Identification of Breast Cancer Subtypes Based on Endoplasmic Reticulum Stress-Related Genes and Analysis of Prognosis and Immune Microenvironment in Breast Cancer Patients.

Introduction: Endoplasmic reticulum stress (ERS) was a response to the accumulation of unfolded proteins and plays a crucial role in the development of tumors, including processes such as tumor cell invasion, metastasis, and immune evasion. However, the specific regulatory mechanisms of ERS in breast cancer (BC) remain unclear. Methods: In this study, we analyzed RNA sequencing data from The Cancer Genome Atlas (TCGA) for breast cancer and identified 8 core genes associated with ERS: ELOVL2, IFNG, MAP2K6, MZB1, PCSK6, PCSK9, IGF2BP1, and POP1. We evaluated their individual expression, independent diagnostic, and prognostic values in breast cancer patients. A multifactorial Cox analysis established a risk prognostic model, validated with an external dataset. Additionally, we conducted a comprehensive assessment of immune infiltration and drug sensitivity for these genes. Results: The results indicate that these eight core genes play a crucial role in regulating the immune microenvironment of breast cancer (BRCA) patients. Meanwhile, an independent diagnostic model based on the expression of these eight genes shows limited independent diagnostic value, and its independent prognostic value is unsatisfactory, with the time ROC AUC values generally below 0.5. According to the results of logistic regression neural networks and risk prognosis models, when these eight genes interact synergistically, they can serve as excellent biomarkers for the diagnosis and prognosis of breast cancer patients. Furthermore, the research findings have been confirmed through qPCR experiments and validation. Conclusion: In conclusion, we explored the mechanisms of ERS in BRCA patients and identified 8 outstanding biomolecular diagnostic markers and prognostic indicators. The research results were double-validated using the GEO database and qPCR.

Pancreatic ß-Cell Identity Change through the Lens of Single-Cell Omics Research.

The main hallmark in the development of both type 1 and type 2 diabetes is a decline in functional ß-cell mass. This decline is predominantly attributed to ß-cell death, although recent findings suggest that the loss of ß-cell identity may also contribute to ß-cell dysfunction. This phenomenon is characterized by a reduced expression of key markers associated with ß-cell identity. This review delves into the insights gained from single-cell omics research specifically focused on ß-cell identity. It highlights how single-cell omics based studies have uncovered an unexpected level of heterogeneity among ß-cells and have facilitated the identification of distinct ß-cell subpopulations through the discovery of cell surface markers, transcriptional regulators, the upregulation of stress-related genes, and alterations in chromatin activity. Furthermore, specific subsets of ß-cells have been identified in diabetes, such as displaying an immature, dedifferentiated gene signature, expressing significantly lower insulin mRNA levels, and expressing increased ß-cell precursor markers. Additionally, single-cell omics has increased insight into the detrimental effects of diabetes-associated conditions, including endoplasmic reticulum stress, oxidative stress, and inflammation, on ß-cell identity. Lastly, this review outlines the factors that may influence the identification of ß-cell subpopulations when designing and performing a single-cell omics experiment.

Gene Expression Profiling to Unfolded Proteins Response as a Risk Modulator of Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease. Despite new methods of diagnostics and treatment as well as extensive biological and immunosuppressive treatment, the etiology of RA is not fully understood. Moreover, the problem of diagnosis and treatment of RA patients is still current and affects a large group of patients. It is suggested that endoplasmic reticulum (ER)-related features may impair adaptation to chronic stress, inferring the risk of rheumatoid arthritis. The main goal in this study was evaluation of changes in mRNA translation to determine chronic ER stress conditions in rheumatoid arthritis patients. The study group consist of 86 individuals including a total of 56 rheumatoid arthritis patients and 30 healthy controls. The expression level of mRNA form blood samples of RA patients as well as controls of the unfolded protein response (UPR)-associated genes (p-eIF2, BCL-2, PERK, ATF4, and BAX) were investigated using real-time qPCR. GAPDH expression was used as a standard control. Considering the median, the expression levels of PERK, BCL-2, p-eIF2, ATF4, and BAX were found to be significantly increased in the blood of RA patients compared with the control group. The p-value for the PERK gene was 0.0000000036, the p-value for the BCL-2 gene was 0.000000014, the p-value for the p-eIF2 gene was 0.006948, the p-value for the ATF4 gene was 0.0000056, and the p-value for the BAX gene was 0.00019, respectively. Thus, it can be concluded that the targeting of the components of the PERK-dependent UPR signaling pathway via small-molecule PERK inhibitors may contribute to the development of novel, innovative treatment strategies against rheumatoid arthritis.

