Identification and bioinformatics analysis of genes associated with pyroptosis in spinal cord injury of rat and mouse.

The mechanism of spinal cord injury (SCI) is highly complex, and an increasing number of studies have indicated the involvement of pyroptosis in the physiological and pathological processes of secondary SCI. However, there is limited bioinformatics research on pyroptosis-related genes (PRGs) in SCI. This study aims to identify and validate differentially expressed PRGs in the GEO database, perform bioinformatics analysis, and construct regulatory networks to explore potential regulatory mechanisms and therapeutic targets for SCI. We obtained high-throughput sequencing datasets of SCI in rats and mice from the GEO database. Differential analysis was conducted using the "limma" package in R to identify differentially expressed genes (DEGs). These genes were then intersected with previously reported PRGs, resulting in a set of PRGs in SCI. GO and KEGG enrichment analyses, as well as correlation analysis, were performed on the PRGs in both rat and mouse models of SCI. Additionally, a protein-protein interaction (PPI) network was constructed using the STRING website to examine the relationships between proteins. Hub genes were identified using Cytoscape software, and the intersection of the top 5 hub genes in rats and mice were selected for subsequent experimentally validated. Furthermore, a competing endogenous RNA (ceRNA) network was constructed to explore potential regulatory mechanisms. The gene expression profiles of GSE93249, GSE133093, GSE138637, GSE174549, GSE45376, GSE171441_3d and GSE171441_35d were selected in this study. We identified 10 and 12 PRGs in rats and mice datasets respectively. Six common DEGs were identified in the intersection of rats and mice PRGs. Enrichment analysis of these DEGs indicated that GO analysis was mainly focused on inflammation-related factors, while KEGG analysis showed that the most genes were enriched on the NOD-like receptor signaling pathway. We constructed a ceRNA regulatory network that consisted of five important PRGs, as well as 24 miRNAs and 34 lncRNAs. This network revealed potential regulatory mechanisms. Additionally, the three hub genes obtained from the intersection were validated in the rat model, showing high expression of PRGs in SCI. Pyroptosis is involved in secondary SCI and may play a significant role in its pathogenesis. The regulatory mechanisms associated with pyroptosis deserve further in-depth research.

PJA1-mediated suppression of pyroptosis as a driver of docetaxel resistance in nasopharyngeal carcinoma.

Chemoresistance is a main reason for treatment failure in patients with nasopharyngeal carcinoma, but the exact regulatory mechanism underlying chemoresistance in nasopharyngeal carcinoma remains to be elucidated. Here, we identify PJA1 as a key E3 ubiquitin ligase involved in nasopharyngeal carcinoma chemoresistance that is highly expressed in nasopharyngeal carcinoma patients with nonresponse to docetaxel-cisplatin-5-fluorouracil induction chemotherapy. We find that PJA1 facilitates docetaxel resistance by inhibiting GSDME-mediated pyroptosis in nasopharyngeal carcinoma cells. Mechanistically, PJA1 promotes the degradation of the mitochondrial protein PGAM5 by increasing its K48-linked ubiquitination at K88, which further facilitates DRP1 phosphorylation at S637 and reduced mitochondrial reactive oxygen species production, resulting in suppression of GSDME-mediated pyroptosis and the antitumour immune response. PGAM5 knockdown fully restores the docetaxel sensitization effect of PJA1 knockdown. Moreover, pharmacological targeting of PJA1 with the small molecule inhibitor RTA402 enhances the docetaxel sensitivity of nasopharyngeal carcinoma in vitro and in vivo. Clinically, high PJA1 expression indicates inferior survival and poor clinical efficacy of TPF IC in nasopharyngeal carcinoma patients. Our study emphasizes the essential role of E3 ligases in regulating chemoresistance and provides therapeutic strategies for nasopharyngeal carcinoma based on targeting the ubiquitin-proteasome system.

ALKBH4 impedes 5-FU Sensitivity through suppressing GSDME induced pyroptosis in gastric cancer.

5-Fluorouracil (5-FU) is the primary treatment option for advanced gastric cancer. However, the current challenge lies in the absence of validated biomarkers to accurately predict the efficacy and sensitivity of 5-FU in individual patients. It has been confirmed that 5-FU can regulate tumor progression by promoting gasdermin E (GSDME, encoded by DFNA5) cleavage to induce pyroptosis. Lysine demethylase ALKBH4 has been shown to be upregulated in a variety of tumors to promote tumor progression. However, its role in gastric cancer is not clear. In this study, we observed a significant upregulation of ALKBH4 expression in gastric cancer tissues compared to adjacent normal tissues, indicating its potential as a predictor for the poor prognosis of gastric cancer patients. On the contrary, GSDME exhibits low expression levels in gastric cancer and demonstrates a negative correlation with poor prognosis among patients diagnosed with gastric cancer. In addition, we also found that high expression of ALKBH4 can inhibit pyroptosis and promote the proliferation of gastric cancer cells. Mechanistically, ALKBH4 inhibits GSDME activation at the transcriptional level by inhibiting H3K4me3 histone modification in the GSDME promoter region, thereby reducing the sensitivity of gastric cancer cells to 5-FU treatment. These findings provide further insight into the regulatory mechanisms of ALKBH4 in the progression of gastric cancer and underscore its potential as a prognostic marker for predicting the sensitivity of gastric cancer cells to 5-FU treatment.

