Changes In mRNA Expression Research Articles

Prospero Homeobox 1 and Doublecortin Correlate with Neural Damage after Ischemic Stroke.

Markers of neuroinflammation during ischemic stroke are well characterized, but additional markers of neural damage are lacking. The study identified associations of behavioral disorders after stroke with histologic neural damage and molecular biological change. Eight-week-old, 25 g male mice of the C57BL/6J strain were subjected to middle cerebral artery occlusion (MCAO) to induce ischemic stroke. The control group was a healthy wild type (WT), and the experimental group were designed as a low severity MCAO1 and a high severity MCAO2 based on post-stroke neurological scoring. All groups underwent behavioral tests, realtime polymerase chain reaction, triphenyltetrazolium chloride (TTC) staining and Hematoxylin and Eosin staining. One-way analysis of variance was used to analyze statistical significance between groups. In TTC staining, MCAO1 showed 29.02% and MCAO2 showed 38.94% infarct volume (p<0.0001). The pro-inflammatory cytokine interleukin (IL)-1β was most highly expressed in MCAO2 (WT 0.44 vs. MCAO1 2.69 vs. MCAO2 5.02, p<0.0001). From the distance to target in the Barnes maze test, WT had a distance of 178 cm, MCAO1 had a distance of 276 cm, and MCAO2 had a distance of 1051 (p=0.0015). The latency to target was 13.3 seconds for WT, 27.9 seconds for MCAO1, and 87.9 seconds for MCAO2 (p=0.0007). Prospero homeobox 1 (Prox1) was most highly expressed in MCAO2 (p=0.0004). Doublecortin (Dcx) was most highly expressed in MCAO2 (p<0.0001). The study demonstrated that histological damage to neural cells and changes in brain mRNA expression were associated with behavioral impairment after ischemic stroke. Prox1 and Dcx may be biomarkers of neural damage associated with long-term cognitive decline, and increased expression at the mRNA level was consistent with neural damage and long-term cognitive dysfunction.

Serotonin Transporter mRNA Expression Is Reduced in the Peripheral Blood Mononuclear Cells of Subjects with Major Depression but Normal in Fibromyalgia.

Fibromyalgia (FM) and major depression disorder (MDD) frequently co-occur. Both disorders may share common serotonergic alterations, although there is less evidence of such alterations in FM. It is also unclear as to whether these alterations are persistent over time or transient. The objectives of this study were to (i) examine the changes in mRNA expression of serotonin transporter (SERT) on the surface of peripheral blood mononuclear cells (PBMCs) in FM, MDD, and the FM + MDD subjects compared to healthy controls, and to (ii) evaluate the effect of drug treatment on SERT expression. PBMCs were isolated from FM, MDD, FM + MDD, and control subjects. SERT expression was analyzed at the mRNA level via quantitative real-time polymerase chain reaction. Statistical analyses were performed using analyses of variance and linear mixed-effects models. SERT mRNA expression was significantly reduced in MDD subjects compared to controls (p < 0.001), but not in FM nor in FM + MDD subjects. Although the drug treatments improved symptoms in FM, MDD, and FM + MDD subjects, they had no significant effect on SERT mRNA expression. These results corroborate the role of the SERT in the pathophysiology of MDD, but not in FM, and show that the decreased mRNA expression of SERT is a persistent, rather than transient, phenomenon.

The RNA binding proteins ZFP36L1 and ZFP36L2 are dysregulated in airway epithelium in human and a murine model of asthma.