Characterization of an endoplasmic reticulum stress-associated lncRNA prognostic signature and the tumor-suppressive role of RP11-295G20.2 knockdown in lung adenocarcinoma.

Endoplasmic reticulum stress (ERS) is commonly induced by accumulating misfolded or unfolded proteins in tumor microenvironment. Long non-coding RNAs (lncRNAs) play important roles in ERS response and lung adenocarcinoma (LUAD) progression. However, the role of ERS-related lncRNAs in LUAD remains unknown. In this study, we aimed to identify ERS-associated lncRNAs with prognostic value in LUAD and characterize their clinical implications. Cox and least absolute shrinkage and selection operator regression analyses identified nine ERS-related lncRNAs with independent prognostic abilities, including five protective factors (CROCCP2, KIAA0125, LINC0996, RPARP-AS1 and TBX5-AS1) and four risk factors (LINC0857, LINC116, RP11-21L23.2 and RP11-295G20.2). We developed an ERS-related lncRNA risk prediction model in predicting overall survival of LUAD patients, which classified TCGA cohorts into high-risk (HS) and low-risk (LS) groups. Comprehensive bioinformatic analyses revealed HS patients featured with late-stage tumors, greater mutation burdens, weaker anti-tumor immunity/responses, and lower sensitivity to targeted drugs compared to LS patients, contributing to tumor progression and a poor prognosis. Functional enrichment analysis implicated these ERS-related lncRNAs in cell migration, cell death, and immunity. Furthermore, expression of the most significantly upregulated risk lncRNA, RP11-295G20.2, was validated at the mRNA level using clinical LUAD samples. Knockdown of RP11-295G20.2 obviously reduced ERS and suppressed proliferation, invasion, and migration of LUAD cells. This novel ERS-related lncRNA signature provides a new biomarker for prognostic prediction, and ERS-associated RP11-295G20.2 serves as a potential therapeutic target in LUAD.

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells.

BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. CONCLUSION: This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

Identification of differentially expressed ER stress-related genes and their association with pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a complex and progressive illness that has a multifaceted origin, significant fatality rates, and profound effects on health. The pathogenesis of PAH is poorly defined due to the insufficient understanding of the combined impact of endoplasmic reticulum (ER) stress and immune infiltration, both of which play vital roles in PAH development. This study aims to identify potential ER stress-related biomarkers in PAH and investigate their involvement in immune infiltration. METHODS: The GEO database was used to download gene expression profiles. Genes associated with ER stress were obtained from the MSigDB database. Weighted gene co-expression network analysis (WGCNA), GO, KEGG, and protein-protein interaction (PPI) were utilized to conduct screening of hub genes and explore potential molecular mechanisms. Furthermore, the investigation also delved into the presence of immune cells in PAH tissues and the correlation between hub genes and the immune system. Finally, we validated the diagnostic value and expression levels of the hub genes in PAH using subject-workup characterization curves and real-time quantitative PCR. RESULTS: In the PAH and control groups, a total of 31 genes related to ER stress were found to be differentially expressed. The enrichment analysis revealed that these genes were primarily enriched in reacting to stress in the endoplasmic reticulum, dealing with unfolded proteins, transporting proteins, and processing proteins within the endoplasmic reticulum. EIF2S1, NPLOC4, SEC61B, SYVN1, and DERL1 were identified as the top 5 hub genes in the PPI network. Immune infiltration analysis revealed that these hub genes were closely related to immune cells. The receiver operating characteristic (ROC) curves revealed that the hub genes exhibited excellent diagnostic efficacy for PAH. The levels of SEC61B, NPLOC4, and EIF2S1 expression were in agreement with the findings of bioinformatics analysis in the PAH group. CONCLUSIONS: Potential biomarkers that could be utilized are SEC61B, NPLOC4, and EIF2S1, as identified in this study. The infiltration of immune cells was crucial to the development and advancement of PAH. This study provided new potential therapeutic targets for PAH.

