mRNA Research Articles

Identifying predictors of treatment response and molecular changes induced by neoadjuvant chemotherapy and endocrine therapy in hormone receptor-positive/HER2-negative breast cancer: the NEOENDO translational study

Predictors of response to neoadjuvant chemotherapy (NACT) and endocrine therapy (NET) in hormone receptor-positive (HoR+)/human epidermal growth factor receptor 2 (HER2)-negative breast cancer (BC) are required. Also, pathological and molecular changes induced by both strategies and their impact on patients' outcomes have not been reported so far. In a cohort of 186 patients with early-stage HoR+/HER2-negative BC treated with NACT or NET, we assessed the association of baseline main clinicopathological features and PAM50 gene expression (GE), intrinsic subtypes (IS) and risk-of-relapse (ROR-P) score with pathological outcomes according to treatment strategy. Molecular NACT/NET-induced changes were described and compared, along with their associations with event-free survival (EFS). Comparison of the two cohorts after propensity score matching (PSM) was used as sensitivity analysis. Molecular changes were confirmed in cell lines. NACT was associated with higher rates of residual cancer burden (RCB)-0/I than NET in the overall population (38.2% versus 13.5%, P < 0.001) and after PSM (P= 0.036). PAM50 non-luminal IS were the only independent and positive predictor of RCB-0/I (P= 0.024) in the NACT cohort, while MMP11 messenger RNA levels were the only independent and negative predictor (P= 0.014) in the NET cohort. Both treatments shifted the tumor types toward less aggressive forms (i.e. PAM50 luminal A/normal-like), lowered the risk of recurrence in terms of ROR-P, up-regulated selected immune genes and PAM50 basal-like-related genes/signature and significantly downregulated proliferation-/luminal-/HER2-related genes/signatures, though NACT more than NET. Molecular findings were confirmed after PSM. A net reduction in proliferation-related genes and ROR-P was confirmed in cell lines with chemotherapy and endocrine therapy. Different baseline molecular features associated with diverse kind of responses (ROR-P downstaging, Ki67 reduction or pathological responses) with NACT and NET. Decreasing ROR-P and transitioning the tumor subtype to resemble normal tissue (i.e. PAM50 normal-like) suggested improved EFS. NACT was more effective in the molecular and dimensional tumor 'downstaging' than NET but baseline molecular features associated with differential responses according to treatment strategy. Examining baseline and post-treatment GE might help tailor more personalized and effective care.

METTL3-VISTA axis-based combination immunotherapy for APC truncation colorectal cancer

ObjectiveAlthough immune checkpoint blockade (ICB) therapy represents a bright spot in antitumor immunotherapy, its clinical benefits in colorectal cancer (CRC) are limited. Therefore, a new target for mediating CRC immunosuppression...

Low-inflammatory lipid nanoparticle-based mRNA vaccine elicits protective immunity against H5N1 high-pathogenicity avian influenza virus with reduced adverse reactions.

Messenger RNA vaccines based on lipid nanoparticles (mRNA-LNPs) are promising vaccine modalities. However, mRNA-LNP vaccines frequently cause adverse reactions such as swelling and fever in humans, partly due to the inflammatory nature of LNP. Modification of the ionizable lipids used in LNP is one approach to avoid these adverse reactions. Herein, we report the development of mRNA-LNP vaccines with better protective immunity and reduced adverse reactions using LNP, which contains a disulfide (SS)-cleavable bond and pH-activated lipid-like materials with oleic acid (ssPalmO) as an ionizable lipid (LNPssPalmO). We used mRNA expressing H5N1 subtype high-pathogenicity avian influenza virus-derived hemagglutinin or neuraminidase to generate mRNA-LNP vaccines against H5N1 influenza. Compared with conventional LNP, mRNA-LNPssPalmO induced comparable antigen-specific antibodies and better interferon-gamma (IFN-γ)-producing T-helper type-1 (Th1) responses in mice. Both mRNA-LNPssPalmO and conventional mRNA-LNP conferred strong protection against homologous H5N1 virus challenge. In addition, mRNA-LNPssPalmO showed better cross-protection against heterologous H5N1 virus challenge compared with conventional mRNA-LNPs. Furthermore, we observed that mRNA-LNPssPalmO induced less inflammatory responses (e.g., inflammatory cytokine production and vascular hyperpermeability) and fewer adverse reactions (e.g., weight loss and fever) compared with conventional mRNA-LNP. These results suggest that mRNA-LNPssPalmO would be a safe alternative to conventional vaccines to overcome mRNA-LNP vaccine hesitancy.