Introduction: Asthma is the most common chronic inflammatory disease of the airways. The airway epithelium is a key driver of the disease, and numerous studies have established genome-wide differences in mRNA expression between health and asthma. However, the underlying molecular mechanisms for such differences remain poorly understood. The human TTP family is comprised of ZFP36, ZFP36L1 and ZFP36L2, and has essential roles in immune regulation by determining the stability and translation of myriad mRNAs encoding for inflammatory mediators. We investigated the expression and possible role of the tristetraprolin (TTP) family of RNA binding proteins (RBPs), poorly understood in asthma. Methods: We analysed the levels of ZFP36, ZFP36L1 and ZFP36L2 mRNA in several publicly available asthma datasets, including single cell RNA-sequencing. We also interrogated the expression of known targets of these RBPs in asthma. We assessed the lung mRNA expression and cellular localization of Zfp36l1 and Zfp36l2 in precision cut lung slices in murine asthma models. Finally, we determined the expression in airway epithelium of ZFP36L1 and ZFP36L2 in human bronchial biopsies and performed rescue experiments in primary bronchial epithelium from patients with severe asthma. Results: We found ZFP36L1 and ZFP36L2 mRNA levels significantly downregulated in the airway epithelium of patients with very severe asthma in different cohorts (5 healthy vs. 8 severe asthma; 36 moderate asthma vs. 37 severe asthma on inhaled steroids vs. 26 severe asthma on oral corticoids). Integrating several datasets allowed us to infer that mRNAs potentially targeted by these RBPs are increased in severe asthma. Zfp36l1 was downregulated in the lung of a mouse model of asthma, and immunostaining of ex vivo lung slices with a dual antibody demonstrated that Zfp36l1/l2 nuclear localization was increased in the airway epithelium of an acute asthma mouse model, which was further enhanced in a chronic model. Immunostaining of human bronchial biopsies showed that airway epithelial cell staining of ZFP36L1 was decreased in severe asthma as compared with mild, while ZFP36L2 was upregulated. Restoring the levels of ZFP36L1 and ZFP36L2 in primary bronchial epithelial cells from patients with severe asthma decreased the mRNA expression of IL6, IL8 and CSF2. Discussion: We propose that the dysregulation of ZFP36L1/L2 levels as well as their subcellular mislocalization contributes to changes in mRNA expression and cytoplasmic fate in asthma.

Dimethyl Fumarate Ameliorated Cardiorenal Anemia Syndrome and Improved Overall Survival in Dahl/Salt-Sensitive Rats.

Cardiovascular disease, chronic kidney disease, and anemia are known to adversely affect each other. Inflammation is commonly involved in these diseases. Cardiorenal anemia syndrome (CRAS) is the name given to this mutually harmful condition. Dimethyl fumarate (DMF) is a Food and Drug Administration-approved antioxidant and anti-inflammatory agent. The purpose of this study was to investigate the effects of DMF on Dahl/salt-sensitive (DS) rats as a CRAS model. Six-week-old DS rats were divided into three groups: the control group, the high-salt (HS) group, and the HS+DMF group. The HS and HS+DMF groups were fed a high-salt diet (8% NaCl) from 6 weeks of age. In the HS+DMF group, DMF (90 mg/kg per day) was orally administered from 6 to 15 weeks of age. Systolic blood pressure was measured every 2 weeks. The heart and renal injuries were assessed with histopathological analysis. The heart and renal expression of mRNAs was assessed by reverse-transcription polymerase chain reaction. DMF significantly improved overall survival, which was shortened by HS in DS rats. Systolic blood pressure increased in the HS group compared with the control group, and DMF tended to suppress this change. DMF ameliorated the cardiac and renal abnormalities confirmed in the HS group by histopathological analysis. Furthermore, the changes in mRNA expressions associated with disease exacerbation in the HS group were suppressed by DMF. DMF also improved anemia. This study suggests that DMF improves overall survival in DS rats through organ-protective effects and is effective against cardiorenal anemia syndrome. SIGNIFICANCE STATEMENT: Dimethyl fumarate was found to improve overall survival in Dahl/salt-sensitive rats, associated with its ability to ameliorate anemia and induce cardioprotective and renoprotective effects through anti-inflammatory and antifibrotic effects.