A novel BAG5 variant impairs the ER stress response pathway, causing dilated cardiomyopathy and arrhythmia.

Pathogenic BAG5 variants recently linked to dilated cardiomyopathy (DCM) prompt further investigation into phenotypic, mutational, and pathomechanistic aspects. We explored the clinical and molecular characteristics of DCM associated with BAG5 variants, uncovering the consistently severe manifestations of the disease and its impact on the endoplasmic reticulum (ER) stress response. The analysis involved three siblings affected by DCM and arrhythmia, along with their four unaffected siblings, their unaffected father, and their mother who exhibited arrhythmia. The parents were consanguineous. Exome and Sanger sequencing identified a novel BAG5 variant, c.444_445delGA (p.Lys149AsnfsTer6), homozygous in affected siblings and heterozygous in parents and unaffected siblings. We generated heterozygous and homozygous Bag5 point mutant knock-in (KI) mice and evaluated cardiac pathophysiology under stress conditions, including tunicamycin (TN) administration. Bag5-/- mice displayed no abnormalities up to 12 months old and showed no anomalies during an exercise stress test. However, following TN injection, Bag5-/- mice exhibited significantly reduced left ventricular fractional shortening (LVFS) and ejection fraction (LVEF). Their cardiac tissues exhibited a notable increase in apoptotic cells, despite non-distinctive changes in CHOP and GRP78 levels. Interestingly, only Bag5 KI male mice demonstrated arrhythmia, which was more pronounced in Bag5-/- than in Bag5+/-males. Here, our study reveals a novel BAG5 mutation causing DCM by impairing the ER stress response, with observed sex-specific arrhythmia differences.

TRIB3 promotes the progression of renal cell carcinoma by upregulating the lipid droplet-associated protein PLIN2.

Abnormal lipid metabolism and lipid accumulation are characteristic hallmarks of renal cell carcinoma (RCC). While there is prior evidence closely linking such lipid accumulation within RCC cells and consequent tumorigenesis, the mechanisms underlying this process remain incompletely understood. In this study, a series of bioinformatics analyses were initially performed by screening RCC databases and gene sets, ultimately leading to the identification of TRIB3 as an oncogene that functions as a central regulator of lipid metabolism. TRIB3 overexpression was observed in both RCC patient tumor tissues and cell lines, and this upregulation was correlated with a worse RCC patient prognosis. When TRIB3 was knocked down, this resulted in a reduction in lipid accumulation and the consequent induction of endoplasmic reticulum (ER) stress-related apoptotic cell death. At the molecular level, interactions between TRIB3 and PLIN2 were found to abrogate TEB4-mediated PLIN2 ubiquitination and consequent degradation, thus maintaining higher PLIN2 expression levels. This simultaneously helps facilitate the accumulation of lipids while preserving ER homeostasis, thus driving accelerated RCC tumor progression. This TRIB3-PLIN2 axis thus represents a promising new target for efforts to treat RCC.

Endoplasmic Reticulum Stress and Unfolded Protein Response in Vernal Keratoconjunctivitis.