Sequentially amplified integration of catalytic DNA circuits for high-performance intracellular imaging of miRNA and interpretation of mRNA-miRNA signalling pathway

The cascaded catalytic circuits are viable tools for improving the signal gain of biosensors, yet their sensing performance is still limited by the signal leakage from complex biological environment and unsatisfying reaction efficiency from inter-reactants steric hindrance. Herein, we proposed a catalytically localized DNA (CLD) circuit for the accurate and high-efficiency imaging of microRNA (miRNA) in living cells by virtue of the sequentially and successively amplified integration of catalytic DNA circuits. The compact CLD circuit was constructed by integrating two elemental catalytic circuits, cell-responsive EDR module and analyte-sensing CHA module, where CHA module was initially caged in EDR module for eliminating the unwanted off-site and off-target signal leakage. Only by cell-specific messenger RNA (mRNA)-activated EDR operation then the elemental CHA circuit could be successively connected to facilitate the highly efficient intramolecular reaction with low steric hindrance, thus leading to accelerated reaction efficiency for miRNA analyte. The multiple molecular recognition and the spatial self-confinement of the smart CLD circuit enable the accurate and high-efficiency imaging of intracellular miRNA. The interaction network of mRNA and miRNA was then investigated in situ through our CLD circuit, which provides a powerful tool for discovering the underlying signal pathways between these different RNAs in living cells.

METTL14 Regulates the m6A Modification of TRAF6 to Suppress Mitochondrial Dysfunction and Ferroptosis in Dopaminergic Neurons via the cGAS-STING Pathway.

The degeneration of dopaminergic (DA) neurons has emerged as a crucial pathological characteristic in Parkinson's disease (PD). To enrich the related knowledge, we aimed to explore the impact of the METTL14-TRAF6-cGASSTING axis in mitochondrial dysfunction and ferroptosis underlying DA neuron degeneration. 1-methyl-4-phenylpyridinium ion (MPP+) was used to treat DA neuron MN9D to develop the PD cell models. Afterward, a cell counting kit, flow cytometer, DCFH-DA fluorescent probe, and Dipyrromethene Boron Difluoride staining were utilized to measure the cell viability, iron concentration, ROS level, and lipid peroxidation, respectively. Meanwhile, the mitochondrial ultrastructure, the activity of mitochondrial respiratory chain complexes, and levels of malondialdehyde and glutathione were monitored. In addition, reverse transcription-quantitative polymerase chain reaction and western blot assays were adopted to measure the expression of related genes. cGAS ubiquitylation and TRAF6 messenger RNA (mRNA) N6-methyladenosine (m6A) levels, the linkages among METTL14, TRAF6, and the cGAS-STING pathway were also evaluated. METTL14 expression was low, and TRAF6 expression was high after MPP+ treatment. In MPP+-treated MN9D cells, METTL14 overexpression reduced ferroptosis, ROS generation, mitochondrial injury, and oxidative stress (OS) and enhanced mitochondrial membrane potentials. TRAF6 overexpression had promoting impacts on mitochondrial dysfunction and ferroptosis in MPP+-treated MN9D cells, which was reversed by further overexpression of METTL14. Mechanistically, METTL14 facilitated the m6A methylation of TRAF6 mRNA to down-regulate TRAF6 expression, thus inactivating the cGAS-STING pathway. METTL14 down-regulated TRAF6 expression through TRAF6 m6A methylation to inactivate the cGAS-STING pathway, thereby relieving mitochondrial dysfunction and ferroptosis in DA neurons.

Construction and validation of a novel prognostic model with palmitoylation-related genes for glioblastoma.

Glioblastoma multiforme (GBM), the most prevalent and aggressive primary brain tumor, poses substantial challenges in both treatment and prognosis. Post-translational modifications, like palmitoylation, are known to have critical roles in the development and progression of glioma. Yet, the molecular mechanisms involved in palmitoylation and its prognostic significance in GBM are still not fully understood. This study aimed to explore prognostic biomarkers for GBM based on palmitoylation-related genes and to construct a prognostic risk model. The messenger ribonucleic acid (mRNA) expressions data and the clinical information were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) to explore palmitoylation-related mechanisms in GBM. The Cox regression analysis was performed to identify prognostic palmitoylation-related genes and the consensus clustering was used for molecular classification. The package "limma" was used for differential gene expression analysis and the least absolute shrinkage and selection operator (LASSO) regression was applied to construct a risk signature. A nomogram model was established using the risk score and clinical variables. Receiver operating characteristic (ROC), calibration curve, and decision curve analysis (DCA) were used to assess the predicted accuracy and clinical benefit of the model. The difference in immune cell infiltration was compared between different risk groups. The drug susceptibility analysis and immunotherapy response prediction were conducted to access the ability of the risk signature in predicting the therapeutic effect. Based on datasets from TCGA, five palmitoylation-related genes were identified as prognostic markers, allowing for the categorization of GBM patients into two subtypes with differing survival rates. Through differential expression analysis, 570 specific genes linked to GBM advancement were uncovered. A total of seven signature genes (COL22A1, IGFBP6, SOD3, UPP1, CA14, TIMP4 and FERMT1) were applied to establish a prognostic risk model, which was demonstrated to be an independent prognostic indicator for patients with GBM. Kaplan-Meier analysis indicted that the GBM patients in low-risk group exhibited a better survival outcome compared the patients in high-risk group. The ROC curve analyses demonstrated that the risk score model was reliable. The nomograms showed excellent predictive ability. Two external cohort of patients from the GSE74187 and GSE83300 in the GEO database confirmed the model's strong predictive performance. The immune infiltration, drug sensitivity and immunotherapy responses were significantly different between the low- and high-risk groups. Our study offers insights into the molecular classification and prognostic assessment of GBM, focusing on palmitoylation-related mechanisms. The prognostic model we constructed provides valuable guidance for tailoring personalized treatment strategies for GBM patients.