S100P as a potential biomarker for immunosuppressive microenvironment in pancreatic cancer: a bioinformatics analysis and in vitro study

BackgroundImmunosuppression is a significant factor contributing to the poor prognosis of cancer. S100P, a member of the S100 protein family, has been implicated in various cancers. However, its role in the tumor microenvironment (TME) of pancreatic cancer remains unclear. This study aimed to investigate the potential impact of S100P on TME characteristics in patients with pancreatic cancer.MethodsMultiple data (including microarray, RNA-Seq, and scRNA-Seq) were obtained from public databases. The expression pattern of S100P was comprehensively evaluated in RNA-Seq data and validated in four different microarray datasets. Prognostic value was assessed through Kaplan-Meier plotter and Cox regression analyses. Immune infiltration levels were determined using the ESTIMATE and ssGSEA algorithms and validated at the single-cell level. Spearman correlation test was used to examine the correlation between S100P expression and immune checkpoint genes, and tumor mutation burden (TMB). DNA methylation analysis was performed to investigate the change in mRNA expression. Reverse transcription PCR (RT-PCR) and immunohistochemical (IHC) were utilized to validate the expression using five cell lines and 60 pancreatic cancer tissues.ResultsThis study found that S100P was differentially expressed in pancreatic cancer and was associated with poor prognosis (P < 0.05). Notably, S100P exhibited a significant negative-correlation with immune cell infiltration, particularly CD8 + T cells. Furthermore, a close association between S100P and immunotherapy was observed, as it strongly correlated with TMB and the expression levels of TIGIT, HAVCR2, CTLA4, and BTLA (P < 0.05). Intriguingly, higher S100P expression demonstrated a negative correlation with methylation levels (cg14323984, cg27027375, cg14900031, cg14140379, cg25083732, cg07210669, cg26233331, and cg22266967), which were associated with CD8 + T cells. In vitro RT-PCR validated upregulated S100P expression across all five pancreatic cancer cell lines, and IHC confirmed high S100P levels in pancreatic cancer tissues (P < 0.05).ConclusionThese findings suggest that S100P could serve as a promising biomarker for immunosuppressive microenvironment, which may provide a novel therapeutic way for pancreatic cancer.

Transcriptional Analysis of lncRNA and Target Genes Induced by Influenza A Virus Infection in MDCK Cells.

The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation-particularly the effect of lncRNA on influenza virus proliferation-is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

MicroRNAs in atrial fibrillation target genes in structural remodelling

We aim to elucidate how miRNAs regulate the mRNA signature of atrial fibrillation (AF), to gain mechanistic insight and identify candidate targets for future therapies. We present combined miRNA–mRNA sequencing using atrial tissues of patient without AF (n = 22), with paroxysmal AF (n = 22) and with persistent AF (n = 20). mRNA sequencing previously uncovered upregulated epithelial to mesenchymal transition, endothelial cell proliferation and extracellular matrix remodelling involving glycoproteins and proteoglycans in AF. MiRNA co-sequencing discovered miRNAs regulating the mRNA expression changes. Key downregulated miRNAs included miR-135b-5p, miR-138-5p, miR-200a-3p, miR-200b-3p and miR-31-5p and key upregulated miRNAs were miR-144-3p, miR-15b-3p, miR-182-5p miR-18b-5p, miR-4306 and miR-206. MiRNA expression levels were negatively correlated with the expression levels of a multitude of predicted target genes. Downregulated miRNAs associated with increased gene expression are involved in upregulated epithelial and endothelial cell migration and glycosaminoglycan biosynthesis. In vitro inhibition of miR-135b-5p and miR-138-5p validated an effect of miRNAs on multiple predicted targets. Altogether, the discovered miRNAs may be explored in further functional studies as potential targets for anti-fibrotic therapies in AF.

Transcriptional and epigenetic changes after dietary and surgical weight loss interventions in an animal model of obesity.