Purpose: Vernal keratoconjunctivitis (VKC) is an ocular allergic disease characterized by a type 2 inflammation, tissue remodeling, and low quality of life for the affected patients. We investigated the involvement of endoplasmic reticulum (ER) stress and unfolded protein response in VKC. Methods: Conjunctival imprints from VKC patients and normal subjects (CTs) were collected, and RNA was isolated, reverse transcribed, and analyzed with the Affymetrix microarray. Differentially expressed genes between VKC patients and CTs were evaluated. Genes related to ER stress, apoptosis, and autophagy were further considered. VKC and CT conjunctival biopsies were analyzed by immunohistochemistry (IHC) with specific antibodies against unfolded protein response (UPR), apoptosis, and inflammation. Conjunctival fibroblast and epithelial cell cultures were exposed to the conditioned medium of activated U937 monocytes and analyzed by quantitative PCR for the expression of UPR, apoptosis, autophagy, and inflammatory markers. Results: ER chaperones HSPA5 (GRP78/BiP) and HYOU1 (GRP170) were upregulated in VKC patients compared to CTs. Genes encoding for ER transmembrane proteins, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), ER-associated degradation (ERAD), and autophagy were upregulated, but not those related to apoptosis. Increased positive reactivity of BiP and ATF6 and unchanged expression of apoptosis markers were confirmed by IHC. Cell cultures in stress conditions showed an overexpression of UPR, proinflammatory, apoptosis, and autophagy markers. Conclusions: A significant overexpression of genes encoding for ER stress, UPR, and pro-inflammatory pathway components was reported for VKC. Even though these pathways may lead to ER homeostasis, apoptosis, or inflammation, ER stress in VKC may predominantly contribute to promote inflammation.

Comprehensive analysis of bulk and single-cell transcriptomic data reveals a novel signature associated with endoplasmic reticulum stress, lipid metabolism, and liver metastasis in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with high probability of recurrence and distant metastasis. Liver metastasis is the predominant metastatic mode developed in most pancreatic cancer cases, which seriously affects the overall survival rate of patients. Abnormally activated endoplasmic reticulum stress and lipid metabolism reprogramming are closely related to tumor growth and metastasis. This study aims to construct a prognostic model based on endoplasmic reticulum stress and lipid metabolism for pancreatic cancer, and further explore its correlation with tumor immunity and the possibility of immunotherapy. METHODS: Transcriptomic and clinical data are acquired from TCGA, ICGC, and GEO databases. Potential prognostic genes were screened by consistent clustering and WGCNA methods, and the whole cohort was randomly divided into training and testing groups. The prognostic model was constructed by machine learning method in the training cohort and verified in the test, TCGA and ICGC cohorts. The clinical application of this model and its relationship with tumor immunity were analyzed, and the relationship between endoplasmic reticulum stress and intercellular communication was further explored. RESULTS: A total of 92 characteristic genes related to endoplasmic reticulum stress, lipid metabolism and liver metastasis were identified in pancreatic cancer. We established and validated a prognostic model for pancreatic cancer with 7 signatures, including ADH1C, APOE, RAP1GAP, NPC1L1, P4HB, SOD2, and TNFSF10. This model is considered to be an independent prognosticator and is a more accurate predictor of overall survival than age, gender, and stage. TIDE score was increased in high-risk group, while the infiltration levels of CD8+ T cells and M1 macrophages were decreased. The number and intensity of intercellular communication were increased in the high ER stress group. CONCLUSIONS: We constructed and validated a novel prognostic model for pancreatic cancer, which can also be used as an instrumental variable to predict the prognosis and immune microenvironment. In addition, this study revealed the effect of ER stress on cell-cell communication in the tumor microenvironment.