Pyroptosis and mitochondrial function participated in miR-654-3p-protected against myocardial infarction.

Myocardial infarction (MI) is one of the leading causes of heart failure with highly complicated pathogeneses. miR-654-3p has been recognized as a pivotal regulator of controlling cell survival. However, the function of miR-654-3p in cardiomyocytes and MI has yet to be reported. This study aimed to identify the role of miR-654-3p in the regulation of myocardial infarction. To understand the contribution of miR-654-3p on heart function, we generated cardiac-specific knockdown and overexpression mice using AAV9 technology in MI injury. Mechanically, we combined cellular and molecular techniques, pharmaceutical treatment, RNA sequencing, and functional testing to elucidate the potential pathological mechanisms. We identified that mice subjected to MI decreased the expression of miR-654-3p in the border and infarcted area. Mice lacking miR-654-3p in the heart showed some inflammation infiltration and myocardial fibrosis, resulting in a mild cardiac injury. Furthermore, we found a deficiency of miR-654-3p in cardiomyocytes resulted in pyroptotic cell death but not other programmed cell death. Intriguingly, miR-654-3p deficiency aggravated MI-induced cardiac dysfunction, accompanied by higher myocardial fibrosis and cardiac enzymes and augmented pyroptosis activation. Cardiac elevating miR-654-3p prevented myocardial fibrosis and inflammation infiltration and decreased pyroptosis profile, thereby attenuating MI-induced cardiac damage. Using RNA sequence and molecular biological approaches, we found overexpression of miR-654-3p in the heart promoted the metabolic ability of the cardiomyocytes by promoting mitochondrial metabolism and mitochondrial respiration function. Our finding identified the character of miR-654-3p in protecting against MI damage by mediating pyroptosis and mitochondrial metabolism. These findings provide a new mechanism for miR-654-3p involvement in the pathogenesis of MI and reveal novel therapeutic targets. miR-654-3p expression was decreased after MI. Mice lacking miR-654-3p in the heart showed some inflammation infiltration and myocardial fibrosis, resulting in a mild cardiac injury. The deficiency of miR-654-3p in cardiomyocytes resulted in pyroptotic cell death. miR-654-3p deficiency aggravated MI-induced cardiac dysfunction, accompanied by higher myocardial fibrosis and cardiac enzymes and augmented pyroptosis activation. Overexpression of miR-654-3p prevented myocardial fibrosis and inflammation infiltration and decreased pyroptosis profile, thereby attenuating MI-induced cardiac damage. Overexpression of miR-654-3p in the heart promoted the metabolic ability of the cardiomyocytes by promoting mitochondrial metabolism and mitochondrial respiration function.

miRNA-206-3p alleviates LPS-induced acute lung injury via inhibiting inflammation and pyroptosis through modulating TLR4/NF-κB/NLRP3 pathway.

Acute lung injury (ALI) is life-threatening. MicroRNAs (miRNAs) are often abnormally expressed in inflammatory diseases and are closely associated with ALI. This study investigates whether miRNA-206-3p attenuates pyroptosis in ALI and elucidates the underlying molecular mechanisms. ALI mouse and cell models were established through lipopolysaccharide (LPS) treatment for 24 h. Subsequently, the models were evaluated based on ultrasonography, the lung tissue wet/dry (W/D) ratio, pathological section assessment, electron microscopy, and western blotting. Pyroptosis in RAW264.7 cells was then assessed via electron microscopy, immunofluorescence, and western blotting. Additionally, the regulatory relationship between miRNA-206-3p and the Toll-like receptor (TLR)4/nuclear factor (NF)-κB/Nod-like receptor protein-3 (NLRP3) pathway was verified. Finally, luciferase reporter gene and RNA pull-down assays were used to verify the targeting relationship between miRNA-206-3p and TLR4. miRNA206-3p levels are significantly decreased in the LPS-induced ALI model. Overexpression of miRNA-206-3p improves ALI, manifested as improved lung ultrasound, improved pathological changes of lung tissue, reduced W/D ratio of lung tissue, release of inflammatory factors in lung tissue, and reduced pyroptosis. Furthermore, overexpression of miRNA-206-3p contributed to reversing the ALI-promoting effect of LPS by hindering TLR4, myeloid differentiation primary response 88 (MyD88), NF-κB, and NLRP3 expression. In fact, miRNA-206-3p binds directly to TLR4. In conclusion, miRNA-206-3p alleviates LPS-induced ALI by inhibiting inflammation and pyroptosis via TLR4/NF-κB/NLRP3 pathway modulation.