Identifying determinants that can predict response to weight loss interventions is imperative for optimizing therapeutic benefit. We aimed to identify changes in DNA methylation and mRNA expression of a subset of target genes following dietary and surgical interventions in high-fat-diet (HFD)-induced obese rats. Forty-two adult Wistar Han male rats were divided into two groups: control rats (n = 7) and obese rats (n = 28), fed a HFD for 10 weeks (t10). Obese rats were randomly subdivided into five intervention groups (seven animals per group): (i) HFD; (ii) very-low-calorie diet (VLCD); (iii) sham surgery, and (iv) sleeve gastrectomy (SG). At week sixteen (t16), animals were sacrificed and tissue samples were collected to analyze changes in DNA methylation and mRNA expression of the selected genes. By type of intervention, the surgical procedures led to the greatest weight loss. Changes in methylation and/or expression of candidate genes occurred proportionally to the effectiveness of the weight loss interventions. Leptin expression, increased sixfold in the visceral fat of the obese rats, was partially normalized after all interventions. The expression of fatty acid synthase (FASN) and monocyte chemoattractant protein 1 (MCP-1) genes, which was reduced 0.5- and 0.15-fold, respectively, in the liver tissue of obese rats, were completely normalized after weight loss interventions, particularly after surgical interventions. The upregulation of FASN and MCP-1 gene expression was accompanied by a significant reduction in promoter methylation, up to 0.5-fold decrease in the case of the FASN (all intervention groups) and a 0.8-fold decrease in the case of the MCP-1 (SG group). Changes in tissue expression of specific genes involved in the pathophysiological mechanisms of obesity can be significantly attenuated following weight loss interventions, particularly surgery. Some of these genes are regulated by epigenetic mechanisms.

Cordycepin from Cordyceps militaris ameliorates diabetic nephropathy via the miR-193b-5p/MCL-1 axis

BackgroundDiabetic nephropathy (DN) is a chronic kidney disease that develops in patients with diabetes mellitus. Cordycepin (CRD), a secondary metabolite produced by Cordyceps militaris, has a variety of bioactive properties. In this study, DN mice and high glucose (HG)-treated HK-2 were used to evaluate the diagnostic value of CRD.MethodsQuantitative real-time PCR (qRT-PCR), western blotting, immunofluorescence analysis, and immunohistochemical staining were used to assess changes in mRNA and protein expression. Oxidative stress was evaluated by detecting the production of reactive oxygen species (ROS) and the activity of antioxidant enzymes. Cell apoptosis was detected by the TUNEL and flow cytometric methods. The interaction of miR-193b-5p and myeloid leukemia 1 (MCL-1) was examined by bioinformatics analysis and luciferase reporter assay. The protective effects of CRD on DN mice were evaluated by examining DN related biochemical indicators and renal histopathology.ResultsIn response to HG, the level of miR-193b-5p was elevated, whilst the level of MCL-1 was downregulated, and CRD therapy reversed this behavior. MCL-1 was further identified to be miR-193b-5p target. CRD attenuated HG-induced cell damage, inflammation and abnormal energy metabolism. Mechanistic investigations on in vitro models confirmed that protective effect of CRD against HG challenge to HK-2 cells is mediated through the regulation of expression of miR-193b-5p/MCL-1 axis. By examining DN related biochemical markers and renal histopathology, the protective effects of CRD on DN mice was assessed.ConclusionsIn summary, CRD decreased oxidative stress and inflammation by increasing miR-193b-5p and inactivating downstream MCL-1 in DN, hinting the pivotal values of CRD and miR-193b-5p in the management of DN.

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.

Adolescent alcohol disrupts development of noradrenergic neurons in the nucleus of the tractus solitarius and enhances stress behaviors in adulthood in mice in a sex specific manner

Alcohol use disorders (AUDs) are common mental health issues worldwide and can lead to other chronic diseases. Stress is a major factor in the development and continuation of AUDs, and adolescent alcohol exposure can lead to enhanced stress-responsivity and increased risk for AUD development in adulthood. The exact mechanisms behind the interaction between adolescence, stress, and alcohol are not fully understood and require further research. In this regard, the nucleus of the tractus solitarius (NTS) provides dense norepinephrine projections to the extended amygdala, providing a key pathway for stress-related alcohol behaviors. While NTS norepinephrine neurons are known to be alcohol sensitive, whether adolescent alcohol disrupts NTS-norepinephrine neuron development and if this is related to altered stress-sensitivity and alcohol preference in adulthood has not previously been examined. Here, we exposed male and female C57Bl/6J mice to the commonly used adolescent intermittent ethanol (AIE) vapor model during postnatal day 28-42 and examined AIE effects on: 1) tyrosine hydroxylase (TH) mRNA expression in the NTS across various ages (postnatal day 21-90), 2) behavioral responses to acute stress in the light/dark box test in adulthood, 3) NTS TH neuron responses to acute stress and ethanol challenges in adulthood, and 4) ethanol conditioned place preference behavior in adulthood. Overall the findings indicate that AIE alters NTS TH mRNA expression and increases anxiety-like behaviors following acute stress exposure in a sex-dependent manner. These mRNA expression and behavioral changes occur in the absence of AIE-induced changes in NTS TH neuron sensitivity to either acute stress or acute alcohol exposure or changes to ethanol conditioned place preference.