Importin 7 enhances protective autophagy induced by nuclear translocation of homeobox A10 in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor. Importin7 (IPO7) is responsible for nucleoplasmic transport of RNAs and proteins, and it has been confirmed to be involved in the development of human cancers. This study aimed to explore the function and mechanism of IPO7 in OSCC. IPO7 expression in tissues and cells was determined by RT-qPCR. Cell proliferative, migratory, and invasive capabilities were detected through transwell assay and colony formation assay. Mice xenograft models were established for evaluating tumor growth. Autophagy was estimated by the LC3 levels in cells through western blot and immunofluorescence (IF). Western blot was utilized to detect the key proteins in PERK/EIF2AK3/ATF4 pathway for assessing the endoplasmic reticulum stress (ERS). The interaction of IPO7 and homeobox A10 (HOXA10) was tested by GST pull-down assay and Co-IP assay. ChIP assay and luciferase reporter assay were utilized to determine the combination of HOXA10 and EIF2AK3. We proved that IPO7 was upregulated in OSCC tissues and cells, and its depletion reduced cell proliferation, migration, invasion and tumor growth. Furthermore, LC3 expression in cells was found to be reduced by IPO7 knockdown. IPO7 promoted OSCC tumor metastasis by activating autophagy. Additionally, we discovered that IPO7 could regulate ERS by activating the PERK/ATF4 pathway. EIF2AK3 upregulation can promote cell autophagy. Furthermore, IPO7 was proven to promote nuclear translocation of HOXA10 in cells. EIF2AK3 promoter can bind to HOXA10. Rescue assay confirmed that HOXA10 upregulation can reverse the effect of IPO7 silencing on OSCC progression. IPO7 can enhance proliferation, migration, invasion, and autophagy by nuclear translocation of HOXA10 and the activation of EIF2AK3/ATF4 pathway in OSCC.

Inhibition of signaling protein ERN1 increases the sensitivity of serine synthesis gene expressions to glucose and glutamine deprivations in U87MG glioblastoma cells.

Objective. Glucose and glutamine supply as well as serine synthesis and endoplasmic reticulum (ER) stress are important factors of glioblastoma growth. Previous studies showed that the knockdown of ERN1 (ER to nucleus signaling 1) suppressed glioblastoma cell proliferation and modified the sensitivity of numerous gene expressions to nutrient deprivations. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of serine synthesis genes in U87MG glioblastoma cells in relation to ERN1 knockdown with the intent to reveal the role of ERN1 signaling pathway on the ER stress-dependent regulation of these gene expressions. Clarification of the regulatory mechanisms of serine synthesis is a great significance for glioblastoma therapy. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed under glucose and glutamine deprivation conditions for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine amino-transferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that the expression level of genes responsible for serine synthesis such as PHGDH, PSAT1, PSPH, and transcription factor ATF4 was up-regulated in U87MG glioblastoma cells under glucose and glutamine deprivations. Furthermore, inhibition of ERN1 significantly enhances the impact of glucose and especially glutamine deprivations on these gene expressions. At the same time, the expression of the SHMT1 gene, which is responsible for serine conversion to glycine, was down-regulated in both nutrient deprivation conditions with more significant changes in ERN1 knockdown glioblastoma cells. Conclusion. Taken together, the results of present study indicate that the expression of genes responsible for serine synthesis is sensitive to glucose and glutamine deprivations in gene-specific manner and that suppression of ERN1 signaling significantly modifies the impact of both glucose and glutamine deprivations on PHGDH, PSAT1, PSPH, ATF4, and SHMT1 gene expressions and reflects the ERN1-mediated genome reprograming introduced by nutrient deprivation condition.

Identification of novel endoplasmic reticulum-related genes and their association with immune cell infiltration in major depressive disorder.

BACKGROUND: Several lines of evidence point to an interaction between genetic predisposition and environmental factors in the onset of major depressive disorder (MDD). This study is aimed to investigate the pathogenesis of MDD by identifying key biomarkers, associated immune infiltration using bioinformatic analysis and human postmortem sample. METHODS: The Gene Expression Omnibus (GEO) database of GSE98793 was adopted to identify hub genes linked to endoplasmic reticulum (ER) stress-related genes (ERGs) in MDD. Another GEO database of GSE76826 was employed to validate the novel target associated with ERGs and immune infiltration in MDD. Moreover, human postmortem sample from MDD patients was utilized to confirm the differential expression analysis of hub genes. RESULTS: We discovered 12 ER stress-related differentially expressed genes (ERDEGs). A LASSO Cox regression analysis helped construct a diagnostic model for these ERDEGs, incorporating immune infiltration analysis revealed that three hub genes (ERLIN1, SEC61B, and USP13) show the significant and consistent expression differences between the two groups. Western blot analysis of postmortem brain samples indicated notably higher expression levels of ERLIN1 and SEC61B in the MDD group, with USP13 also tending to increase compared to control group. LIMITATIONS: The utilization of the MDD gene chip in this analysis was sourced from the GEO database, which possesses a restricted number of pertinent gene chip samples. CONCLUSIONS: These findings indicate that ERDEGs especially including ERLIN1, SEC61B, and USP13 associated the infiltration of immune cells may be potential diagnostic indicators for MDD.

