Changes In Expression Research Articles

RNA-Seq analysis reveals the gene expression changes of Bemisia tabaci in response to destruxin A treatment

ABSTRACT Bemisia tabaci is an important agricultural pest that has been causing significant economic losses to crops across the globe. Destruxins are secondary metabolites of entomopathogenic fungi which can be used as a potential biopesticide against B. tabaci. However, little is known about the molecular mechanism involved in the regulation of the immune response of B. tabaci post-destruxin application. In this study, we explored the molecular responses of B. tabaci exposed to destruxin A (DA) using RNA-Seq. A total of 1702, 616, and 555 differentially expressed genes (DEGs) were identified in B. tabaci after 4, 8, and 12 h of DA treatment. In addition, 39 putative detoxification-related DEGs including 29 cytochrome P450s (P450s), 5 glutathione S-transferases (GSTs), and 5 carboxylesterases (CarEs) were also identified. Quantitative real-time PCR analysis indicated that the expression profiles of 19 random DEGs were consistent with the RNA-Seq results. These findings serve as valuable information for a better understanding of the interaction and molecular mechanisms involved in the defense response of B. tabaci against DA.

Temporal Transcriptomic Profiling Reveals Dynamic Changes in Gene Expression of Giant Freshwater Prawn upon Acute Saline-Alkaline Stresses.

Bicarbonate and sulfate are among two primary ion constituents of saline-alkaline water, with excessive levels potentially causing metabolic disorders in crustaceans, affecting their molting and interrupting development. As an economically important crustacean species, the molecular adaptive mechanism of giant freshwater prawn Macrobrachium rosenbergii in response to the stress of bicarbonate and sulfate remains unexplored. To investigate the mechanism underlying NaHCO3, Na2SO4, and mixed NaHCO3, Na2SO4 stresses, M. rosenbergii larvae were exposed to the above three stress conditions, followed by total RNA extraction and high-throughput sequencing at eight distinct time points (0, 4, 8, 12, 24, 48, 72, and 96h). Subsequent analysis revealed 13, 16, and 13 consistently identified differentially expressed genes (DEGs) across eight time points under three stress conditions. These consistently identified DEGs were significantly involved in the Gene Ontology (GO) terms of chitin-based cuticle development, protein-carbohydrate complex, structural constituent of cuticle, carnitine biosynthetic process, extracellular matrix, and polysaccharide catabolic process, indicating that alkaline stresses might potentially impact the energy metabolism, growth, and molting of M. rosenbergii larvae. Particularly, the transcriptome data revealed that DEGs associated with energy metabolism, immunity, and amino acid metabolism were enriched across multiple time points under three stress conditions. These DEGs are linked to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including glycolysis/glucogenesis, amino sugar and nucleotide sugar metabolism, and lysine degradation. Consistent enrichment findings across the three stress conditions support conclusions above. Together, these insights are instrumental in enhancing our understanding of the molecular mechanisms underlying the alkaline response in M. rosenbergii larvae. Additionally, they offer valuable perspectives on the regulatory mechanisms of freshwater crustaceans amid saline-alkaline water development.

Conserved role of hnRNPL in alternative splicing of epigenetic modifiers enables B cell activation.

The multifunctional RNA-binding protein hnRNPL is implicated in antibody class switching but its broader function in B cells is unknown. Here, we show that hnRNPL is essential for B cell activation, germinal center formation,and antibody responses. Upon activation, hnRNPL-deficient B cells show proliferation defects and increased apoptosis. Comparative analysis of RNA-seq data from activated B cells and another eight hnRNPL-depleted cell types reveals common effects on MYC and E2F transcriptional programs required for proliferation. Notably, while individual gene expression changes are cell type specific, several alternative splicing events affecting histone modifiers like KDM6A and SIRT1, are conserved across cell types. Moreover, hnRNPL-deficient B cells show global changes in H3K27me3 and H3K9ac. Epigenetic dysregulation after hnRNPL loss could underlie differential gene expression and upregulation of lncRNAs, and explain common and cell type-specific phenotypes, such as dysfunctional mitochondria and ROS overproduction in mouse B cells. Thus, hnRNPL is essential for the resting-to-activated B cell transition by regulating transcriptional programs and metabolism, at least in part through the alternative splicing of several histone modifiers.