Isoforskolin modulates AQP4-SPP1-PIK3C3 related pathway for chronic obstructive pulmonary disease via cAMP signaling

BackgroundCyclic adenosine monophosphate (cAMP) levels are directly activated by adenylate cyclase (AC) and play an anti-inflammatory role in chronic obstructive pulmonary disease (COPD). Previously, we have shown that isoforskolin (ISOF) can effectively activate AC1 and AC2 in vitro, improve pulmonary ventilation and reduce the inflammatory response in COPD model rats, supporting that ISOF may be a potential drug for the prevention and treatment of COPD, but the mechanism has not been explored in detail.MethodsThe potential pharmacological mechanisms of ISOF against COPD were analyzed by network pharmacology and multi-omics based on pharmacodynamic study. To use specific agonists, inhibitors and/or SiRNA for gene regulation function studies, combined qPCR, WB were applied to detect changes in mRNA and protein expression of important targets PIK3C3, AKT, mTOR, SPP1 and AQP4 which related to ISOF effect on COPD. And the key inflammatory factors detected by ELISA.ResultsBioinformatics suggested that the anti-COPD pharmacological mechanism of ISOF was related to PI3K-AKT signaling pathway, and suggested target protein like PIK3C3, AQP4, SPP1, AKT, mTOR. Using the AQP4 inhibitor,or inhibiting SPP1 expression by siRNA-SPP1 could block the PIK3C3-AKT-mTOR pathway and ameliorate chronic inflammation. ISOF showed cAMP-promoting effect then suppressed AQP4 expression, together with decreased level of IL-1β, IL-6, and IL-8.ConclusionsThese findings demonstrate ISOF controlled the cAMP-regulated PIK3C3-AKT-mTOR pathway, thereby alleviating inflammatory development in COPD. The cAMP/AQP4/PIK3C3 axis also modulate Th17/Treg differentiation, revealed potential therapeutic targets for this disease.

Genome-Wide Profiling of Transcriptome and DNA Methylome in Human Embryonic Stem Cells Exposed to Extractable Organic Matter from PM2.5.

Increasing evidence indicates that PM2.5 exposure disrupts early embryonic development, but the mechanisms remain unclear. We hypothesized that PM2.5 cause abnormal embryonic development by interfering with DNA methylation and mRNA expression. In this study, we observed that human embryonic stem cells (hESCs) treated with extractable organic matters (EOM) from PM2.5 concentrations above 100 μg/mL exhibited reduced viability. While EOM within non-cytotoxicity concentrations did not affect the expression levels of pluripotency genes, it did enhance cellular proliferation, as indicated by increased Edu incorporation and the upregulation of cell cycle genes (Cdk2, Mdm2). Additionally, EOM significantly influenced the transcriptome patterns in hESCs. Notably, the differentially expressed genes were found to be significantly enriched in processes such as extracellular matrix organization, cell-cell junction organization, chromatin organization, and DNA methylation. Furthermore, we observed whole genomic-wide DNA methylation changes. Through a cross-analysis of changes in DNA methylation and mRNA expression, we identified an enrichment of terms related to the VEGFR signaling pathway and extracellular matrix. The gene signal transduction networks revealed that crucial hubs were implicated in cell growth and division. In conclusion, our findings demonstrate that PM2.5 induce significant alterations in transcriptome and DNA methylome in hESCs, leading to aberrant cell proliferation. This research provides novel insights into the molecular mechanisms underlying the developmental toxicity of PM2.5.