Caspase-mediated processing of TRBP regulates apoptosis during viral infection.

RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.

Identification of a novel RNA transcript TISPL upregulated by stressors that stimulate ATF4.

Cells sense, respond, and adapt to environmental conditions that cause stress. In a previous study using HeLa cells, we isolated reporter cells responding to the endoplasmic reticulum (ER) stress inducers, thapsigargin and tunicamycin, using a highly sensitive promoter trap vector system. Splinkerette PCR and 5' rapid amplification of cDNA ends (5' RACE) identified a novel transcript that is upregulated by ER stress. Its endogenous expression increased approximately 10-fold in response to thapsigargin and tunicamycin within 1 h, but was down-regulated after 4 h. Because the transcript starts from an intron of a long noncoding RNA known as LINC-PINT, we designated the newly identified transcript TISPL (transcript induced by stressors from LINC-PINTlocus). TISPL was also expressed under several other stress conditions. It was particularly increased > 10-fold upon glucose starvation and 7-fold by arsenite exposure. Furthermore, in silico analyses, including a ChIP-atlas search, revealed that there is an ATF4-binding region with a c/ebp-Atf response element (CARE) downstream of the transcription start site of TISPL. Based on these results, we hypothesized that TISPL may be induced by the phospho-eIF2α and ATF4- axis of the integrated stress response pathway, which is known to be activated by the stress conditions listed above. As expected, knockout of ATF4 abolished the stress-induced upregulation of TISPL. Our results indicate that TISPL may be a useful biomarker for detecting stress conditions that activate ATF4. Our highly sensitive trap vector system proved beneficial in discovering new biomarkers.

Comprehensive molecular and cellular characterization of endoplasmic reticulum stress-related key genes in renal ischemia/reperfusion injury.

Background: Renal ischemia-reperfusion injury (RIRI) is an inevitable complication in the process of kidney transplantation and lacks specific therapy. The study aims to determine the underlying mechanisms of RIRI to uncover a promising target for efficient renoprotection. Method: Four bulk RNA-seq datasets including 495 renal samples of pre- and post-reperfusion were collected from the GEO database. The machine learning algorithms were utilized to ascertain pivotal endoplasmic reticulum stress genes. Then, we incorporated correlation analysis and determined the interaction pathways of these key genes. Considering the heterogeneous nature of bulk-RNA analysis, the single-cell RNA-seq analysis was performed to investigate the mechanisms of key genes at the single-cell level. Besides, 4-PBA was applied to inhibit endoplasmic reticulum stress and hence validate the pathological role of these key genes in RIRI. Finally, three clinical datasets with transcriptomic profiles were used to assess the prognostic role of these key genes in renal allograft outcomes after RIRI. Results: In the bulk-RNA analysis, endoplasmic reticulum stress was identified as the top enriched pathway and three endoplasmic reticulum stress-related genes (PPP1R15A, JUN, and ATF3) were ranked as top performers in both LASSO and Boruta analyses. The three genes were found to significantly interact with kidney injury-related pathways, including apoptosis, inflammatory response, oxidative stress, and pyroptosis. For oxidative stress, these genes were more strongly related to oxidative markers compared with antioxidant markers. In single-cell transcriptome, the three genes were primarily upregulated in endothelium, distal convoluted tubule cells, and collecting duct principal cells among 12 cell types of renal tissues in RIRI. Furthermore, distal convoluted tubule cells and collecting duct principal cells exhibited pro-inflammatory status and the highest pyroptosis levels, suggesting their potential as main effectors of three key genes for mediating RIRI-associated injuries. Importantly, inhibition of these key genes using 4-phenyl butyric acid alleviated functional and histological damage in a mouse RIRI model. Finally, the three genes demonstrated highly prognostic value in predicting graft survival outcomes. Conclusion: The study identified three key endoplasmic reticulum stress-related genes and demonstrated their prognostic value for graft survival, providing references for individualized clinical prevention and treatment of postoperative complications after renal transplantation.