Quantifying 3′UTR length from scRNA-seq data reveals changes independent of gene expression

Although more than half of all genes generate transcripts that differ in 3′UTR length, current analysis pipelines only quantify the amount but not the length of mRNA transcripts. 3′UTR length is determined by 3′ end cleavage sites (CS). We map CS in more than 200 primary human and mouse cell types and increase CS annotations relative to the GENCODE database by 40%. Approximately half of all CS are used in few cell types, revealing that most genes only have one or two major 3′ ends. We incorporate the CS annotations into a computational pipeline, called scUTRquant, for rapid, accurate, and simultaneous quantification of gene and 3′UTR isoform expression from single-cell RNA sequencing (scRNA-seq) data. When applying scUTRquant to data from 474 cell types and 2134 perturbations, we discover extensive 3′UTR length changes across cell types that are as widespread and coordinately regulated as gene expression changes but affect mostly different genes. Our data indicate that mRNA abundance and mRNA length are two largely independent axes of gene regulation that together determine the amount and spatial organization of protein synthesis.

Construction and Validation of a Rat Model of Acute Necrotizing Pancreatitis-Associated Intestinal Injury.

Acute pancreatitis (AP) is an acute inflammatory reaction of the pancreatic tissue, which involves auto-digestion, oedema, haemorrhage, and necrosis. AP can be categorized into mild, moderately severe and severe AP, with severe pancreatitis also referred to as acute necrotizing pancreatitis (ANP). ANP is characterized by the accumulation of necrotic material in the peritoneal cavity. This can result in intestinal injury. However, the mechanism of ANP-associated intestinal injury remains unclear. We established an ANP-associated intestinal injury rat model (ANP-IR model) by injecting pancreatitis-associated ascites fluid (PAAF) and necrotic pancreatic tissue at various proportions into the triangular area formed by the left renal artery and ureter. The feasibility of the ANP-IR model was verified by comparing the similar changes in indicators of intestinal inflammation and barrier function between the two rat models. In addition, we detected changes in apoptosis levels and YAP protein expression in the ileal tissues of rats in each group and validated them in vitro in rat epithelial crypt cells (IEC-6) to further explore the potential injury mechanisms of ANP-associated intestinal injury. We also collected clinical data from patients with ANP to validate the effects of PAAF and pancreatic necrosis on intestinal injury. Our findings offer a theoretical basis for restricting the buildup of peritoneal necrosis in individuals with ANP, thus promoting the restoration of intestinal function and enhancing treatment efficacy. The use of the ANP-IR model in further studies can help us better understand the mechanism and treatment of ANP-associated intestinal injury.

Macrophages modulate fibrosis during newt lens regeneration

BackgroundPrevious studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated.MethodsHere we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery.ResultsMacrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process.ConclusionsTogether, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs.

Decreased mRNA expression of NR1H3 and ABCA1 in pulmonary tuberculosis patients from population of Punjab, India.

Mycobacterium tuberculosis (MTB) is the causative organism of tuberculosis. Cholesterol is a crucial carbon source required for the survival of MTB in host cells. Transcription factor NR1H3 along with its important target genes ABCA1 and ApoE play important role in removal of extra cholesterol from cells. Changes in the gene expression of NR1H3, ABCA1 and ApoE can affect cholesterol homeostasis and thus the survival of MTB in host cells.Therefore, the present study was designed to analyze the mRNA expression of NR1H3, ABCA1 and ApoE in pulmonary TB (PTB) patients from the population of Punjab, India. In this study, mRNA expression of the transcription factor NR1H3 and its target genes ABCA1 and ApoE was analyzed in 89 subjects, including 41 PTB patients and 48 healthy controls (HCs) by real-time quantitative PCR. It was found that the mRNA expression of both NR1H3 and ABCA1 genes was significantly lower in TB patients than in HCs (p < 0.001). Even after sex-wise stratification of the subjects, mRNA expression of NR1H3 and ABCA1 was found to be down-regulated in both male and female TB patients. No significant difference was observed in expression of ApoE (p = 0.98). The present study found that the mRNA expression of NR1H3 and ABCA1 is down-regulated in TB patients from Punjab state of India.

