IRGM is a novel regulator of PD-L1 via promoting S6K1-mediated phosphorylation of YBX1 in hepatocellular carcinoma.

Immunity-related GTPase M (IRGM), an Interferon-inducible protein, functions as a pivotal immunoregulator in multiple autoimmune diseases and infection. However, the role of IRGM in hepatocellular carcinoma (HCC) development remains unveiled. Here, we found interferon-γ (IFN-γ) treatment in HCC drastically triggered the expression of IRGM, and the high level of IRGM indicated poor prognosis in HCC patients. Functionally, IRGM promoted the malignant progression of HCC. Single-cell sequencing revealed that IRGM inhibition promoted the infiltration of CD8+ cytotoxic T lymphocytes (CTLs) with significant downregulation of PD-L1 expression in HCC. Furthermore, Immunoprecipitation-Mass Spectrometry assay revealed that IRGM interacted with transcription factor YBX1, which facilitated PD-L1 transcription. Mechanistically, IRGM promoted the interaction of YBX1 and phosphokinase S6K1, increasing phosphorylation and nuclear localization of YBX1, transcription of PD-L1. Additionally, the combination of IRGM inhibition with α-PD1 demonstrated a stronger anti-tumor effect compared to the single application of α-PD1. In summary, IRGM is a novel regulator of PD-L1, which suppresses CD8+ CTLs infiltration and function in HCC, resulting in cancer progression. This study may raise a novel therapeutic strategy combined with immune checkpoint inhibitors (ICIs) against HCC.

N6 -methyladenosine-modified circSTX6 promotes hepatocellular carcinoma progression by regulating the HNRNPD/ATF3 axis and encoding a 144 amino acid polypeptide.

BACKGROUND: Circular RNAs (circRNAs) play a significant role in the initiation and progression of various cancers, including hepatocellular carcinoma (HCC). Circular syntaxin 6 (circSTX6, also known as hsa_circ_0007905) has been identified as a microRNA (miRNA) sponge in pancreatic adenocarcinoma. However, its full range of functions in terms of protein scaffold and translation remain largely unexplored in the context of HCC. METHODS: The expression of circSTX6 and its encoded protein was examined in HCC tumour tissues. N6 -methyladenosine (m6 A) on circSTX6 was verified and quantified by methylated RNA immunoprecipitation (Me-RIP), RIP and dual luciferase reporter assays. The biological functions of circSTX6 and its encoded protein in HCC were clarified by in vitro and in vivo experiments. Mechanistically, the interaction between circSTX6 and heterogeneous nuclear ribonucleoprotein D (HNRNPD) was investigated by RNA pull-down, RIP and fluorescence in situ hybridization (FISH)/IF. The regulatory effects of circSTX6 and HNRNPD on activating transcription factor 3 (ATF3) mRNA were determined by mRNA stability and RIP assays. Furthermore, the presence of circSTX6-encoded protein was verified by mass spectrometry. RESULTS: CircSTX6 and its encoded 144 amino acid polypeptide, circSTX6-144aa, were highly expressed in HCC tumour tissues and served as independent risk factors for overall survival in HCC patients. The expression of circSTX6 was regulated by METTL14 in an m6 A-dependent manner. Functionally, circSTX6 accelerated HCC proliferation and tumourigenicity and reinforced tumour metastasis in vitro and in vivo. Mechanistically, circSTX6 acted as a sponge for HNRNPD protein, facilitating its binding to ATF3 mRNA, consequently promoting ATF3 mRNA decay. Meanwhile, circSTX6-144aa promoted HCC proliferation, migration and invasion independent of circSTX6 itself. CONCLUSION: Collectively, our study reveals that m6 A-modified circSTX6 drives malignancy in HCC through the HNRNPD/ATF3 axis, while its encoded circSTX6-144aa contributes to HCC progression independent of circSTX6. CirSTX6 and its encoded protein hold promise as potential biomarkers and therapeutic targets in HCC.

Amino acid metabolism in health and disease.