Molecular signaling and mechanisms of low-level laser-induced gene expression in cells involved in orthodontic tooth movement

The study aimed to observe molecular signaling, including reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm), to evaluate the alteration of gene expression by low-level laser therapy (LLLT) and the correlation between its mechanisms and the NF-kB pathway in cells involved in orthodontic tooth movement. Osteoblast-like cells (MG63), immortalized periodontal ligament cells (iPDL), and M1 macrophage-like cells were irradiated by 980-nm LLLT with energy densities of 1 and 10J/cm2 ΔΨm and intracellular ROS were monitored using fluorescent probes. The changes of mRNA expression were assessed using reverse transcription polymerase chain reaction (RT-PCR). NF-kB inhibitor, ROS scavenger, and ΔΨm suppressor were used to analyze signals associated with the regulation of gene expression. Finally, Western blot analysis was performed to confirm NF-kB signaling after LLLT. We found the increases of ΔΨm and ROS in all three cell types after LLLT, but no significant difference was observed between 1 and 10J/cm2 LLLT. Regarding gene expression, some target genes were upregulated in MG63 6h, 12h, and 1 day after LLLT and in iPDL cells 12h and 1 day after LLLT. However, no changes occurred in M1 cells. The inhibitor that significantly reduced most changes in gene expression was NF-kB inhibitor. Western blot analysis showed the increase in p-IkBα level after LLLT in iPDL and MG63, but not in M1. The 980-nm LLLT increased ΔΨm and ROS production in all three cell types. However, changes in gene regulation were found only in MG63 and iPDL cells, which related to the NF-kB pathway.

THU482 The Cholesterol Metabolite, 27-hydroxycholesterol, Enhances The Secretion Of Cancer Promoting Extracellular Vesicles By Impairing Lysosomal Integrity

Abstract Disclosure: A. Das Gupta: None. N. Krawczynska: None. H. Vidana Gamage: None. H. Kim: None. J. Park: None. J.E. Sorrells: None. S.A. Boppart: None. E.R. Nelson: None. Breast cancer is a highly prevalent cancer among women and mortality is typically associated with metastatic disease. Elevated circulating cholesterol has been associated with breast cancer recurrence in pre-clinical as well as clinical models. The cholesterol metabolite, 27-hydroxycholesterol (27HC), has been identified to establish an immunosuppressive tumor microenvironment via Liver X Receptor (LXR) in myeloid immune cells. Interestingly, 27HC treatment of different myeloid immune cell types results in increased secretion of extracellular vesicles (EVs) with altered cargo. Importantly, EVs from 27HC treated myeloid cells promote tumor growth and metastasis in murine models of breast cancer. Given that these altered EVs are involved in cancer progression, it is imperative that we understand how their biogenesis and secretion are regulated. The formation of EVs involves maturation of endosomes to form multivesicular bodies containing intraluminal vesicles. These MVBs are either targeted to the lysosomes for degradation or directed towards the plasma membrane to release EVs. Immunofluorescence microscopy experiments revealed that 27HC increases the size and number of CD63+ multivesicular bodies (MVBs) but does not alter EEA1+ endosomes in RAW 264.7 cells. Quantitative PCR analysis revealed no significant changes in mRNA expression of genes associated with EV biogenesis machinery. However, we observed that 27HC increased the size and number of LAMP1+ lysosomes. This led to the hypothesis that 27HC increases EV secretion by skewing MVBs away from lysosomal degradation and towards secretion as EVs by i) impairing lysosomal integrity, and/or ii) altering the post-translational modifications on the MVBs. To study the effect of 27HC on lysosomal function, we found that 27HC increased in lysosomal pH, suggesting a loss of lysosomal integrity. Treatment of cells with a lysosomal proton pump inhibitor phenocopies the 27HC mediated increase in EV secretion. Post-translational modifications such as ISGylation and NEDDylation have been previously implicated in modulating EV secretion and cargo, and we have observed that 27HC decreases overall ISGylation and NEDDylation of proteins in cells. Thus, 27HC increases the size of multivesicular bodies, impairs lysosomal integrity, and alters levels of ISG- and NEDDylated proteins in cells, and a combination of these modulations leads to the increase in EV secretion. Collectively, our studies reveal novel mechanisms by which EV secretion is modulated by 27HC. By extension, this axis represents a novel avenue for intervening in EV secretion and ultimately in the development of breast cancer therapeutics. Funding: Department of Defense Breast Cancer Research Program Era of Hope Scholar Award (BC200206), and National Cancer Institute (R01CA234025). Presentation: Thursday, June 15, 2023

Altered 5-methylcytosine modification of mRNA is involved in the pathogenesis of pre-eclampsia.