N-acetyltransferase 10 as a novel prognostic biomarker in papillary renal cell carcinoma: a machine learning and experimental validation study.

N-acetyltransferase 10 (NAT10) is a lysine acetyltransferase known for catalyzing the N4-acetylcytidine (ac4C) modifications on RNAs. Recent studies have associated NAT10 with the pathogenesis of various cancers. However, its specific function and prognostic significance in papillary renal cell carcinoma (pRCC) remain poorly understood. This study aimed to explore NAT10's prognostic value and mechanisms in pRCC. NAT10 expression and prognostic associations in pancancer were analyzed using The Cancer Genome Atlas (TCGA). Single-cell RNA (scRNA) sequencing data from a previous study were used to characterize NAT10 expression at the single-cell level in pRCC. Pathway enrichment analysis, including gene set variance analysis (GSVA) and overrepresentation analysis, was conducted to investigate the potential mechanisms through which NAT10 exerts its effects. Immune cell infiltration analysis, conducted through the ESTIMATE and CIBERSORT algorithms, was performed to examine the association of NAT10 with the tumor microenvironment (TME). A NAT10-related prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) Cox regression on genes that were both positively correlated with NAT10 and were identified as NAT10-mediated ac4C targets by ac4C RNA immunoprecipitation sequencing The model's performance was validated in the TCGA training set (n=285), with 42 events (deaths) and 243 censored cases, and in the GSE2748 external validation set (n=28), with 13 events (deaths) and 15 censored cases. The association between NAT10-related risk scores and immunotherapy response was assessed via the IMvigor210 cohort. Finally, the aberrant expression of NAT10 was confirmed through immunohistochemistry data from the Human Protein Atlas database and our experimental validations from quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses. NAT10 is upregulated in multiple cancers, including pRCC, and higher NAT10 expression correlates with advanced stages and poorer prognosis. Single-cell RNA-sequencing data confirmed elevated NAT10 expression in malignant pRCC cells. Pathway enrichment and immune cell infiltration analyses indicated that NAT10 is associated with malignancy-related pathways and a disordered TME. A prognostic model was constructed using LASSO Cox regression, with 18 core genes being identified, and demonstrated high predictive accuracy for survival. The model achieved AUC values of 0.97, 0.93, and 0.82 for 1-, 3-, and 5-year survival in the TCGA training set, respectively, while all AUC values in the GSE2748 external validation set were 1. Higher NAT10-related risk scores were linked to poorer immunotherapy response in the IMvigor210 cohort. NAT10's prognostic significance was validated across various cancers, with elevated expression at both the messenger RNA and protein levels confirmed through immunohistochemistry and experimental validation in RCC cell lines. Our findings suggest that NAT10 is aberrantly expressed in pRCC, is associated with poor prognosis, and contributes to pRCC progression through multiple pathways, offering new insights into the personalized treatment of patients with pRCC.

PARP1 promotes tumor proliferation in lenalidomide-resistant multiple myeloma via the downregulation of microRNA-192-5p-AKT signaling.