Amino acids are the building blocks of protein synthesis. They are structural elements and energy sources of cells necessary for normal cell growth, differentiation and function. Amino acid metabolism disorders have been linked with a number of pathological conditions, including metabolic diseases, cardiovascular diseases, immune diseases, and cancer. In the case of tumors, alterations in amino acid metabolism can be used not only as clinical indicators of cancer progression but also as therapeutic strategies. Since the growth and development of tumors depend on the intake of foreign amino acids, more and more studies have targeted the metabolism of tumor-related amino acids to selectively kill tumor cells. Furthermore, immune-related studies have confirmed that amino acid metabolism regulates the function of effector T cells and regulatory T cells, affecting the function of immune cells. Therefore, studying amino acid metabolism associated with disease and identifying targets in amino acid metabolic pathways may be helpful for disease treatment. This article mainly focuses on the research of amino acid metabolism in tumor-oriented diseases, and reviews the research and clinical research progress of metabolic diseases, cardiovascular diseases and immune-related diseases related to amino acid metabolism, in order to provide theoretical basis for targeted therapy of amino acid metabolism.

ST6GALNAC4 promotes hepatocellular carcinogenesis by inducing abnormal glycosylation.

Hepatocellular carcinoma (HCC) is one of the most lethal tumor types worldwide. Glycosylation has shown promise in the study of tumor mechanisms and treatment. The glycosylation status of HCC and the underlying molecular mechanisms are still not fully elucidated. Using bioinformatic analysis we obtained a more comprehensive characterization of glycosylation of HCC. Our analysis presented that high glycosylation levels might correlate with tumor progression and poor prognosis. Subsequent Experiments identified key molecular mechanisms for ST6GALNAC4 promoting malignant progression by inducing abnormal glycosylation. We confirmed the contribution of ST6GALNAC4 to proliferation, migration, and invasion in vitro and in vivo. Mechanistic studies revealed that ST6GALNAC4 may be induced abnormal TGFBR2 glycosylation, resulting in the higher protein levels of TGFBR2 and TGF[Formula: see text] pathway increased activation. Our study also provided a further understand of immunosuppressive function of ST6GALNAC4 through T antigen-galectin3+ TAMs axis. This study has provided one such possibility that galectin3 inhibitors might be an acceptable treatment choice for HCC patients with high T antigen expression.

MiR-652-3p promotes malignancy and metastasis of cancer cells via inhibiting TNRC6A in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) was one of the most prevalent life-threatening cancers. Metastasis is the leading cause of cancer-related death in HCC. MiRNAs play essential roles in cancer metastasis. METHODS: Expression of miR-652-3p in HCC was assessed. Function experiments of miR-652-3p and trinucleotide repeat-containing gene 6A protein (TNRC6A) were performed both in vitro and in vivo. mRNA sequencing, PCR, and western blot were performed to verify the target genes and pathway of miR-652-3p. The lung metastasis and xenograft cancer model in nude mice was established to investigate the effects of the miR-652-3p and TRNC6A on tumor metastasis in vivo. The relationship between the expression of the miR-652-3p, TNRC6A and the prognosis of HCC patients was analyzed. RESULTS: Upregulated miR-652-3p was found in the tumor tissues of HCC, especially in metastatic HCC patients. Overexpression of miR-652-3p promoted and knockdown of miR-652-3p suppressed HCC metastasis both in vitro and in vivo. MiR-652-3p promoted HCC metastasis via regulating the EMT pathway. TNRC6A was identified as a direct target of miR-652-3p, and the knockdown of TNRC6A restored repressed EMT and HCC metastasis caused by the inhibition of miR-652-3p. Clinical results revealed that high expression of miR-652-3p and low expression of TNRC6A were positively correlated to shortened overall survival and disease-free survival in HCC patients. CONCLUSIONS: MiR-652-3p promotes EMT and HCC metastasis by inhibiting TNRC6A expression in HCC. MiR-652-3p and TNRC6A may serve as potential biomarkers to predict prognosis in HCC patients with metastasis.

Shikonin regulates autophagy via the AMPK/mTOR pathway and reduces apoptosis of human umbilical cord mesenchymal stem cells to improve survival in tissues surrounding brain contusion.