5-Methylcytosine (m5 C) is a prevalent RNA modification in messenger RNAs (mRNAs). Despite its abundance, its role in the decidua of pre-eclampsia (PE)remains elusive. In this study, we utilized methylated RNA immunoprecipitation sequencing (MeRIP-seq)and RNA-sequencing (RNA-seq) to map m5 C peaks and mRNA expression profile in the decidua of human early-onset PE (EPE), late-onset PE (LPE), and normal pregnancy (NP). Gene Ontology and Kyoto Encyclopedia of Genes and Genomespathway analyses elucidated potential roles of the differentially methylated mRNAs (DMGs) and differentially expressed mRNAs in decidualization pathways. Integrative analysis of MeRIP-seq and RNA-seq data pinpointed 50 candidate genes linked to PE, marked by both differentially methylated m5 C peaks and congruent expression changes. To validate these observations, we selected nine genes for verification via quantitative PCR. Our results underscore the precision and reproducibility of our bioinformatics approach. Importantly, we propose that changes in m5 C modification and expression of relevant mRNA might influence the pathogenesis of PE by hampering decidualization. This work shines light on the distinct mRNA m5 C modification patterns and expression profiles in the decidua of PE, implicating pivotal signaling disruptions and decidualization impediments in the onset of PE.

Effects of gestational haloperidol exposure on mRNA expressions related to glutamate and GABA receptors in offspring

Antipsychotic treatment is vital for patients with schizophrenia even in the perinatal period, but the impact at the molecular biological level on offspring is unclear. The aim of the present study was to investigate the effects of intraperitoneal haloperidol injection to pregnant mice on glutamate and GABA receptors in the brain of offspring mice. Eight-week-old pregnant mice were treated with either intraperitoneal haloperidol or normal saline injection, and their offspring were defined as F1 mice. In addition, eight-week-old male mice were used as acute mice that were intraperitoneally injected with haloperidol or normal saline for 20 days. mRNA expression levels were measured by RT-qPCR. Western blotting was performed of the frontal lobes of F1 mice. In the hippocampi of F1 mice, Grik3 (p = 0.023) and Gabra3 (p = 0.004) mRNA expression levels were significantly higher in the haloperidol group than in the control group, whereas Gria2 (p < 0.001) and Grin2a (p < 0.001) mRNA expression levels were significantly lower in the haloperidol group than in the control group. Gria2 (p = 0.015), and Grik3 (p = 0.037), and Grin2a (p = 0.012) mRNA expression levels were significantly lower in the haloperidol group than in the control group in the frontal lobes of F1 mice. In the hippocampi of acute mice, Grik3 (p = 0.049) and Gabra3 (p = 0.007) mRNA expression levels were significantly decreased in the haloperidol group. Fetal exposure to haloperidol can affect glutamate and GABA receptors through mRNA expression changes in the brain of offspring.

A primary luminal/HER2 negative breast cancer patient with mismatch repair deficiency

Here, we present the case of a 47-year-old woman diagnosed with luminal B breast cancer subtype and provide an in-depth analysis of her gene mutations, chromosomal alterations, mRNA and protein expression changes. We found a point mutation in the FGFR2 gene, which is potentially hyper-activating the receptor function, along with over-expression of its ligand FGF20 due to genomic amplification. The patient also harbors somatic and germline mutations in some mismatch repair (MMR) genes, with a strong MMR mutational signature. The patient displays high microsatellite instability (MSI) and tumor mutational burden (TMB) status and increased levels of CTLA-4 and PD-1 expression. Altogether, these data strongly implicate that aberrant FGFR signaling, and defective MMR system might be involved in the development of this breast tumor. In addition, high MSI and TMB in the context of CTLA-4 and PD-L1 positivity, suggest the potential benefit of immune checkpoint inhibitors. Accurate characterization of molecular subtypes, based on gene mutational and expression profiling analyses, will be certainly helpful for individualized treatment and targeted therapy of breast cancer patients, especially for those subtypes with adverse outcome.