Lenalidomide-based therapies are recommended as first-line treatment for multiple myeloma (MM) patients, regardless of the transplant eligibility. Resistance to lenalidomide is a clinical problem that urgently needs to be addressed. The expression of poly(ADP-ribose) polymerase 1 (PARP1) is abnormally high in a variety of tumor tissues including MM. However, in lenalidomide-resistant MM, it is not yet known whether the abnormally high expression of PARP1 is involved in the occurrence of drug resistance, and whether the inhibition of PARP1 can reverse lenalidomide resistance. The aim of this study was to investigate the mechanism of PARP1 promoting lenalidomide-resistant in MM patients. Samples of bone marrow from patients with MM who were sensitive or resistant to lenalidomide were collected. The expression levels of PARP1 at the messenger RNA and protein levels were detected through polymerase chain reaction and western blot. MM cell lines were cultivated in vitro, cell lines resistant to lenalidomide were screened out, and the expression levels of PARP1 in the resistant cell lines were detected. The apoptosis level was also detected in the lenalidomide-resistant MM cell lines treated with a PARP1 inhibitor. The proliferation rates of the two groups of cells at different time points were evaluated by mono-methyl terephthalate (MMT) experiments. Finally, the effect of PARP1 on the proliferation of lenalidomide-resistant MM through the microRNA-192-5p-AKT signaling pathway was analyzed. In the lenalidomide-resistant cell lines, the expression level of PARP1 was higher, the proliferation more rapid, and the apoptosis rate was lower than lenalidomide-sensitive cell lines. Additionally, the activated AKT pathway was suppressed by downregulating the expression of microRNA-192-5p. MM resistance can be inhibited to some extent by impacting PARP1. PARP1 is involved in the production of lenalidomide resistance in MM, and could serve as a potential target for the treatment of MM in the future.

Innovations in mRNA-based cancer immunotherapy: Challenges and future directions

Messenger RNA (mRNA) vaccines have quickly become a potent tool in cancer immunotherapy, with the potential to personalise cancer treatment and get around many of the drawbacks of conventional therapies. With an emphasis on their processes, technological developments, and clinical applications, this study examines the scientific breakthroughs and difficulties surrounding mRNA cancer vaccines. We begin by investigating the ways in which mRNA vaccines work to stimulate the immune system, encode antigens specific to tumours, and elicit a potent anti-tumor response. We also look at the variety of mRNA vaccines which are currently being deployed and emphasize their successes in clinical studies, especially when combined with immune checkpoint inhibitors. The effectiveness and safety of these vaccines have been significantly increased by technological developments, such as enhancements in mRNA stability, design, and delivery systems (such as lipid nanoparticles and polyplexes). Emerging technologies like circular RNA and self-amplifying present promising opportunities for enduring and more potent therapies. But there are still limitations with mRNA vaccines, such as stability, immunogenicity, degradation, and effective in vivo delivery. Concerns about possible adverse effects and safety in long-term applications are also covered in this review. Despite these obstacles, novel approaches are being developed to boost antigen expression, optimize delivery systems, and improve mRNA stability, establishing mRNA vaccines as a crucial component of tailored cancer immunotherapy. These developments provide the foundation for upcoming advances in mRNA vaccine technology and represent a major breakthrough in the fight against cancer.

Elucidating the molecular and immune interplay between head and neck squamous cell carcinoma and diffuse large B-cell lymphoma through bioinformatics and machine learning.

Head and neck squamous cell carcinoma (HNSCC) contributes significantly to global health challenges, presenting primarily in the oral cavity, pharynx, nasopharynx, and larynx. HNSCC has a high propensity for lymphatic metastasis. Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin lymphoma, exhibits significant heterogeneity and aggressive behavior, leading to high mortality rates. Epstein-Barr virus (EBV) is notably associated with DLBCL and certain types of HNSCC. The purpose of this study is to elucidate the molecular and immune interplay between HNSCC and DLBCL using bioinformatics and machine learning (ML) to identify shared biomarkers and potential therapeutic targets. Differentially expressed genes (DEGs) were identified using the "limma" package in R from the HNSCC dataset in The Cancer Genome Atlas (TCGA) database, and relevant modules were selected through weighted gene co-expression network analysis (WGCNA) from a DLBCL dataset in the Gene Expression Omnibus (GEO) database. Based on their intersection genes, functional enrichment analyses were conducted using Gene Ontology (GO), Disease Ontology, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Protein-protein interaction (PPI) networks and ML algorithms were employed to screen for biomarkers. The prognostic value of these biomarkers was evaluated using Kaplan-Meier (K-M) survival analysis and receiver operating characteristic (ROC) curve analyses. The Human Protein Atlas (HPA) database facilitated the examination of messenger RNA (mRNA) and protein expressions. Further analyses of mutations, immune infiltration, drug predictions, and pan-cancer impacts were performed. Additionally, single-cell RNA sequencing (scRNA-seq) data analysis at the cell type level was conducted to provide deeper insights into the tumor microenvironment. From 2,040 DEGs and 1,983 module-related genes, 85 shared genes were identified. PPI analysis with six algorithms proposed 21 prospective genes, followed ML examination yielded 16 candidates. Survival and ROC analyses pinpointed four hub genes-ACACB, MMP8, PAX5, and TNFAIP6-as significantly associated with patient outcomes, demonstrating high predictive capabilities. Evaluations of mutations and immune infiltration, coupled with drug prediction and a comprehensive cancer analysis, highlighted these biomarkers' roles in tumor immune response and treatment efficacy. The scRNA-seq data analysis revealed an increased abundance of fibroblasts, epithelial cells and mononuclear phagocyte system (MPs) in HNSCC tissues compared to lymphoid tissues. MMP8 showed higher expression in five cell types in HNSCC tissues, while TNFAIP6 and PAX5 exhibited higher expression in specific cell types. Leveraging bioinformatics and ML, this study identified four pivotal genes with significant diagnostic capabilities for DLBCL and HNSCC. The survival analysis corroborates their diagnostic accuracy, supporting the development of a diagnostic nomogram to assist in clinical decision-making.