Identification and validation of an endoplasmic-reticulum-stress-related gene signature as an effective diagnostic marker of endometriosis.

Background: Endometriosis is one of the most common benign gynecological diseases and is characterized by chronic pain and infertility. Endoplasmic reticulum (ER) stress is a cellular adaptive response that plays a pivotal role in many cellular processes, including malignant transformation. However, whether ER stress is involved in endometriosis remains largely unknown. Here, we aimed to explore the potential role of ER stress in endometriosis, as well as its diagnostic value. Methods: We retrieved data from the Gene Expression Omnibus (GEO) database. Data from the GSE7305 and GSE23339 datasets were integrated into a merged dataset as the training cohort. Differentially expressed ER stress-related genes (DEG-ERs) were identified by integrating ER stress-related gene profiles downloaded from the GeneCards database with differentially expressed genes (DEGs) in the training cohort. Next, an ER stress-related gene signature was identified using LASSO regression analysis. The receiver operating characteristic curve was used to evaluate the discriminatory ability of the constructed model, which was further validated in the GSE51981 and GSE105764 datasets. Online databases were used to explore the possible regulatory mechanisms of the genes in the signature. Meanwhile, the CIBERSORT algorithm and Pearson correlation test were applied to analyze the association between the gene signature and immune infiltration. Finally, expression levels of the signature genes were further detected in clinical specimens using qRT-PCR and validated in the Turku endometriosis database. Results: In total, 48 DEG-ERs were identified in the training cohort. Based on LASSO regression analysis, an eight-gene-based ER stress-related gene signature was constructed. This signature exhibited excellent diagnostic value in predicting endometriosis. Further analysis indicated that this signature was associated with a compromised ER stress state. In total, 12 miRNAs and 23 lncRNAs were identified that potentially regulate the expression of ESR1, PTGIS, HMOX1, and RSAD2. In addition, the ER stress-related gene signature indicated an immunosuppressive state in endometriosis. Finally, all eight genes showed consistent expression trends in both clinical samples and the Turku database compared with the training dataset. Conclusions: Our work not only provides new insights into the impact of ER stress in endometriosis but also provides a novel biomarker with high clinical value.

Endoplasmic reticulum stress and therapeutic strategies in metabolic, neurodegenerative diseases and cancer.

The accumulation of unfolded or misfolded proteins within the endoplasmic reticulum (ER), due to genetic determinants and extrinsic environmental factors, leads to endoplasmic reticulum stress (ER stress). As ER stress ensues, the unfolded protein response (UPR), comprising three signaling pathways-inositol-requiring enzyme 1, protein kinase R-like endoplasmic reticulum kinase, and activating transcription factor 6 promptly activates to enhance the ER's protein-folding capacity and restore ER homeostasis. However, prolonged ER stress levels propels the UPR towards cellular demise and the subsequent inflammatory cascade, contributing to the development of human diseases, including cancer, neurodegenerative disorders, and diabetes. Notably, increased expression of all three UPR signaling pathways has been observed in these pathologies, and reduction in signaling molecule expression correlates with decreased proliferation of disease-associated target cells. Consequently, therapeutic strategies targeting ER stress-related interventions have attracted significant research interest. In this review, we elucidate the critical role of ER stress in cancer, metabolic, and neurodegenerative diseases, offering novel therapeutic approaches for these conditions.

Dual regulation of NEMO by Nrf2 and miR-125a inhibits ferroptosis and protects liver from endoplasmic reticulum stress-induced injury.