KIF23 promotes cervical cancer progression via inhibiting NLRP3-mediated pyroptosis.

BACKGROUND: Cervical cancer (CC), closely linked to persistent human papillomavirus infection, represents a major health problem for women worldwide. The objective of this study is to elucidate KIF23's role in the development of CC and its regulatory mechanism. METHODS: The bioinformatics methods were utilized to extract pyroptosis-associated differentially expressed genes (DEGs) and pivot genes from the GSE9750 and GSE63678 datasets, followed by immune infiltration analysis and quantification of these genes' expression. The effects of kinesin family member 23 (KIF23) were verified through functional experiments in vitro and a mouse xenograft model. The NLPR3 activator, nigericin, was applied for further analyzing the potential regulatory mechanism of KIF23 in CC. RESULTS: A total of 8 pyroptosis-related DEGs were screened out, among which 4 candidate core genes were identified as candidate hub genes and confirmed upregulation in CC tissues and cells. These genes respectively showed a positive correlation with the infiltration of distinct immune cells or tumor purity. Downregulation of KIF23 could suppress the proliferation, migration, and invasion abilities in CC cells and tumorigenesis through enhancing pyroptosis. Conversely, KIF23 overexpression accelerated the malignant phenotypes of CC cells and inhibited pyroptosis activation, which was blocked by nigericin treatment. CONCLUSIONS: KIF23 may play an oncogenic role in CC progression via inhibition of the NLRP3-mediated pyroptosis pathway.

Prognostic and chemotherapeutic implications of a novel four-gene pyroptosis model in head and neck squamous cell carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers. Chemotherapy remains one dominant therapeutic strategy, while a substantial proportion of patients may develop chemotherapeutic resistance; therefore, it is particularly significant to identify the patients who could achieve maximum benefits from chemotherapy. Presently, four pyroptosis genes are reported to correlate with the chemotherapeutic response or prognosis of HNSCC, while no study has assessed the combinatorial predicting efficacy of these four genes. Hence, this study aims to evaluate the predictive value of a multi-gene pyroptosis model regarding the prognosis and chemotherapeutic responsiveness in HNSCC. Methods: By utilizing RNA-sequencing data from The Cancer Genome Atlas database and the Gene Expression Omnibus database, the pyroptosis-related gene score (PRGscore) was computed for each HNSCC sample by performing a Gene Set Variation Analysis (GSVA) based on four genes (Caspase-1, Caspase-3, Gasdermin D, Gasdermin E). The prognostic significance of the PRGscore was assessed through Cox regression and Kaplan-Meier survival analyses. Additionally, chemotherapy sensitivity stratified by high and low PRGscore was examined to determine the potential association between pyroptosis activity and chemosensitivity. Furthermore, chemotherapy sensitivity assays were conducted in HNSCC cell lines in vitro. Results: As a result, our study successfully formulated a PRGscore reflective of pyroptotic activity in HNSCC. Higher PRGscore correlates with worse prognosis. However, patients with higher PRGscore were remarkably more responsive to chemotherapy. In agreement, chemotherapy sensitivity tests on HNSCC cell lines indicated a positive association between overall pyroptosis levels and chemosensitivity to cisplatin and 5-fluorouracil; in addition, patients with higher PRGscore may benefit from the immunotherapy. Overall, our study suggests that HNSCC patients with higher PRGscore, though may have a less favorable prognosis, chemotherapy and immunotherapy may exhibit better benefits in this population.

ANGPTL2 knockdown induces autophagy to relieve alveolar macrophage pyroptosis by reducing LILRB2-mediated inhibition of TREM2.