Shikonin has been reported to regulate autophagy via the AMP-activated protein kinase (AMPK)/mTOR signalling pathway and decrease apoptosis in transplanted human umbilical cord mesenchymal stem cells (HUMSCs). In the present study, HUMSCs were exposed to oxygen glucose deprivation (OGD) in vitro for 12 h, and TUNEL fluorescence staining was used to detect apoptosis. Differences in autophagy and AMPK/mTOR pathway-related protein expression following treatment with shikonin were quantitatively analyzed by western blotting. Green fluorescent protein-labelled stem cells were implanted into traumatic brain injury-model mice and the survival of HUMSCs was observed after 7 days. Shikonin increased the number of cells in brain tissue surrounding the contusion 7 days after transplantation. Furthermore, shikonin treatment decreased apoptosis, increased the expression of autophagy-related proteins, increased phosphorylated AMPK expression and downregulated phosphorylated mTOR expression. In addition, the autophagy inhibitor 3-methyladenine attenuated these effects and aggravated apoptosis. Subsequently, shikonin upregulated autophagy and protected HUMSCs in the area surrounding contused brain tissue. Shikonin may regulate autophagy via the AMPK/mTOR signalling pathway and protect transplanted HUMSCs from apoptosis induced by hypoxia/ischemia.

Pyridoxal Isonicotinoyl Hydrazone Improves Neurological Recovery by Attenuating Ferroptosis and Inflammation in Cerebral Hemorrhagic Mice.

Ferroptosis and inflammation induced by cerebral hemorrhage result in an excessive inflammatory response and irreversible neuronal injury. Alleviating ferroptosis might be an effective way to prevent neuroinflammatory injury and promote neural functional recovery. Pyridoxal isonicotinoyl hydrazine (PIH), a lipophilic iron-chelating agent, has been reported to reduce excess iron-induced cytotoxicity. However, whether PIH could ameliorate the effects of hemorrhagic stroke is not completely understood. In the present study, the preventive effects of PIH in an intracerebral hemorrhage (ICH) mouse model were investigated. Neurological score, rotarod test, and immunofluorescence around the hematoma were assessed to evaluate the effects of PIH on hemorrhagic injury. The involvement of ferroptosis and inflammation was also examined in vitro to explore the underlying mechanism. Results showed that administration of PIH prevented neuronal cell death and reduced lipid peroxidation in Erastin-treated PC-12 cells. In vivo, mice treated with PIH after ICH attenuated neurological deficit scores. Additionally, we found PIH reduced ROS production, iron accumulation, and lipid peroxidation around the hematoma peripheral tissue. Meanwhile, ICH mice treated with PIH showed an upregulation of the key ferroptosis enzyme, glutathione peroxidase 4, and downregulation of cyclooxygenase-2. Moreover, PIH administration inhibited proinflammatory polarization and reduced interleukin-1 beta and tumor necrosis factor alpha in ICH mice. Collectively, these results demonstrated that PIH protects mice against hemorrhage stroke, which was associated with mitigation of inflammation and ferroptosis.

ANXA1 as a Prognostic and Immune Microenvironmental Marker for Gliomas Based on Transcriptomic Analysis and Experimental Validation.

The tumor microenvironment (TME) plays an important role in the growth and invasion of glioma. This study aimed to analyze the composition of the immune microenvironment in glioma samples and analyze the important differentially expressed genes to identify novel immune-targeted therapy for glioma. We downloaded transcriptomic data of 669 glioma samples from The Cancer Genome Atlas database. CIBERSORT and ESTIMATE methods were used to calculate the proportion of tumor-infiltrating immune cells and ratio of immune and stromal components in the TME. The differentially expressed genes (DEGs) were screened by comparing the genes expressed by both stromal and immune cells. Annexin A1 (ANXA1) was determined to be an important prognostic indicator through the common overlap of univariate Cox regression analysis and protein-protein interaction network analysis. The proportion of tumor-infiltrating immune cells, calculated by CIBERSORT algorithm, had a significant difference in distribution among the high and low ANXA1 expression groups, indicating that ANXA1 could be an important immune marker of TME. Furthermore, ANXA1 level was positively correlated with the histopathological factors and negatively related to the survival of glioma patients based on the analysis of multiple databases. Finally, in vitro experiments verified that antagonizing ANXA1 expression promoted cell apoptosis and inhibited the invasion and migration capacities of glioma cells. Therefore, ANXA1 due to its immune-related functions, can be an important prognostic indicator and immune microenvironmental marker for gliomas. Further studies are warranted to confirm ANXA1 as a potential immunotherapeutic target for gliomas.