Suppression of the TGF-β signaling exacerbates degeneration of auditory neurons in kanamycin-induced ototoxicity in mice

Transforming growth factor-β (TGF-β) signaling plays a significant role in multiple biological processes, including inflammation, immunity, and cell death. However, its specific impact on the cochlea remains unclear. In this study, we aimed to investigate the effects of TGF-β signaling suppression on auditory function and cochlear pathology in mice with kanamycin-induced ototoxicity. Kanamycin and furosemide (KM-FS) were systemically administered to 8-week-old C57/BL6 mice, followed by immediate topical application of a TGF-β receptor inhibitor (TGF-βRI) onto the round window membrane. Results showed significant TGF-β receptor upregulation in spiral ganglion neurons (SGNs) after KM-FA ototoxicity, whereas expression levels in the TGF-βRI treated group remained unchanged. Interestingly, despite no significant change in cochlear TGF-β expression after KM-FS ototoxicity, TGF-βRI treatment resulted in a significant decrease in TGF-β signaling. Regarding auditory function, TGF-βRI treatment offered no therapeutic effects on hearing thresholds and hair cell survival following KM-FS ototoxicity. However, SGN loss and macrophage infiltration were significantly increased with TGF-βRI treatment. These results imply that inhibition of TGF-β signaling after KM-FS ototoxicity promotes cochlear inflammation and SGN degeneration.

Distinct roles of SOX9 in self-renewal of progenitors and mesenchymal transition of the endothelium.

Regenerative capabilities of the endothelium rely on vessel-resident progenitors termed endothelial colony forming cells (ECFCs). This study aimed to investigate if these progenitors are impacted by conditions (i.e., obesity or atherosclerosis) characterized by increased serum levels of oxidized low-density lipoprotein (oxLDL), a known inducer of Endothelial-to-Mesenchymal Transition (EndMT). Our investigation focused on understanding the effects of EndMT on the self-renewal capabilities of progenitors and the associated molecular alterations. In the presence of oxLDL, ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Additionally, ECFCs displayed a dramatic loss in self-renewal capacity in the presence of oxLDL. RNA-sequencing analysis of ECFCs exposed to oxLDL validated gene expression changes suggesting EndMT and identified SOX9 as one of the highly differentially expressed genes. ATAC sequencing analysis identified SOX9 binding sites associated with regions of dynamic chromosome accessibility resulting from oxLDL exposure, further pointing to its importance. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo were reversed by the silencing of SOX9 in ECFCs or the endothelial-specific conditional knockout of Sox9 in murine models. Overall, our findings support that EndMT affects vessel-resident endothelial progenitor's self-renewal. SOX9 activation is an early transcriptional event that drives the mesenchymal transition of endothelial progenitor cells. The identification of the molecular network driving EndMT in vessel-resident endothelial progenitors presents a new avenue in understanding and preventing a range of condition where this process is involved.

Transcript and protein signatures derived from shared molecular interactions across cancers are associated with mortality

BackgroundCharacterization of shared cancer mechanisms have been proposed to improve therapy strategies and prognosis. Here, we aimed to identify shared cell–cell interactions (CCIs) within the tumor microenvironment across multiple solid cancers and assess their association with cancer mortality.MethodsCCIs of each cancer were identified by NicheNet analysis of single-cell RNA sequencing data from breast, colon, liver, lung, and ovarian cancers. These CCIs were used to construct a shared multi-cellular tumor model (shared-MCTM) representing common CCIs across cancers. A gene signature was identified from the shared-MCTM and tested on the mRNA and protein level in two large independent cohorts: The Cancer Genome Atlas (TCGA, 9185 tumor samples and 727 controls across 22 cancers) and UK biobank (UKBB, 10,384 cancer patients and 5063 controls with proteomics data across 17 cancers). Cox proportional hazards models were used to evaluate the association of the signature with 10-year all-cause mortality, including sex-specific analysis.ResultsA shared-MCTM was derived from five individual cancers. A shared gene signature was extracted from this shared-MCTM and the most prominent regulatory cell type, matrix cancer-associated fibroblast (mCAF). The signature exhibited significant expression changes in multiple cancers compared to controls at both mRNA and protein levels in two independent cohorts. Importantly, it was significantly associated with mortality in cancer patients in both cohorts. The highest hazard ratios were observed for brain cancer in TCGA (HR [95%CI] = 6.90[4.64–10.25]) and ovarian cancer in UKBB (5.53[2.08–8.80]). Sex-specific analysis revealed distinct risks, with a higher mortality risk associated with the protein signature score in males (2.41[1.97–2.96]) compared to females (1.84[1.44–2.37]).ConclusionWe identified a gene signature from a comprehensive shared-MCTM representing common CCIs across different cancers and revealed the regulatory role of mCAF in the tumor microenvironment. The pathogenic relevance of the gene signature was supported by differential expression and association with mortality on both mRNA and protein levels in two independent cohorts.