Acute lung injury (ALI) is featured with a robust inflammatory response. Angiopoietin-like protein 2 (ANGPTL2), a pro-inflammatory protein, is complicated with various disorders. However, the role of ANGPTL2 in ALI remains to be further explored. The mice and MH-S cells were administrated with lipopolysaccharide (LPS) to evoke the lung injury in vivo and in vitro. The role and mechanism of ANGPTL was investigated by haematoxylin-eosin, measurement of wet/dry ratio, cell count, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling, reverse transcription quantitative polymerase chain reaction, immunofluorescence, enzyme-linked immunosorbent assay, detection of autophagic flux and western blot assays. The level of ANGPTL2 was upregulated in lung injury. Knockout of ANGPTL2 alleviated LPS-induced pathological symptoms, reduced pulmonary wet/dry weight ratio, the numbers of total cells and neutrophils in BALF, apoptosis rate and the release of pro-inflammatory mediators, and modulated polarization of alveolar macrophages in mice. Knockdown of ANGPTL2 downregulated the level of pyroptosis indicators, and elevated the level of autophagy in LPS-induced MH-S cells. Besides, downregulation of ANGPTL2 reversed the LPS-induced the expression of leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) and triggering receptor expressed on myeloid cells 2 (TREM2), which was reversed by the overexpression of LILRB2. Importantly, knockdown of TREM2 reversed the levels of autophagy- and pyroptosis-involved proteins, and the contents of pro-inflammatory factors in LPS-induced MH-S cells transfected with si ANGPTL2, which was further inverted with the treatment of rapamycin. Therefore, ANGPTL2 silencing enhanced autophagy to alleviate alveolar macrophage pyroptosis via reducing LILRB2-mediated inhibition of TREM2.

Correlations between the modification patterns mediated by pyroptosis-related genes, tumor microenvironment, and immunotherapy in soft tissue sarcoma.

Soft tissue sarcoma (STS) incidence, progression, and metastasis are tightly linked to the tumor microenvironment (TME). The modification patterns mediated by pyroptosis-related genes (PRGs) in STS are unknown regarding the immune cell infiltration landscape of TME, immunotherapy effect, and prognostic value. First, we downloaded STS samples from the Cancer Genome Atlas (TCGA) and gene-expression omnibus (GEO) databases. Based on 52 PRGs, 2 pyroptosis modification patterns were analyzed, and the associations of pyroptosis modification patterns with immune cell infiltration in the TME were elucidated systematically. To quantify PRG modification patterns in STS patients, we generated a pyroptosis scoring system using principal component analysis (PCA). We identified 2 distinct pyroptosis modification patterns in STS. Compared to PRG cluster A, the prognosis of cluster B was better. These 2 pyroptosis modification patterns corresponded to different characteristics of immune cell infiltration in the TME and biological behaviors. In the pyroptosis scoring system, a high pyroptosis score was connected to higher immune cell infiltration, stronger immune surveillance, immune-killing effects on tumor cells, and better clinical benefits. The results from 3 anti-PD1/PD-L1-treated immune cohorts demonstrated that higher pyroptosis scores are also closely connected to better immunotherapy results. We demonstrated that pyroptosis modification is essential to the STS microenvironment. Moreover, the pyroptosis score is a reliable and independent prognostic factor in STS patients, enabling a richer understanding of the STS microenvironment and the screening of immunotherapy candidates, predicting the immunotherapeutic effects for individual STS patients, and guiding the use of chemotherapy drugs.

Knockdown of CENPM activates cGAS-STING pathway to inhibit ovarian cancer by promoting pyroptosis.

OBJECTIVE: We aimed to screen novel gene signatures for ovarian cancer (OC) and explore the role of biomarkers in OC via regulating pyroptosis using bioinformatics analysis. METHODS: Differentially expressed genes (DEGs) of OC were screened from GSE12470 and GSE16709 datasets. Hub genes were determined from protein-protein interaction networks after bioinformatics analysis. The role of Centromeric protein M (CENPM) in OC was assessed by subcutaneous tumor experiment using hematoxylin-eosin and immunohistochemical staining. Tumor metastasis was evaluated by detecting epithelial-mesenchymal transition-related proteins. The proliferation, migration, and invasion were determined using cell counting kit and transwell assay. Enzyme-linked immunosorbent assay was applied to measure inflammatory factors. The mRNA and protein expression were detected using real-time quantitative PCR and western blot. RESULTS: We determined 9 hub genes (KIFC1, PCLAF, CDCA5, KNTC1, MCM3, OIP5, CENPM, KIF15, and ASF1B) with high prediction value for OC. In SKOV3 and A2780 cells, the expression levels of hub genes were significantly up-regulated, compared with normal ovarian cells. CENPM was selected as a key gene. Knockdown of CENPM suppressed proliferation, migration, and invasion of OC cells. Subcutaneous tumor experiment revealed that CENPM knockdown significantly suppressed tumor growth and metastasis. Additionally, pyroptosis was promoted in OC cells and xenograft tumors after CENPM knockdown. Furthermore, CENPM knockdown activated cGAS-STING pathway and the pathway inhibitor reversed the inhibitory effect of CENPM knockdown on viability, migration, and invasion of OC cells. CONCLUSION: CENPM was a novel biomarker of OC, and knockdown of CENPM inhibited OC progression by promoting pyroptosis and activating cGAS-STING pathway.

Super enhancer-associated circRNA-circLrch3 regulates hypoxia-induced pulmonary arterial smooth muscle cells pyroptosis by formation of R-loop with host gene.