Identification of mRNA biomarkers in extremely early hypertensive intracerebral hemorrhage (HICH)

IntroductionHypertensive intracerebral hemorrhage (HICH) stands out as a critical complication of primary hypertension. Consequently, investigating messenger RNA (mRNA) biomarkers becomes imperative, offering potential targets. This study is conducted for elucidating the expression profile of blood mRNA biomarkers in HICH.MethodsTwenty-five HICH patients were constituted the HICH group.Twenty-two healthy volunteers recruited and comprised the control group. Peripheral blood cells were extracted to identify candidate mRNA. The identified differential expressions of genes between the two groups were validated, and the potential associations between these differentially expressed genes and adverse events were analyzed. GO and KEGG enrichment of DEGs, Weighted Gene Co-expression Network and Protein Interaction Network were established. target mRNA was screened.ResultsThe study identified 3163 differentially expressed genes in HICH. 8 candidate mRNA (SPI1, HK3, HCK, SYK, CD14, FCER1G, CYBB, FGR) were pinpointed. Associations with pathways affecting HICH development included HIF-1 signaling, NF-kappa B signaling, and C-type lectin receptor signaling. In the HICH group, higher expressions of HK3, HCK, SYK, CD14, FCER1G, CYBB, and FGR, and lower SPI1 expression compared to the control group. HICH patients experienced high rates of complications: pulmonary infection (84%), epilepsy (16%), enlarged hematoma (20%), gastrointestinal bleeding (48%), malnutrition (84%), and lower limb deep vein thrombosis (DVT) (12%). Factors contributing to pulmonary infection included age and elevated expression of HCK, SYK, CD14, and FGR. SPI1 was associated with epilepsy, while its lower expression correlated with hematoma enlargement. Gastrointestinal bleeding was linked to increased cerebral hemorrhage. Malnutrition was associated with higher age, and expressions of HK3, HCK, SYK, CD14, FCER1G, CYBB, and FGR. Patients with lower limb DVT had elevated expressions of the identified genes.ConclusionIn hypertensive intracerebral hemorrhage, there are elevated expressions of HK3, HCK, SYK, CD14, FCER1G, CYBB, and FGR, along with reduced expression of SPI1. Furthermore, age, along with elevated expressions of HCK, SYK, CD14, and FGR, serves as influencing factors contributing to pulmonary infection in patients.

METTL14 Promotes Lipopolysaccharide-Induced Myocardial Damage via m6A-Dependent Stabilization of TRPM7 mRNA.

Sepsis-induced myocardial injury (SIMI) is a vital pathological component of severe sepsis and septic shock. As a prevalent internal mRNA modification in eukaryotic cells, N6-methyladenosine (m6A) modification is implicated in sepsis and immune disorders. Methyltransferase-like 14 (METTL14), a core subunit of the methyltransferase complex that catalyzes messenger RNA m6A modification, is involved in the regulation of human cardiomyocyte cell line (AC16) injury. This study aimed to explore the role and mechanism of METTL14 in lipopolysaccharide (LPS) -induced myocardial injury.Cell viability and apoptosis were analyzed via 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. The Tumor necrosis factor alpha (TNF-α) and Interleukin-1beta (IL-1β) levels were analyzed via Enzyme linked immunosorbent assay (ELISA). Caspase-3 activity, reactive oxygen species activity, malondialdehyde level, and glutathione level were assessed using special assay kits. The levels of transient receptor potential melastatin 7 (TRPM7) and METTL14 mRNA were determined via Real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the protein levels of TRPM7, METTL14, phospho-p65 (p-p65), total p65 (p65), p-IκBα, and total IκBα (IκBα) were examined via western blot assay.LPS treatment repressed AC16 cell viability and induced cell apoptosis, inflammatory response, oxidative stress, and ferroptosis in vitro. METTL14 and TRPM7 were upregulated in LPS-treated AC16 cells. At the molecular level, METTL14 could increase the stability of TRPM7 mRNA via m6A methylation. Moreover, METTL14 deficiency could abolish LPS-triggered AC16 cell injury and ferroptosis via TRPM7 regulation.METTL14 knockdown reversed LPS-caused myocardial cell damage mainly by regulating the stability of TRPM7 mRNA, providing a novel therapeutic target for septic cardiomyopathy treatment.