Gene expression of iron transporters, markers of vascularization and structural integrity in placenta of mothers with and without anemia.

To compare the mRNA expression of placental iron transporters (TfR-1 and FPN), markers of placental vascularization (VEGF and sFLT1) and marker of structural integrity (LMN-A) in term women with and without iron deficiency anemia. A total of 30 pregnant women were enrolled; 15 cases of iron deficiency anemia (Hb 7-10.9 gm/dL) and 15 gestational age matched healthy controls (Hb ≥ 11 gm/dL). Peripheral venous blood was collected for assessment of hemoglobin levels and serum iron profile. Placental tissue was used for assessing the mRNA expression of TfR-1, FPN, VEGF, sFLT-1 and LMN-A via real time PCR. Placental expression of TfR-1, VEGF and LMN-A was increased in pregnant women with anemia compared to healthy pregnant controls. Placental expression of sFLT-1 was decreased in pregnant women with anemia compared to healthy pregnant controls. There was no change in the placental expression of FPN. The increased expression of TfR-1, VEGF and LMN-A in cases of iron deficiency anemia are most likely to be compensatory in nature to help maintain adequate fetal iron delivery. Compensatory changes in the placenta aimed at buffering transport of iron to the fetus are seen in pregnant women with anemia compared to healthy pregnant controls.

Catecholamine treatment induces reversible heart injury and cardiomyocyte gene expression

BackgroundCatecholamines are commonly used as therapeutic drugs in intensive care medicine to maintain sufficient organ perfusion during shock. However, excessive or sustained adrenergic activation drives detrimental cardiac remodeling and may lead to heart failure. Whether catecholamine treatment in absence of heart failure causes persistent cardiac injury, is uncertain. In this experimental study, we assessed the course of cardiac remodeling and recovery during and after prolonged catecholamine treatment and investigated the molecular mechanisms involved.ResultsC57BL/6N wild-type mice were assigned to 14 days catecholamine treatment with isoprenaline and phenylephrine (IsoPE), treatment with IsoPE and subsequent recovery, or healthy control groups. IsoPE improved left ventricular contractility but caused substantial cardiac fibrosis and hypertrophy. However, after discontinuation of catecholamine treatment, these alterations were largely reversible. To uncover the molecular mechanisms involved, we performed RNA sequencing from isolated cardiomyocyte nuclei. IsoPE treatment resulted in a transient upregulation of genes related to extracellular matrix formation and transforming growth factor signaling. While components of adrenergic receptor signaling were downregulated during catecholamine treatment, we observed an upregulation of endothelin-1 and its receptors in cardiomyocytes, indicating crosstalk between both signaling pathways. To follow this finding, we treated mice with endothelin-1. Compared to IsoPE, treatment with endothelin-1 induced minor but longer lasting changes in cardiomyocyte gene expression. DNA methylation-guided analysis of enhancer regions identified immediate early transcription factors such as AP-1 family members Jun and Fos as key drivers of pathological gene expression following catecholamine treatment.ConclusionsThe results from this study show that prolonged catecholamine exposure induces adverse cardiac remodeling and gene expression before the onset of left ventricular dysfunction which has implications for clinical practice. The observed changes depend on the type of stimulus and are largely reversible after discontinuation of catecholamine treatment. Crosstalk with endothelin signaling and the downstream transcription factors identified in this study provide new opportunities for more targeted therapeutic approaches that may help to separate desired from undesired effects of catecholamine treatment.