BACKGROUND: Pulmonary hypertension (PH) is a complex vascular disorder, characterized by pulmonary vessel remodeling and perivascular inflammation. Pulmonary arterial smooth muscle cells (PASMCs) pyroptosis is a novel pathological mechanism implicated of pulmonary vessel remodeling. However, the involvement of circRNAs in the process of pyroptosis and the underlying regulatory mechanisms remain inadequately understood. METHODS: Western blotting, PI staining and LDH release were used to explore the role of circLrch3 in PASMCs pyroptosis. Moreover, S9.6 dot blot and DRIP-PCR were used to assess the formation of R-loop between circLrch3 and its host gene Lrch3. Chip-qPCR were used to evaluate the mechanism of super enhancer-associated circLrh3, which is transcriptionally activated by the transcription factor Tbx2. RESULTS: CircLrch3 was markedly upregulated in hypoxic PASMCs. CircLrch3 knockdown inhibited hypoxia induced PASMCs pyroptosis in vivo and in vitro. Mechanistically, circLrch3 can form R-loop with host gene to upregulate the protein and mRNA expression of Lrch3. Furthermore, super enhancer interacted with the Tbx2 at the Lrch3 promoter locus, mediating the augmented transcription of circLrch3. CONCLUSION: Our findings clarify the role of a super enhancer-associated circLrch3 in the formation of R-loop with the host gene Lrch3 to modulate pyroptosis in PASMCs, ultimately promoting the development of PH.

Exosomal long non-coding RNA AU020206 alleviates macrophage pyroptosis in atherosclerosis by suppressing CEBPB-mediated NLRP3 transcription.

Recent studies have suggested exosomes (EXO) as potential therapeutic tools for cardiovascular diseases, including atherosclerosis (AS). This study investigates the function of bone marrow stem cell (BMSC)-derived exosomes (EXO) on macrophage pyroptosis in AS and explores the associated mechanism. BMSC-EXO were isolated from healthy mice and identified. RAW264.7 cells (mouse macrophages) were exposed to oxLDL to simulate an AS condition. BMSC-EXO treatment enhanced viability and reduced lactate dehydrogenase release of macrophages. An animal model of AS was established using ApoE-/- mice. BMSC-EXO treatment suppressed plaque formation as well as macrophage and lipid infiltration in mouse aortic tissues. Moreover, BMSC-EXO decreased concentrations of pyroptosis-related markers interleukin (IL)-1ß, IL-18, cleaved-caspase-1 and gasdermin D in vitro and in vivo. Long non-coding RNA AU020206 was carried by the BMSC-EXO, and it bound to CCAAT enhancer binding protein beta (CEBPB) to block CEBPB-mediated transcriptional activation of NLR family pyrin domain containing 3 (NLRP3). Functional assays revealed that silencing of AU020206 aggravated macrophage pyroptosis and exacerbated AS symptoms in mice. These exacerbations were blocked upon CEBPB silencing but then restored after NLRP3 overexpression. In conclusion, this study demonstrates that AU020206 delivered by BMSC-EXO alleviates macrophage pyroptosis in AS by blocking CEBPB-mediated transcriptional activation of NLRP3.

Epigenetic regulation of diverse cell death modalities in cancer: a focus on pyroptosis, ferroptosis, cuproptosis, and disulfidptosis.

Tumor is a local tissue hyperplasia resulted from cancerous transformation of normal cells under the action of various physical, chemical and biological factors. The exploration of tumorigenesis mechanism is crucial for early prevention and treatment of tumors. Epigenetic modification is a common and important modification in cells, including DNA methylation, histone modification, non-coding RNA modification and m6A modification. The normal mode of cell death is programmed by cell death-related genes; however, recent researches have revealed some new modes of cell death, including pyroptosis, ferroptosis, cuproptosis and disulfidptosis. Epigenetic regulation of various cell deaths is mainly involved in the regulation of key cell death proteins and affects cell death by up-regulating or down-regulating the expression levels of key proteins. This study aims to investigate the mechanism of epigenetic modifications regulating pyroptosis, ferroptosis, cuproptosis and disulfidptosis of tumor cells, explore possible triggering factors in tumor development from a microscopic point of view, and provide potential targets for tumor therapy and new perspective for the development of antitumor drugs or combination therapies.

Unveiling the role of PD-L1 in vascular endothelial dysfunction: Insights into the mtros/NLRP3/caspase-1 mediated pyroptotic pathway.