Dexamethasone: a cheap and convenient way to alleviate subsequent stenosis after thermal injury on ureter.

Ureteral thermal injury (UTI) occurs when the ureter is exposed to high temperatures during surgical procedures, leading to tissue damage and ureteral stricture. Dexamethasone (DEX) has been widely used to treat a variety of diseases for its anti-inflammatory and immunosuppressant effects. However, its role in treating UTI remains unclear. The aim of this study was to determine the efficacy of treatment in mice with UTI. Thirty C57BL/6J mice were randomly divided into three groups (n=10 per group): (I) control group, (II) UTI group, (III) UTI + DEX group. UTI mouse models were constructed using a fast-pack pro-obturation pack handpiece instrument, and the therapeutic effect of DEX (2.5 mg/kg) on UTI was investigated via intraperitoneal injection for two weeks. Pathological changes in the ureter, renal function, extracellular matrix protein deposition, and inflammatory markers in the kidneys were evaluated in mouse models. Ureteral histopathological changes were more severe in the UTI group than in the control group; however, DEX treatment alleviated these changes to a certain extent. Mice in the UTI group exhibited elevated renal function. Although DEX alleviated renal function deterioration, the difference was not statistically significant. Moreover, in comparison to the control group, kidney samples from the mice in the UTI group revealed increased messenger RNA (mRNA) levels of inflammation markers, indicating a progressive inflammation response, while DEX significantly reversed interleukin-1β (IL-1β) expression at the protein level. Additionally, fibrosis-related markers were markedly increased in the kidneys of UTI mice. These findings were significantly reversed by DEX treatment, revealing its anti-inflammatory and anti-fibrotic activities. DEX reduces the severity of renal fibrosis and inflammation after UTI and exerts a significant renal protective effect.

Comprehensive Analysis of circRNA and mRNA Revealing Potential Mechanism Underlying Neuroinflammation in BV2 Cells

Background: The significance of circular RNAs (circRNAs) in diabetic complications has been established. However, their role in basal and diabetic states, as well as cognitive dysfunction, requires further investigation. Methods: BV-2 microglial cells were exposed to high glucose (50mM) and insulin (2μM) for 48 hours. The levels of interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factoralpha (TNF-α) were assessed through quantitative polymerase chain reaction (qPCR), western blot, and ELISA. CircRNA and messenger RNA (mRNA) sequencing were performed, and the data were analyzed. Differentially expressed circRNAs and mRNAs were identified using qPCR. The circRNA-miRNA interaction was predicted using Miranda and TargetScan software, and their levels were quantified by qPCR. Results: The results demonstrated a significant increase in mRNA and protein levels of IL-1β, IL-6, and TNF-α in BV2 cells treated with glucose and insulin. Five circRNAs (four upregulated and one downregulated) were identified in both glucose and insulin groups compared to the control. Further qPCR analysis revealed marked increases in the levels of chr17:40159331- 40159711+ and chr2:72800499-72801858- (mmu_circ_0010164) in both treatment groups. Competitive endogenous RNA networks showed significant upregulation of mRNA levels of mitochondrial transcription termination factor 1b (Mterf1b) and G protein subunit gamma 4 (Gng4), accompanied by a decrease in mmu-miR-6918-3p and mmu-miR-7043-3p levels in the glucose and insulin groups compared to the control. Knockdown of mmu_circ_0010164 significantly inhibited the inflammatory response induced by glucose and insulin in BV-2 microglial cells. Conclusion: These findings indicate that both glucose and insulin can elicit inflammatory responses in BV2 cells through the modulation of mmu_circ_0010164 levels. The underlying mechanism may involve potential downstream targets of mmu_circ_0010164, specifically mmu-miR-7043-3p/Gng4 and mmu-miR-6918-3p/Mterf1b. This provides novel insights into the treatment of glucose-induced neuroinflammation.

Adenosine relaxes vagina smooth muscle through the cyclic guanosine monophosphate- and cyclic guanosine monophosphate-dependent pathways.