Rationale: The surge of severe liver damage underscores the necessity for identifying new targets and therapeutic agents. Endoplasmic reticulum (ER) stress induces ferroptosis with Gα12 overexpression. NF-κB essential modulator (NEMO) is a regulator of inflammation and necroptosis. Nonetheless, the regulatory basis of NEMO de novo synthesis and its impact on hepatocyte ferroptosis need to be established. This study investigated whether Nrf2 transcriptionally induces IKBKG (the NEMO gene) for ferroptosis inhibition and, if so, how NEMO induction protects hepatocytes against ER stress-induced ferroptosis. Methods: Experiments were conducted using human liver tissues, hepatocytes, and injury models, incorporating NEMO overexpression and Gα12 gene modulations. RNA sequencing, immunoblotting, immunohistochemistry, reporter assays, and mutation analyses were done. Results: NEMO downregulation connects closely to ER and oxidative stress, worsening liver damage via hepatocyte ferroptosis. NEMO overexpression protects hepatocytes from ferroptosis by promoting glutathione peroxidase 4 (GPX4) expression. This protective role extends to oxidative and ER stress. Similar shifts occur in nuclear factor erythroid-2-related factor-2 (Nrf2) expression alongside NEMO changes. Nrf2 is newly identified as an IKBKG (NEMO gene) transactivator. Gα12 changes, apart from Nrf2, impact NEMO expression, pointing to post-transcriptional control. Gα12 reduction lowers miR-125a, an inhibitor of NEMO, while overexpression has the opposite effect. NEMO also counters ER stress, which triggers Gα12 overexpression. Gα12's significance in NEMO-dependent hepatocyte survival is confirmed via ROCK1 inhibition, a Gα12 downstream kinase, and miR-125a. The verified alterations or associations within the targeted entities are validated in human liver specimens and datasets originating from livers subjected to exposure to other injurious agents. Conclusions: Hepatic injury prompted by ER stress leads to the suppression of NEMO, thereby facilitating ferroptosis through the inhibition of GPX4. IKBKG is transactivated by Nrf2 against Gα12 overexpression responsible for the increase of miR-125a, an unprecedented NEMO inhibitor, resulting in GPX4 induction. Accordingly, the induction of NEMO mitigates ferroptotic liver injury.

XAF1 overexpression inhibits the malignant progression and cisplatin resistance of NSCLC by activating endoplasmic reticulum stress.

BACKGROUND: XIAP-associated factor 1 (XAF1) has been found to participate in the progression of multiple human cancers. Nevertheless, its role as well as the reaction mechanism in non-small cell lung cancer (NSCLC) still remains obscure. METHODS: In this study, the protein expression of XAF1 in NSCLC cell lines was evaluated using western blot. With the employment of CCK-8 assay, EdU staining, wound healing and transwell, capabilities of NSCLC cells to proliferate, migrate and invade were assessed. Cell apoptotic level and cell cycle were resolved utilizing flow cytometry. Western blot was applied for the estimation of apoptosis- and endoplasmic reticulum (ER) stress-related proteins. RESULTS: It was discovered that XAF1 expression was conspicuously reduced in NSCLC cell lines. XAF1 overexpression suppressed H1299 cell proliferative, invasive and migrative capabilities, but exhibited promotive effects on cell cycle arrest. Meanwhile, XAF1 overexpression inhibited cisplatin resistance in H1299 and H1299/DDP cells by promoting cell apoptosis and enhanced the expression levels of ER stress-related proteins CHOP, GRP78 and ATF4. What's more, 4-PBA treatment reversed the impacts of XAF1 overexpression on the proliferative, invasive, migrative and apoptotic capabilities of H1299 cells, as well as cell cycle and cisplatin resistance. CONCLUSION: In conclusion, XAF1 overexpression impeded the advancement of NSCLC and repressed cisplatin resistance of NSCLC cells through inducing ER stress, which indicated that XAF1 might be a novel targeted-therapy for NSCLC.