Chronic secondary orofacial pain as the first symptom of chronic non-bacterial osteomyelitis in the mandible: a case report and literature review

Chronic non-bacterial osteomyelitis clinically manifests with pain, swelling and limitation of movement. When it affects the jaws, it can cause pain, similar to other orofacial conditions, requiring a correct differential diagnosis. The objective of this study was to report a clinical case of chronic secondary orofacial pain as the first manifestation of an inflammatory bone disease located in the mandible, in addition to discussing the diagnostic hypotheses, the pathophysiological aspects and the therapy. A 21-year-old male patient came to the service complaining of pain in the mandible, preauricular and temporal regions bilaterally with one year of evolution, associated with anterior open bite and psychological impairment. So, it was chronic orofacial pain secondary to non-bacterial osteomyelitis with a high rate of bone remodeling. Elevation of pro-inflammatory cytokines can lead to an increased power cycle of nociceptive signaling, chronic inflammation, and bone loss. Pro-inflammatory cytokines that originate in neuronal and glial cells can trigger effects such as chronic hyperexcitability, changes in the phenotypic expression of nociceptors and abnormal processing of noxious signals. The patient is still undergoing treatment and without pain. Careful assessment, ordering tests and multidisciplinary investigation are essential in the care of patients with chronic and complex orofacial pain.

Long-term combination therapy with metformin and oxymetholone in a Fanconi anemia mouse model.

Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3weeks to 18months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p=.01) and hemoglobin levels (p<.05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p=.001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.

Comparative toxicological assessment of two bisphenols using a systems approach: Evaluation of the behavioral and transcriptomic responses of Danio rerio to Bisphenol A and Tetrabromobisphenol A.

The zebrafish (Danio rerio) is becoming a critical component of New Approach Methods (NAMs) in chemical risk assessment. As a whole organism in vitro NAM, the zebrafish model offers significant advantages over individual cell-line testing, including toxicokinetic and toxicodynamic competencies. A transcriptomic approach not only allows for insight into mechanism of action for both apical endpoints and unobservable adverse outcomes, but also changes in gene expression induced by lower, environmentally relevant concentrations. In this study, we used a larval zebrafish model to assess the behavioral and transcriptomic alterations caused by sub-phenotypic concentrations of two chemicals with the same structural backbone, the endocrine disrupting chemicals: Bisphenol A and Tetrabromobisphenol A. Following assessment of behavioral toxicity, we used a transcriptomic approach to identify molecular pathways associated with previously described phenotypes. We also determined the transcriptomic Point of Departure (POD) for each chemical by modelling gene expression changes as continuous systems which allows for the identification of a single concentration at which toxic effects can be predicted. This can then be investigated with confirmatory cell-based testing in an integrated approach to testing and assessment (IATA) to determine risk to human health and the environment with greater confidence. This paper demonstrates the impact of using a multi-faceted approach for evaluating the physiological and neurotoxic effects of exposure to structurally related chemicals. By comparing phenotypic effects with transcriptomic outcomes, we were able to differentiate, characterize and rank the toxicities of related bisphenols, which demonstrates methodological advantages unique to the larval zebrafish NAM.

Insertion of short L1 sequences generates inter-strain histone acetylation differences in the mouse

BackgroundGene expression divergence between populations and between individuals can emerge from genetic variations within the genes and/or in the cis regulatory elements. Since epigenetic modifications regulate gene expression, it is conceivable that epigenetic variations in cis regulatory elements can also be a source of gene expression divergence.ResultsIn this study, we compared histone acetylation (namely, H3K9ac) profiles in two mouse strains of different subspecies origin, C57BL/6 J (B6) and MSM/Ms (MSM), as well as their F1 hybrids. This identified 319 regions of strain-specific acetylation, about half of which were observed between the alleles of F1 hybrids. While the allele-specific presence of the interferon regulatory factor 3 (IRF3) binding sequence was associated with allele-specific histone acetylation, we also revealed that B6-specific insertions of a short 3′ fragment of LINE-1 (L1) retrotransposon occur within or proximal to MSM-specific acetylated regions. Furthermore, even in hyperacetylated domains, flanking regions of non-polymorphic 3′ L1 fragments were hypoacetylated, suggesting a general activity of the 3′ L1 fragment to induce hypoacetylation. Indeed, we confirmed the binding of the 3′ region of L1 by three Krüppel-associated box domain-containing zinc finger proteins (KZFPs), which interact with histone deacetylases. These results suggest that even a short insertion of L1 would be excluded from gene- and acetylation-rich regions by natural selection. Finally, mRNA-seq analysis for F1 hybrids was carried out, which disclosed a link between allele-specific promoter/enhancer acetylation and gene expression.ConclusionsThis study disclosed a number of genetic changes that have changed the histone acetylation levels during the evolution of mouse subspecies, a part of which is associated with gene expression changes. Insertions of even a very short L1 fragment can decrease the acetylation level in their neighboring regions and thereby have been counter-selected in gene-rich regions, which may explain a long-standing mystery of discrete genomic distribution of LINEs and SINEs.

