study provides valuable insights into the structure, organization, evolution, and expression profiles of the Hsp family and contributes to a better understanding of plant stress mechanisms. These findings have important implications for developing crops that can withstand environmental stress conditions better.

Leptin receptor expression in blood mononuclear cells of lactating women is associated with infant body weight: Potential role as a molecular biomarker.

Molecular biomarkers of maternal leptin resistance associated with infant weight are needed. To evaluate gene expression of leptin receptor (LEPR), suppressor of cytokine signalling 3 (SOCS3) and insulin receptor in peripheral blood mononuclear cells (PBMCs) of lactating women and their relationship with infant body weight and adiposity. At day 10 postpartum, maternal gene expression in PBMCs as well as leptin and insulin concentrations in plasma and milk were assessed (n = 68). Infant weight and BMI z-scores, skinfolds and arm circumference were obtained at 10 days and/or at 3 months old. In mothers with pre-pregnancy overweight or obesity (OW/OB), LEPR expression was reduced (p = 0.013) whereas plasma and milk leptin and milk insulin concentrations were elevated. LEPR expression was positively related with infant weight z-score (Beta (95% CI): 0.40 (0.17, 0.63), p = 0.001) but not with leptin concentrations. SOCS3 expression was positively related with infant weight z-score (Beta (95% CI): 0.28 (0.04, 0.51), p = 0.024) and arm circumference (Beta (95% CI): 0.57 (0.32, 0.82), p < 0.001). Relationships remained significant after adjusting for maternal and infant confounders. LEPR and SOCS3 gene expression in PBMCs are novel maternal molecular biomarkers that reflect leptin resistance and are associated with infant body weight and adiposity.

YY1-regulated lncRNA SOCS2-AS1 suppresses HCC cell stemness and progression via miR-454-3p/CPEB1

BackgroundCancer stem cells are one fundamental reason for the high recurrence rate of hepatocellular carcinoma (HCC) and its resistance to treatment. This study explored the mechanism by which SOCS2-AS1 affects HCC cell stemness. MethodsStem cells of HCC cell lines Huh7 and SNU-398 were sorted as NANOG-positive by flow cytometry. Stem cell sphere formation ability was detected. Stem cell viability, migration, invasion, and apoptosis were assessed by colony formation assays, Transwell assays, wound-healing assays, and TUNEL assays, respectively. The binding sites for SOCS2-AS1, miR-454-3p, miR-454-3p, and CPEB1 mRNA were assessed by dual-luciferase reporter assays. Quantitative real-time PCR (qPCR) and Western blot studies were performed to evaluate gene expression levels. ChIP and EMSA assays were conducted to confirm that YY1 binds with the SOCS2-AS1 promoter. A subcutaneous xenograft model was used to verify results in vivo. Tumor tissues were analyzed by H&E and TUNEL staining. ResultsSOCS2-AS1 was expressed at low levels in NANOG+ HCC stem cells, and HCC patients with a high level of SOCS2-AS1 expression had a higher survival rate. SOCS2-AS1 inhibited HCC cell stemness, migration, and invasion, and increased the cisplatin sensitivity of HCC cells by regulating miR-454-3p/CPEB1. YY1 was confirmed as a transcription factor of SOCS2-AS1, and served to inhibit SOCS2-AS1 transcription. YY1 knockdown suppressed HCC stemness via SOCS2-AS1. The role of SOCS2-AS1 was confirmed in a subcutaneous xenograft model, and SOCS2-AS1 overexpression enhanced the inhibitory effect of cisplatin on HCC in vivo. ConclusionsYY1-regulated lncRNA SOCS2-AS1 suppresses HCC cell stemness and progression via miR-454-3p/CPEB1.

Baseline tumor gene expression signatures correlate with chemoimmunotherapy treatment responsiveness in canine B cell lymphoma.

Pet dogs develop spontaneous diffuse large B cell lymphoma (DLBCL), and veterinary clinical trials have been employed to treat canine DLBCL and to inform clinical trials for their human companions. A challenge that remains is selection of treatment to improve outcomes. The dogs in this study were part of a larger clinical trial evaluating the use of combinations of doxorubicin chemotherapy, anti-CD20 monoclonal antibody, and one of three small molecule inhibitors: KPT-9274, TAK-981, or RV1001. We hypothesized that significant differential expression of genes (DEGs) in the tumors at baseline could help predict which dogs would respond better to each treatment based on the molecular pathways targeted by each drug. To this end, we evaluated gene expression in lymph node aspirates from 18 trial dogs using the NanoString nCounter Canine Immuno-oncology (IO) Panel. We defined good responders as those who relapsed after 90 days, and poor responders as those who relapsed prior to 90 days. We analyzed all dogs at baseline and compared poor responders to good responders, and found increased CCND3 correlated with poor prognosis and increased CD36 correlated with good prognosis, as is observed in humans. There was minimal DEG overlap between treatment arms, prompting separate analyses for each treatment cohort. Increased CREBBP and CDKN1A for KPT-9274, increased TLR3 for TAK-981, and increased PI3Kδ, AKT3, and PTEN, and decreased NRAS for RV1001 were associated with better prognoses. Trends for selected candidate biomarker genes were confirmed via qPCR. Our findings emphasize the heterogeneity in DLBCL, similarities and differences between canine and human DLBCL, and ultimately identify biomarkers that may help guide the choice of chemoimmunotherapy treatment in dogs.

Guselkumab Modulates Differentially Expressed Genes in Blood of Patients With Psoriatic Arthritis: Results from Two Phase 3, Randomized, Placebo-Controlled Trials.

To evaluate gene expression in blood of patients with psoriatic arthritis (PsA) versus healthy controls and identify changes associated with guselkumab treatment. Whole blood transcriptome profiling via paired-end RNA sequencing was conducted using samples from DISCOVER-1 and DISCOVER-2 at baseline (n=673) and at weeks 4 and 24 from a representative subgroup that received placebo or guselkumab (n=227 [longitudinal PsA cohort]). Baseline samples were compared with demographically matched healthy controls (n=21). Guselkumab-mediated changes in gene expression were assessed in participants from the longitudinal PsA cohort who did versus did not achieve at least 20% improvement in American College of Rheumatology response criteria (ACR20) or at least 75% improvement in Psoriasis Area and Severity Index (PASI75). Differential gene expression was analyzed using edgeR. At baseline, 355 upregulated and 314 downregulated genes (PsA-associated genes) were identified in patients with PsA versus healthy controls. Upregulated genes were related to neutrophil, mononuclear cell, and CD11b+ gene sets. No cell type-specific gene sets were identified among downregulated genes. Most PsA-associated genes were modulated by guselkumab treatment. At week 24, genes downregulated by guselkumab were enriched with neutrophil, monocyte, eosinophil, and macrophage gene sets; genes upregulated by guselkumab were enriched with B cell, T cell, and natural killer cell gene sets. Reductions in expression of upregulated PsA-associated gene sets were more pronounced in ACR20 and PASI75 responders than in nonresponders. These findings suggest a dysregulation of immune cell profiles in blood from patients in the baseline PsA cohort that approached levels in healthy controls after guselkumab treatment.