In males, adenosine (ADO) is known to relax penile smooth muscles, although its role in the vagina is not yet fully elucidated. This study investigated the effect of ADO on vagina smooth muscle activity, using a validated female Sprague-Dawley rat model. Contractility studies, using noradrenaline-precontracted vaginal strips, tested the effects of ADORA1/3 antagonists and ADORA2A/2B antagonists and agonists. Increasing doses of ADO were tested after in vivo or in vitro treatment with Nω-nitro-L-arginine-methyl-ester hydrochloride (L-NAME) or with guanylate or adenylate cyclase inhibitors. Immunopositivity for ADORA2A and ADORA2B was assessed, and messenger RNA (mRNA) analysis was performed. Cyclic ADO monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were quantified both in rat vagina smooth muscle cells (rvSMCs) and in vaginal tissues with increasing doses of ADO. Demonstrating ADO's role in the relaxing/contractile mechanism in distal vagina smooth muscle. All ADO receptors mRNAs were expressed in vaginal tissue, with a prevalent content of ADORA2B. A high expression of genes regulating ADO catabolism (ADK) and de novo synthesis (NT5E) was found. In vaginal strips, ADO induced relaxation with IC50 = 144.7μM and a flat pseudo-Hill coefficient value = -0.42, indicating an activity on heterogeneous receptors. Blocking ADORA1/3 shifted ADO response to the left and with a steeper slope. ADORA2A/2B agonists showed a higher potency than ADO in inducing relaxation. Immunolocalization confirmed the presence of ADORA2A/2B in vaginal musculature, in the blood vessels endothelium, and in the epithelium. ADO stimulation of vagina tissues induced a significant increase in cAMP and cGMP contents. Experiments on rvSMCs confirmed that ADO time- and dose-dependently stimulated cAMP production in these cells. However, ADORA2A/2B antagonists, although reducing the ADO-induced relaxation, did not completely block it. A similar inhibition was obtained by blocking adenylate cyclase. Overall, these findings suggest that ADO relaxation involves other pathways, eg, nitric oxide (NO)/cGMP. Accordingly, blocking NO formation through L-NAME substantially blunted ADO responsiveness, as it does the block of cGMP formation through 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Simultaneous incubation with cGMP and cAMP blockers completely inhibited ADO responsiveness. The study highlights ADO's role in regulating vaginal smooth muscle activity, suggesting its potential effect on the vagina. This is the first study on ADO in the vagina, although the results are preliminary and limited to the rat model. These results show that ADO acts as a vaginal relaxing modulator through selective activation of receptors involving not only cAMP but also cGMP.

Advancing Therapeutic and Vaccine Proteins: Switching from Recombinant to Ribosomal Delivery-A Humanitarian Cause.

Recombinant therapeutic and vaccine proteins have revolutionized healthcare, but there remain challenges, as many are awaiting development due to their slow development speed and high development cost. Cell-free in vivo ribosomes offer one choice, but they come with similar constraints. The validation of in vivo messenger RNA (mRNA) technology has been accomplished for COVID-19 vaccines. The bioreactors inside the body, the ribosomes, deliver these proteins at a small cost, since these are chemical products and do not require extensive analytical and regulatory exercises. In this study, we test and validate the final product. A smaller fraction of the recombinant protein cost is needed, removing both constraints. Although thousands of in vivo mRNA products are under development, their regulatory classification remains unresolved: do they qualify as chemical drugs, biological drug, or gene therapy items? These questions will soon be resolved. Additionally, how would the copies of approved in vivo mRNA protein products be brought in, and how would they be treated: as new drugs, generic drugs, or new biological drugs? Researchers are currently working to answer these questions. Regardless, these products' cost of goods (COGs) remains much smaller than that of ex vivo mRNA or recombinant products. This is necessary to meet the needs of the approximately 6.5 billion people around the world who do not have access to biological drugs; these products will indeed serve the dire needs of humanity. Given the minor cost of establishing the manufacturing of these products, it will also prove financially attractive to investors.

An mRNA-encoded dominant-negative inhibitor of transcription factor RUNX1 suppresses vitreoretinal disease in experimental models.

Messenger RNA (mRNA)-based therapies are a promising approach to medical treatment. Except for infectious diseases, no other disease has mRNA-based therapies available. The eye is an ideal model for mRNA therapeutic development because it requires limited dosing. Proliferative vitreoretinopathy (PVR) is a blinding condition caused by retinal detachment that now lacks available medical treatment, with surgery as the only treatment option. We previously implicated runt-related transcription factor-1 (RUNX1) as a driver of epithelial-to-mesenchymal transition (EMT) in PVR and as a critical mediator of aberrant ocular angiogenesis when up-regulated. On the basis of these findings, an mRNA was designed to express a dominant-negative inhibitor of RUNX1 (RUNX1-Trap). We show that RUNX1-Trap delivered in polymer-lipidoid complexes or lipid nanoparticles sequestered RUNX1 in the cytosol and strongly reduced proliferation in primary cell cultures established from fibrotic membranes derived from patients with PVR. We assessed the preclinical efficacy of intraocular delivery of mRNA-encoded RUNX1-Trap in a rabbit model of PVR and in a laser-induced mouse model of aberrant angiogenesis often used to study wet age-related macular degeneration. mRNA-encoded RUNX1-Trap suppressed ocular pathology, measured as pathological scores in the rabbit PVR model and leakage and lesion size in the laser-induced choroidal neovascularization mouse model. mRNA-encoded RUNX1-Trap also strongly reduced proliferation in a human ex vivo explant model of PVR. These data demonstrate the therapeutic potential of mRNA-encoded therapeutic molecules with dominant-negative properties, highlighting the potential of mRNA-based therapies beyond standard gene supplementation approaches.