Differentially Expressed mRNA in Streptozotocin-Induced Diabetic Bladder Using RNA Sequencing Analysis.

To detect elements governing the pathogenesis of diabetic cystopathy (DC), mRNA sequencing was carried out for bladder tissues from normal rats and those with induced diabetes mellitus (DM). This research therefore offers possible underlying molecular pathways for the advancement of DC in relation to differential mRNA expression, together with visceral functional and architectural alterations noted in individuals with this condition. An intraperitoneal injection of streptozotocin (STZ) was utilized to provoke DM in male Sprague-Dawley rats. Dysregulation and significant variations between normal rats and those with induced DM were then identified by a fold change of ≥ 1.5 with a false discovery rate P < 0.05. Hierarchical clustering/heat map and Gene Ontology/DAVID reference sources were generated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction analysis were then performed. The diabetic rodent group exhibited a greater residual urine volume (4.0 ± 0.4 mL) than their control counterparts (0.7 ± 0.2 mL, P < 0.01) at 12 weeks after diagnosis of diabetes. Expression analysis revealed 16 upregulated and 4 downregulated genes in STZDM bladder samples. A notable increase in expression was seen in PTHLH, TNFAIP6, PRC1, MAPK10, LOC686120, CASQ2, ACTG2, PDLIM3, FCHSD1, DBN1, NKD2, PDLIM7, ATF4, RBPMS2, ITGB1 and HSPB8. A notable decrease in expression was seen in SREBLF1, PBGFR1, PBLD1 and CELF1. Major genetic themes associated with mRNA upregulation and downregulation ware identified via Gene Ontology analysis and KEGG pathways. Protein to protein interaction analysis detected PDLIM3, PDLIM7, ITGB1, ACTG2 as core high frequency nodes within the network. Changes in mRNA expression together with biological process and pathways that contribute to the etiologies underlying visceral impairment of the bladder in DM are evident. Our strategy is promising for recognizing mRNAs exclusive to the bladder in DM that might offer useful targets for diagnosis and treatment.

Tubuloside A, a phenylethanoid glycoside, alleviates diclofenac induced hepato-nephro oxidative injury via Nrf2/HO-1.

The most prominent adverse effects of nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DF) are hepato-renal damage. Natural antioxidants can be preferred as an alternative and/or combination to improve this damage. This present study was conducted to evaluate the protective effect of Tubuloside A (TA) against diclofenac (DF)-induced hepato-renal damage. TA (1 mg/kg, ip) was administered to male Sprague-Dawley rats for 5 days, and DF (50 mg/kg, ip) was administered on Days 4 and 5. Plasma aspartate amino transferase, alanine amino transferase, alkaline phosphatase, blood urea nitrogen and creatinine were measured to evaluate liver and kidney functions. Additionally, oxidative stress parameters (malondialdehyde, glutathione, superoxide dismutase, catalase, and 8-oxo-7,8-dihydro-2'-deoxyguanosine) in blood, liver, and kidney tissues, changes in mRNA expression of genes involved in the Nrf2/HO-1 signalling pathway (Nrf2, HO-1, NQO-1, IL-6, iNOS, Cox-2, TNF-α, IL1-β and NFκB) and apoptotic process (Bcl-2, Cas-3 and Bax) in liver and kidney tissues were determined. Additionally, tissue sections were evaluated histopathologically. Biochemical, histopathological, and molecular results demonstrated the hepato-renal toxic effects of DF, and TA treatment protected the liver and kidney from DF-induced damage. This provides an explanation for the hepato-nephro damage caused by DF and offers new ideas and drug targets together with TA for the prevention and treatment of DF injury.