MicroRNA-875-5p inhibits the growth and metastasis of cervical cancer cells by promoting autophagy and apoptosis and inhibiting the epithelial-mesenchymal transition

IntroductionMicroRNA-875-5p (miR-875-5p) is a cancer-related microRNA. It has been demonstrated that miR−875−5p participates in the development of various types of cancer such as hepatocellular carcinoma, gastric carcinoma, prostate and bladder cancer. Previous research suggested that miR-875 is implicated in the development of cervical cancer cells. However, the exact role and function of miR−875−5p in cervical cancer remain unexplored. It is important to examine the role and function of miR-875-5p and the associated signaling pathway, as the findings may have diagnostic and therapeutic significance. Thus, in this study, we investigated the effect of miR-875-5p on the growth and metastasis of cervical cancer cells and the possible underlying mechanisms.MethodsReverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-875-5p in cervical cancer cells and normal cervical epithelium. After overexpression or co-expression of miR-875-5p in cells, the changes in cell function were analyzed. Western blot was used to detect the expression changes of epithelial-mesenchymal transition (EMT) -related proteins and autophagy-related proteins.ResultsFunctional studies demonstrated that miR-875-5p overexpression significantly inhibited the proliferation, migration, invasion, and EMT, and promotes apoptosis and autophagy of cervical cancer cells., while miR-875-5p knockdown promoted the proliferation, migration, invasion, and EMT, and inhibited apoptosis and autophagy cervical cancer cells. Furthermore, Western blot results showed that overexpression of miR-875-5p downregulated the expressions of N-cadherin, Snail, Vimentin and microtubule-associated protein 1 light chain 3B I (LC3B I). Conversely, miR-875-5p upregulated the expression of E-cadherin.ConclusionIn conclusion, our findings suggest that miR-875-5p functions as a tumor inhibitor suppressing the growth and metastasis of cervical cancer. Overexpression of miR-875-5p inhibits malignant behavior and promotes autophagy and apoptosis in cervical cancer cells. These findings advance our understanding of the role and function of miR-875-5p in cervical cancer and could facilitate the development of early genetic markers or biomarkers and therapeutic targets for cervical cancer.

Retinal microglia express more MHC class I and promote greater T-cell-driven inflammation than brain microglia

IntroductionMacrophage function is determined by microenvironment and origin. Brain and retinal microglia are both derived from yolk sac progenitors, yet their microenvironments differ. Utilizing single-cell RNA sequencing (scRNA-seq) data from mice, we tested the hypothesis that retinal and brain microglia exhibit distinct transcriptional profiles due to their unique microenvironments.MethodsEyes and brains from 2-4 month wildtype mice were combined (20 eyes; 3 brains) to yield one biologically diverse sample per organ. Each tissue was digested into single cell suspensions, enriched for immune cells, and sorted for scRNA-seq. Analysis was performed in Seurat v3 including clustering, integration, and differential expression. Multi-parameter flow cytometry was used for validation of scRNA-seq results. Lymphocytic choriomeningitis virus (LCMV) Clone 13, which produces a systemic, chronic, and neurotropic infection, was used to validate scRNA-seq and flow cytometry results in vivo.ResultsCluster analysis of integrated gene expression data from eye and brain identified 6 Tmem119+P2ry12+ microglial clusters. Differential expression analysis revealed that eye microglia were enriched for more pro-inflammatory processes including antigen processing via MHC class I (14.0-fold, H2-D1 and H2-K1) and positive regulation of T-cell immunity (8.4-fold) compared to brain microglia. Multi-parameter flow cytometry confirmed that retinal microglia expressed 3.2-fold greater H2-Db and 263.3-fold more H2-Kb than brain microglia. On Day 13 and 29 after LCMV infection, CD8+ T-cell density was greater in the retina than the brain.DiscussionOur data demonstrate that the microenvironment of retina and brain differs, resulting in microglia-specific gene expression changes. Specifically, retinal microglia express greater MHC class I by scRNA-seq and multi-parameter flow cytometry, resulting in a possibly enhanced capability to stimulate CD8+ T-cell inflammation during LCMV infection. These results may explain tissue-specific differences between retina and brain during systemic viral infections and CD8+ T-cell driven autoimmune disease.