Transcriptome-wide mapping of internal mRNA N7-methylguanosine in sporulated and unsporulated oocysts of Eimeria tenella reveals stage-specific signatures

BackgroundGrowing evidence indicates that N7-methylguanosine (m7G) modification plays critical roles in epigenetic regulation. However, no data regarding m7G modification are currently available in Eimeria tenella, a highly virulent species causing coccidiosis in chickens.MethodsIn the present study, we explore the distribution of internal messenger RNA (mRNA) m7G modification in sporulated and unsporulated oocysts of E. tenella as well as its potential biological functions during oocyst development using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and mRNA sequencing (mRNA-seq), and the mRNA-seq and MeRIP-seq data were verified by the quantitative reverse transcription polymerase chain reaction (RT–qPCR) and MeRIP–qPCR, respectively.ResultsOur data showed that m7G peaks were detected throughout the whole mRNA body, and the coding DNA sequence (CDS) region displayed the most methylation modification. Compared with unsporulated oocysts, 7799 hypermethylated peaks and 1945 hypomethylated peaks were identified in sporulated oocysts. Further combined analysis of differentially methylated genes (DMGs) and differentially expressed genes (DEGs) showed that there was a generally positive correlation between m7G modification levels and gene transcript abundance. Unsurprisingly, the mRNA-seq and MeRIP-seq data showed good consistency with the results of the RT–qPCR and MeRIP–qPCR, respectively. Gene Ontology (GO) and pathway enrichment analysis of DEGs with altered m7G-methylated peaks were involved in diverse biological functions and pathways, including DNA replication, RNA transport, spliceosome, autophagy-yeast, and cAMP signaling pathway.ConclusionsAltogether, our findings revealed the potential significance of internal m7G modification in E. tenella oocysts, providing some directions and clues for later in-depth research.Graphical abstract

MiR-574-5p in epigenetic regulation and Toll-like receptor signaling.

miR-574-5p is an unusual microRNA (miRNA) that is often upregulated or downregulated following exposure to irradiation or toxic chemicals; bacterial, parasitic or viral infection; and a variety of other disease conditions. Canonically, miR-574-5p epigenetically regulates the expression of many messenger RNAs (mRNAs) through miRNA-mediated posttranscriptional regulation, thereby affecting cellular physiology or pathophysiology and contributing to the pathogenesis or progression of a variety of diseases. However, recent studies have established that in addition to serving as a fine-tuning repressor of gene expression, miR-574-5p also stimulates gene expression as an endogenous ligand for Toll-like receptor-8/7 (TLR8/7). Indeed, the binding of miR-574-5p to TLR8/7 triggers the TLR signaling pathway, leading to the induction of interferons, inflammatory cytokines and autoimmune signaling. These findings suggest that miR-574-5p is not only an important epigenetic regulator of gene expression, but also an important regulator of immune and inflammatory responses. Abnormal miR-574-5p-TLR8/7 signaling has been shown to be tightly associated with inflammation-related cancers and a number of autoimmune disorders. miR-574-5p can serve as a potential biomarker for many diseases. Most importantly, miR-574-5p is a promising therapeutic target for the treatment or prevention of human disorders, especially infectious diseases, cancers and autoimmune diseases.

Chemotherapeutic agents and leucine deprivation induce codon-biased aberrant protein production in cancer.

Messenger RNA (mRNA) translation is a tightly controlled process frequently deregulated in cancer. Key to this deregulation are transfer RNAs (tRNAs), whose expression, processing and post-transcriptional modifications are often altered in cancer to support cellular transformation. In conditions of limiting levels of amino acids, this deregulated control of protein synthesis leads to aberrant protein production in the form of ribosomal frameshifting or misincorporation of non-cognate amino acids. Here, we studied leucine, an essential amino acid coded by six different codons. Surprisingly, we found that leucine deprivation leads to ribosomal stalling and aberrant protein production in various cancer cell types, predominantly at one codon, UUA. Similar effects were observed after treatment with chemotherapeutic agents, implying a shared mechanism controlling the downstream effects on mRNA translation. In both conditions, a limitation in the availability of tRNALeu(UAA) for protein production was shown to be the cause for this dominant effect on UUA codons. The induced aberrant proteins can be processed and immune-presented as neoepitopes and can direct T-cell killing. Altogether, we uncovered a novel mode of interplay between DNA damage, regulation of tRNA availability for mRNA translation and aberrant protein production in cancer that could be exploited for anti-cancer therapy.