Ablation of GSDMD Improves Outcome of Ischemic Stroke Through Blocking Canonical and Non-canonical Inflammasomes Dependent Pyroptosis in Microglia.

Ischemia/reperfusion (I/R) injury is a significant cause of mortality and long-term disability worldwide. Recent evidence has proved that pyroptosis, a novel cell death form, contributes to inflammation-induced neuron death and neurological function impairment following ischemic stroke. Gasdermin D (GSDMD) is a newly discovered key molecule of cell pyroptosis, but its biological function and precise role in ischemic stroke are still unclear. The present study investigates the cleavage activity of GSDMD, localization of pyroptotic cells, and global neuroinflammation in gsdmd -/- mice after I/R. The level of cell pyroptosis around the infarcted area was significantly increased in the acute phase of cerebral I/R injury. The ablation of GSDMD reduced the infraction volume and improved neurological function against cerebral I/R injury. Furthermore, we confirmed I/R injury induced cell pyroptosis mainly in microglia. Knockdown of GSDMD effectively inhibited the secretion of mature IL-1ß and IL-18 from microglia cells but did not affect the expression of caspase-1/11 in vitro and in vivo. In summary, blocking GSDMD expression might serve as a potential therapeutic strategy for ischemic stroke.

Melatonin Enhances the Therapeutic Effect of Plasma Exosomes Against Cerebral Ischemia-Induced Pyroptosis Through the TLR4/NF-κB Pathway.

INTRODUCTION: Ischemic stroke-induced inflammation and inflammasome-dependent pyroptotic neural death cause serious neurological injury. Nano-sized plasma exosomes have exhibited therapeutic potential against ischemia and reperfusion injury by ameliorating inflammation. To enhance its therapeutic potential in patients with ischemic injury, we isolated exosomes from melatonin-treated rat plasma and assessed the neurological protective effect in a rat model of focal cerebral ischemia. METHODS: Basal plasma exosomes and melatonin-treated plasma exosomes were isolated and intravenously injected into a rat model of focal cerebral ischemia. Neurological recovery was evaluated by determining the modified neurological severity score (mNSS), infarct volume, and brain water content. Pyroptosis in the ischemic cortex was detected through dUTP nick-end labeling (TUNEL) assay, lactate dehydrogenase (LDH) release, and gasdermin D (GSDMD) cleavage. NLRP3 inflammasome assembly and global inflammatory cytokine secretion were detected by enzyme-linked immunosorbent assay (ELISA) and Western blot assay. In immunized Sprague-Dawley rats, microglia pyroptosis was determined through a positive percentage of IBA1+ and caspase-1 (p20)+ cells. Finally, the microRNA (miRNA) profiles in melatonin-treated plasma exosomes were analyzed by exosome miRNA microarray analysis. RESULTS: Melatonin treatment enhanced plasma exosome therapeutic effects against ischemia-induced inflammatory responses and inflammasome-mediated pyroptosis. In addition, we confirmed that ischemic stroke-induced pyroptotic cell death occurred in the microglia and neuron, while the administration of melatonin-treated exosomes further effectively decreased the infarct volume and improved recovery of function via regulation of the TLR4/NF-κB signaling pathway. Finally, the altered miRNA profiles in the melatonin-treated plasma exosomes demonstrated the regulatory mechanisms involved in neurological recovery after ischemic injury. CONCLUSION: This study suggests that nano-sized plasma exosomes with melatonin pretreatment might be a more effective strategy for patients with ischemic brain injury. Further exploration of key molecules in the plasma exosome may provide increased therapeutic value for cerebral ischemic injury.

VX765 Attenuates Pyroptosis and HMGB1/TLR4/NF-κB Pathways to Improve Functional Outcomes in TBI Mice.