BACKGROUND: Programmed death ligand-1(PD-L1) has been postulated to play a crucial role in the regulation of barrier functions of the vascular endothelium, yet how this novel molecule mediates dysfunction in endothelial cells (ECs) during acute lung injury (ALI) remains largely unknown. METHODS: PD-L1 siRNA and plasmids were synthesized and applied respectively to down- or up-regulate PD-L1 expression in human lung microvascular endothelial cells (HMVECs). RNA sequencing was used to explore the differentially expressed genes following PD-L1 overexpression. The expression levels of tight junction proteins (ZO-1 and occludin) and the signaling pathways of NLRP-3/caspase-1/pyroptosis were analyzed. A mouse model of indirect ALI was established through hemorrhagic shock (HEM) followed by cecal ligation and puncture (CLP), enabling further investigation into the effects of intravenous delivery of PD-L1 siRNA. RESULTS: A total of 1502 differentially expressed genes were identified, comprising 532 down-regulated and 970 up-regulated genes in ECs exhibiting PD-L1overexpression. Enrichment of PD-L1-correlated genes were observed in the NOD-like receptor signaling pathway and the TNF signaling pathway. Western blot assays confirmed that PD-L1 overexpression elevated the expression of NLRP3, cleaved-caspase-1, ASC and GSDMD, and concurrently diminished the expression of ZO-1 and occludin. This overexpression also enhanced mitochondrial oxidative phosphorylation and mitochondrial reactive oxygen species (mtROS) production. Interestingly, mitigating mitochondrial dysfunction with mitoQ partially countered the adverse effects of PD-L1 on the functionality of ECs. Furthermore, intravenous administration of PD-L1 siRNA effectively inhibited the activation of the NLRP3 inflammasome and pyroptosis in pulmonary ECs, subsequently ameliorating lung injury in HEM/CLP mice. CONCLUSION: PD-L1-mediated activation of the inflammasome contributes significantly to the disruption of tight junction and induction of pyroptosis in ECs, where oxidative stress associated with mitochondrial dysfunction serves as a pivotal mechanism underpinning these effects.

A novel pyroptosis-related gene signature exhibits distinct immune cells infiltration landscape in Wilms' tumor.

BACKGROUND: Wilms' tumor (WT) is the most common renal tumor in childhood. Pyroptosis, a type of inflammation-characterized and immune-related programmed cell death, has been extensively studied in multiple tumors. In the current study, we aim to construct a pyroptosis-related gene signature for predicting the prognosis of Wilms' tumor. METHODS: We acquired RNA-seq data from TARGET kidney tumor projects for constructing a gene signature, and snRNA-seq data from GEO database for validating signature-constructing genes. Pyroptosis-related genes (PRGs) were collected from three online databases. We constructed the gene signature by Lasso Cox regression and then established a nomogram. Underlying mechanisms by which gene signature is related to overall survival states of patients were explored by immune cell infiltration analysis, differential expression analysis, and functional enrichment analysis. RESULTS: A pyroptosis-related gene signature was constructed with 14 PRGs, which has a moderate to high predicting capacity with 1-, 3-, and 5-year area under the curve (AUC) values of 0.78, 0.80, and 0.83, respectively. A prognosis-predicting nomogram was established by gender, stage, and risk score. Tumor-infiltrating immune cells were quantified by seven algorithms, and the expression of CD8( +) T cells, B cells, Th2 cells, dendritic cells, and type 2 macrophages are positively or negatively correlated with risk score. Two single nuclear RNA-seq samples of different histology were harnessed for validation. The distribution of signature genes was identified in various cell types. CONCLUSIONS: We have established a pyroptosis-related 14-gene signature in WT. Moreover, the inherent roles of immune cells (CD8( +) T cells, B cells, Th2 cells, dendritic cells, and type 2 macrophages), functions of differentially expressed genes (tissue/organ development and intercellular communication), and status of signaling pathways (proteoglycans in cancer, signaling pathways regulating pluripotent of stem cells, and Wnt signaling pathway) have been elucidated, which might be employed as therapeutic targets in the future.

Mitochondrial (mt)DNA-cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling promotes pyroptosis of macrophages via interferon regulatory factor (IRF)7/IRF3 activation to aggravate lung injury during severe acute pancreatitis.