Single-cell analysis defines highly specific leukemia-induced neutrophils and links MMP8 expression to recruitment of tumor associated neutrophils during FGFR1 driven leukemogenesis

BackgroundLeukemias driven by activated, chimeric FGFR1 kinases typically progress to AML which have poor prognosis. Mouse models of this syndrome allow detailed analysis of cellular and molecular changes occurring during leukemogenesis. We have used these models to determine the effects of leukemia development on the immune cell composition in the leukemia microenvironment during leukemia development and progression.MethodsSingle cell RNA sequencing (scRNA-Seq) was used to characterize leukemia associated neutrophils and define gene expression changes in these cells during leukemia progression.ResultsscRNA-Seq revealed six distinct subgroups of neutrophils based on their specific differential gene expression. In response to leukemia development, there is a dramatic increase in only two of the neutrophil subgroups. These two subgroups show specific gene expression signatures consistent with neutrophil precursors which give rise to immature polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Analysis of gene expression in these precursor cells identified pathways that were specifically upregulated, the most pronounced of which involved matrix metalloproteinases Mmp8 and Mmp9, during leukemia progression. Pharmacological inhibition of MMPs using Ilomastat preferentially restricted in vitro migration of neutrophils from leukemic mice and led to a significantly improved survival in vivo, accompanied by impaired PMN-MDSC recruitment. As a result, levels of T-cells were proportionally increased. In clinically annotated TCGA databases, MMP8 was shown to act as an independent indicator for poor prognosis and correlated with higher neutrophil infiltration and poor pan-cancer prognosis.ConclusionWe have defined specific leukemia responsive neutrophil subgroups based on their unique gene expression profile, which appear to be the precursors of neutrophils specifically associated with leukemia progression. An important event during development of these neutrophils is upregulation MMP genes which facilitated mobilization of these precursors from the BM in response to cancer progression, suggesting a possible therapeutic approach to suppress the development of immune tolerance.

Hyper-physiologic mechanical cues, as an osteoarthritis disease-relevant environmental perturbation, cause a critical shift in set points of methylation at transcriptionally active CpG sites in neo-cartilage organoids

BackgroundOsteoarthritis (OA) is a complex, age-related multifactorial degenerative disease of diarthrodial joints marked by impaired mobility, joint stiffness, pain, and a significant decrease in quality of life. Among other risk factors, such as genetics and age, hyper-physiological mechanical cues are known to play a critical role in the onset and progression of the disease (Guilak in Best Pract Res Clin Rheumatol 25:815–823, 2011). It has been shown that post-mitotic cells, such as articular chondrocytes, heavily rely on methylation at CpG sites to adapt to environmental cues and maintain phenotypic plasticity. However, these long-lasting adaptations may eventually have a negative impact on cellular performance. We hypothesize that hyper-physiologic mechanical loading leads to the accumulation of altered epigenetic markers in articular chondrocytes, resulting in a loss of the tightly regulated balance of gene expression that leads to a dysregulated state characteristic of the OA disease state.ResultsWe showed that hyper-physiological loading evokes consistent changes in CpGs associated with expression changes (ML-tCpGs) in ITGA5, CAV1, and CD44, among other genes, which together act in pathways such as anatomical structure morphogenesis (GO:0009653) and response to wound healing (GO:0042060). Moreover, by comparing the ML-tCpGs and their associated pathways to tCpGs in OA pathophysiology (OA-tCpGs), we observed a modest but particular interconnected overlap with notable genes such as CD44 and ITGA5. These genes could indeed represent lasting detrimental changes to the phenotypic state of chondrocytes due to mechanical perturbations that occurred earlier in life. The latter is further suggested by the association between methylation levels of ML-tCpGs mapped to CD44 and OA severity.ConclusionOur findings confirm that hyper-physiological mechanical cues evoke changes to the methylome-wide landscape of chondrocytes, concomitant with detrimental changes in positional gene expression levels (ML-tCpGs). Since CAV1, ITGA5, and CD44 are subject to such changes and are central and overlapping with OA-tCpGs of primary chondrocytes, we propose that accumulation of hyper-physiological mechanical cues can evoke long-lasting, detrimental changes in set points of gene expression that influence the phenotypic healthy state of chondrocytes. Future studies are necessary to confirm this hypothesis.