BACKGROUND: Traumatic brain injury (TBI) refers to temporary or permanent damage to brain function caused by penetrating objects or blunt force trauma. TBI activates inflammasome-mediated pathways and other cell death pathways to remove inactive and damaged cells, however, they are also harmful to the central nervous system. The newly discovered cell death pattern termed pyroptosis has become an area of interest. It mainly relies on caspase-1-mediated pathways, leading to cell death. METHODS: Our research focus is VX765, a known caspase-1 inhibitor which may offer neuroprotection after the process of TBI. We established a controlled cortical impact (CCI) mouse model and then controlled the degree of pyroptosis in TBI with VX765. The effects of caspase-1 inhibition on inflammatory response, pyroptosis, blood-brain barrier (BBB), apoptosis, and microglia activation, in addition to neurological deficits, were investigated. RESULTS: We found that TBI led to NOD-like receptors (NLRs) as well as absent in melanoma 2 (AIM2) inflammasome-mediated pyroptosis in the damaged cerebral cortex. VX765 curbed the expressions of indispensable inflammatory subunits (caspase-1 as well as key downstream proinflammatory cytokines such as interleukin- (IL-) 1ß and IL-18). It also inhibited gasdermin D (GSDMD) cleavage and apoptosis-associated spot-like protein (ASC) oligomerization in the injured cortex. In addition to the above, VX765 also inhibited the inflammatory activity of the high-mobility cassette -1/Toll-like receptor 4/nuclear factor-kappa B (HMGB1/TLR4/NF-kappa B) pathway. By inhibiting pyroptosis and inflammatory mediator expression, we demonstrated that VX765 can decrease blood-brain barrier (BBB) leakage, apoptosis, and microglia polarization to exhibit its neuroprotective effects. CONCLUSION: In conclusion, VX765 can counteract neurological damage after TBI by reducing pyroptosis and HMGB1/TLR4/NF-κB pathway activities. VX765 may have a good therapeutic effect on TBI.

VX-765 enhances autophagy of human umbilical cord mesenchymal stem cells against stroke-induced apoptosis and inflammatory responses via AMPK/mTOR signaling pathway.

INTRODUCTION: To investigate the protective effect of VX-765 on human umbilical mesenchymal stem cells (HUMSCs) in stroke and its mechanism. MATERIALS AND METHODS: Mouse models of ischemic stroke were established using the distal middle cerebral artery occlusion (dMCAO) method. The dMCAO mice were accordingly transplanted with HUMSCs, VX-765-treated HUMSCs, or VX-765 + MHY185-treated HUMSCs. The HUMSCs were inserted with green fluorescent protein (GFP) for measurement of transplantation efficiency which was determined by immunofluorescence assay. Oxygen-glucose deprivation (OGD) was applied to mimic ischemic environment in vitro experiments, and the HUMSCs herein were transfected with AMPK inhibitor Compound C or autophagy inhibitor 3-MA. MTT assay was used to test the toxicity of VX-765. TUNEL staining and ELISA were applied to measure the levels of apoptosis and inflammatory cytokines (IL-1ß, IL-6, and IL-10), respectively. The expressions of autophagy-associated proteins, AMPK, and mTOR were detected by Western blotting. TTC staining was applied to reveal the infarct lesions in the brain of dMCAO mice. RESULTS: The pro-inflammatory cytokines, TUNEL-positive cells, and p-mTOR were decreased while the anti-inflammatory cytokine, autophagy-related proteins, and p-AMPK were increased in HUMSCs treated with VX-765 under OGD condition. Different expression patterns were found with the above factors after transfection of 3-MA or Compound C. The pro-inflammatory cytokines, TUNEL-positive cells, and infarct sections were decreased while the anti-inflammatory cytokine and autophagy-related proteins were increased in dMCAO mice transplanted with VX-765-treated HUMSCs compared to those transplanted with HUMSCs only. The autophagy was inhibited while p-mTOR was up-regulated after transfection of MHY. CONCLUSION: VX-765 protects HUMSCs against stroke-induced apoptosis and inflammatory responses by activating autophagy via the AMPK/mTOR signaling pathway in vivo and in vitro.