BACKGROUND: Macrophage proinflammatory activation contributes to the pathology of severe acute pancreatitis (SAP) and, simultaneously, macrophage functional changes, and increased pyroptosis/necrosis can further exacerbate the cellular immune suppression during the process of SAP, where cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) plays an important role. However, the function and mechanism of cGAS-STING in SAP-induced lung injury (LI) remains unknown. METHODS: Lipopolysaccharide (LPS) was combined with caerulein-induced SAP in wild type, cGAS -/- and sting -/- mice. Primary macrophages were extracted via bronchoalveolar lavage and peritoneal lavage. Ana-1 cells were pretreated with LPS and stimulated with nigericin sodium salt to induce pyroptosis in vitro. RESULTS: SAP triggered NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation-mediated pyroptosis of alveolar and peritoneal macrophages in mouse model. Knockout of cGAS/STING could ameliorate NLRP3 activation and macrophage pyroptosis. In addition, mitochondrial (mt)DNA released from damaged mitochondria further induced macrophage STING activation in a cGAS- and dose-dependent manner. Upregulated STING signal can promote NLRP3 inflammasome-mediated macrophage pyroptosis and increase serum interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α levels and, thus, exacerbate SAP-associated LI (SAP-ALI). Downstream molecules of STING, IRF7, and IRF3 connect the mtDNA-cGAS-STING axis and the NLRP3-pyroptosis axis. CONCLUSIONS: Negative regulation of any molecule in the mtDNA-cGAS-STING-IRF7/IRF3 pathway can affect the activation of NLRP3 inflammasomes, thereby reducing macrophage pyroptosis and improving SAP-ALI in mouse model.

Competitive adsorption of microRNA-532-3p by circular RNA SOD2 activates Thioredoxin Interacting Protein/NLR family pyrin domain containing 3 pathway and promotes pyroptosis of non-alcoholic fatty hepatocytes.

OBJECTIVE: There is a growing body of evidence indicating that pyroptosis, a programmed cell death mechanism, plays a crucial role in the exacerbation of inflammation and fibrosis in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Circular RNAs (circRNAs), functioning as vital regulators within NAFLD, have been shown to mediate the process of cell pyroptosis. This study aims to elucidate the roles and mechanisms of circRNAs in NAFLD. METHODS: Utilizing a high-fat diet (HFD)-induced rat model for in vivo experimentation and hepatocytes treated with palmitic acid (PA) for in vitro models, we identified circular RNA SOD2 (circSOD2) as our circRNA of interest through analysis with the circMine database. The expression levels of associated genes and pyroptosis-related proteins were determined using quantitative real-time polymerase chain reaction and Western blotting, alongside immunohistochemistry. Serum liver function markers, cellular inflammatory cytokines, malondialdehyde, lactate dehydrogenase levels, and mitochondrial membrane potential, were assessed using enzyme-linked immunosorbent assay, standard assay kits, or JC-1 staining. Flow cytometry was employed to detect pyroptotic cells, and lipid deposition in liver tissues was observed via Oil Red O staining. The interactions between miR-532-3p/circSOD2 and miR-532-3p/Thioredoxin Interacting Protein (TXNIP) were validated through dual-luciferase reporter assays and RNA immunoprecipitation experiments. RESULTS: Our findings demonstrate that, in both in vivo and in vitro NAFLD models, there was an upregulation of circSOD2 and TXNIP, alongside a downregulation of miR-532-3p. Mechanistically, miR-532-3p directly bound to the 3'-UTR of TXNIP, thereby mediating inflammation and cell pyroptosis through targeting the TXNIP/NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway. circSOD2 directly interacted with miR-532-3p, relieving the suppression on the TXNIP/NLRP3 signaling pathway. Functionally, the knockdown of circSOD2 or TXNIP improved hepatocyte pyroptosis; the deletion of miR-532-3p reversed the effects of circSOD2 knockdown, and the deletion of TXNIP reversed the effects of circSOD2 overexpression. Furthermore, the knockdown of circSOD2 significantly mitigated the progression of NAFLD in vivo. CONCLUSION: circSOD2 competitively sponges miR-532-3p to activate the TXNIP/NLRP3 inflammasome signaling pathway, promoting pyroptosis in NAFLD.

DNMT1 methylation of LncRNA-ANRIL causes myocardial fibrosis pyroptosis by interfering with the NLRP3/Caspase-1 pathway.

Myocardial fibrosis is a common pathological manifestation that occurs in various cardiac diseases. The present investigation aims to reveal how DNMT1/lncRNA-ANRIL/NLRP3 influences fibrosis and cardiac fibroblast pyroptosis. Here, we used ISO to induce myocardial fibrosis in mice, and LPS and ATP to induce myocardial fibroblast pyroptosis. The results showed that DNMT1, Caspase-1, and NLRP3 expression were significantly increased in fibrotic murine myocardium and pyroptotic cardiac fibroblasts, whereas LncRNA-ANRIL expression was decreased. DNMT1 overexpression decreased the level of LncRNA-ANRIL while increasing the levels of NLRP3 and Caspase-1. Contrarily, silencing DNMT1 increased the LncRNA-ANRIL and decreased the levels of NLRP3 and Caspase-1. Silencing LncRNA-ANRIL increased the levels of NLRP3 and Caspase-1. The present findings suggest that DNMT1 can methylate LncRNA-ANRIL during the development of myocardial fibrosis and CFs cell scorching, resulting in low LncRNA-ANRIL expression, thereby influencing myocardial fibrosis and cardiac fibroblast pyroptosis.

Transcriptomic analysis reveals molecular characterization and immune landscape of PANoptosis-related genes in atherosclerosis.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease characterized by abnormal lipid deposition in the arteries. Programmed cell death is involved in the inflammatory response of atherosclerosis, but PANoptosis, as a new form of programmed cell death, is still unclear in atherosclerosis. This study explored the key PANoptosis-related genes involved in atherosclerosis and their potential mechanisms through bioinformatics analysis. METHODS: We evaluated differentially expressed genes (DEGs) and immune infiltration landscape in atherosclerosis using microarray datasets and bioinformatics analysis. By intersecting PANoptosis-related genes from the GeneCards database with DEGs, we obtained a set of PANoptosis-related genes in atherosclerosis (PANoDEGs). Functional enrichment analysis of PANoDEGs was performed and protein-protein interaction (PPI) network of PANoDEGs was established. The machine learning algorithms were used to identify the key PANoDEGs closely linked to atherosclerosis. Receiver operating characteristic (ROC) analysis was used to assess the diagnostic potency of key PANoDEGs. CIBERSORT was used to analyze the immune infiltration patterns in atherosclerosis, and the Spearman method was used to study the relationship between key PANoDEGs and immune infiltration abundance. The single gene enrichment analysis of key PANoDEGs was investigated by GSEA. The transcription factors and target miRNAs of key PANoDEGs were predicted by Cytoscape and online database, respectively. The expression of key PANoDEGs was validated through animal and cell experiments. RESULTS: PANoDEGs in atherosclerosis were significantly enriched in apoptotic process, pyroptosis, necroptosis, cytosolic DNA-sensing pathway, NOD-like receptor signaling pathway, lipid and atherosclerosis. Four key PANoDEGs (ZBP1, SNHG6, DNM1L, and AIM2) were found to be closely related to atherosclerosis. The ROC curve analysis demonstrated that the key PANoDEGs had a strong diagnostic potential in distinguishing atherosclerotic samples from control samples. Immune cell infiltration analysis revealed that the proportion of initial B cells, plasma cells, CD4 memory resting T cells, and M1 macrophages was significantly higher in atherosclerotic tissues compared to normal tissues. Spearman analysis showed that key PANoDEGs showed strong correlations with immune cells such as T cells, macrophages, plasma cells, and mast cells. The regulatory networks of the four key PANoDEGs were established. The expression of key PANoDEGs was verified in further cell and animal experiments. CONCLUSIONS: This study evaluated the expression changes of PANoptosis-related genes in atherosclerosis, providing a reference direction for the study of PANoptosis in atherosclerosis and offering potential new avenues for further understanding the pathogenesis and treatment strategies of atherosclerosis.

Transcriptomics confirms IRF1 as a key regulator of pyroptosis in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a retinal microvascular complication caused by hyperglycemia, which can lead to visual impairment or blindness. Pyroptosis is a type of inflammation-related programmed cell death, activated by caspase-1, resulting in the maturation of IL-1ß and IL-18 and the rupture of the cell membrane. RNA sequencing (RNA-seq) is a high-throughput sequencing technique that reveals the presence and quantity of RNA in the genome at a specific time point, i.e., the transcriptome. RNA-seq can analyze gene expression levels, splicing variants, mutations, fusions, editing and other post-transcriptional modifications, as well as gene expression differences between different samples or conditions. It has been widely used in biological and medical research, clinical diagnosis and new drug development. This study aimed to establish an in vitro model of diabetic retinopathy by culturing human retinal endothelial cells (HREC) with high glucose (30 mmol/L), and to detect their transcriptome expression by RNA-seq, screen for key genes related to pyroptosis, and validate the sequencing results by subsequent experiments. METHODS: We used RNA-seq to detect the transcriptome expression differences between HREC cells cultured with high glucose and control group, and identified differentially expressed genes by GO/KEGG analysis. We constructed a PPI network and determined the key genes by Cytoscape software and CytoHubba plugin. We validated the expression of related factors by Western Blot, qPCR and ELISA. RESULTS: We performed GO and KEGG analysis on the RNA-seq data and found differentially expressed genes. We used Cytoscape and CytoHubba plugin to screen out IRF1 as the key gene, and then detected the expression of IRF1 in HREC under high glucose and control group by Western Blot and qPCR. We found that the expression of Caspase-1, GSDMD and IL-1ß proteins in HREC under high glucose increased, while the expression of these proteins decreased after the inhibition of IRF1 by siRNA. ELISA showed that the secretion of IL-1ß in HREC under high glucose increased, while the inhibition of IRF1 reduced the secretion of IL-1ß. These results indicate that IRF1 plays an important role in DR, and provides a new target and strategy for the prevention and treatment of this disease.