Expression Of Hexokinase Research Articles

Identification of HK3 as a promising immunomodulatory and prognostic target in sepsis-induced acute lung injury

BackgroundSepsis is a life-threatening global disease with a significant impact on human health. Acute lung injury (ALI) has been identified as one of the primary causes of mortality in septic patients. This study aimed to identify candidate genes involved in sepsis-induced ALI through a comprehensive approach combining bioinformatics analysis and experimental validation. MethodsThe datasets GSE65682 and GSE32707 obtained from the Gene Expression Omnibus database were merged to screen for sepsis-induced ALI related differentially expressed genes (DEGs). Functional enrichment and immune infiltration analyses were conducted on DGEs, with the construction of protein-protein interaction (PPI) networks to identify hub genes. In vitro and in vivo models of sepsis-induced ALI were used to study the expression and function of hexokinase 3 (HK3) using various techniques including Western blot, real-time PCR, immunohistochemistry, immunofluorescence, Cell Counting Kit-8, Enzyme-linked immunosorbent assay, and flow cytometry. ResultsThe results of bioinformatics analysis have identified HK3, MMP9, and S100A8 as hub genes with diagnostic and prognostic significance for sepsis-induced ALI. The HK3 has profound effects on sepsis-induced ALI and exhibits a correlation with immune regulation. Experimental results showed increased HK3 expression in lung tissue of septic mice, particularly in bronchial and alveolar epithelial cells. In vitro studies demonstrated upregulation of HK3 in lipopolysaccharide (LPS)-stimulated lung epithelial cells, with cytoplasmic localization around the nucleus. Interestingly, following the knockdown of HK3 expression, lung epithelial cells exhibited a significant decrease in proliferation activity and glycolytic flux, accompanied by an increase in cellular inflammatory response, oxidative stress, and cell apoptosis. ConclusionsIt was observed for the first time that HK3 plays a crucial role in the progression of sepsis-induced ALI and may be a valuable target for immunomodulation and therapy.Bioinformatics analysis identified HK3, MMP9, and S100A8 as hub genes with diagnostic and prognostic relevance in sepsis-induced ALI. Experimental findings showed increased HK3 expression in the lung tissue of septic mice, particularly in bronchial and alveolar epithelial cells. In vitro experiments demonstrated increased HK3 levels in lung epithelial cells stimulated with LPS, with cytoplasmic localization near the nucleus. Knockdown of HK3 expression resulted in decreased proliferation activity and glycolytic flux, increased inflammatory response, oxidative stress, and cell apoptosis in lung epithelial cells.

Real-time resolution studies of the regulation of lactate production by hexokinases binding to mitochondria in single cells.

During hypoxia accumulation of lactate may be a key factor in acidosis-induced tissue damage. Binding of hexokinase (HK) to the outer membrane of mitochondria may have a protective effect under these conditions. We have investigated the regulation of lactate metabolism by hexokinases (HKs), using HEK293 cells in which the endogenous hexokinases have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were also transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown HEK cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. Upon inhibition of the mitochondrial electron transfer chain by NaCN this effect was reversed as a rapid increase in lactate developed which was followed by a slow and sustained increase in the continued presence of the inhibitor. Incubation of the HKI/HKII double knockdown HEK cells with the inhibitor of the malic enzyme, ME1*, blocked the delayed accumulation of lactate evoked by NaCN. With replacement by overexpression of HKI or HKII the accumulation of intracellular lactate evoked by NaCN was prevented. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN) abolished the protective effect of HK expression, with NaCN causing again a sustained increase in lactate. The effect of HK was dependent on HK's catalytic activity and interaction with the mitochondrial outer membrane (MOM). Based on these data we propose that transformation of glucose into G6P by HK activates the pentose phosphate pathway which increases the production of NADPH, which then blocks the activity of the malic enzyme to transform malate into pyruvate and lactate.

Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages

To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1β (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-β mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1β, TNF-α and TGF-β in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-β mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

ALKBH5 facilitates the progression of infantile hemangioma by increasing FOXF1 expression in a m6A-YTHDF2 dependent manner to activate HK-2 signaling.

Alkylation repair homolog protein 5 (ALKBH5) is reported to participate in infantile hemangioma (IH) progression. However, the underlying mechanism of ALKBH5 in IH remains unclear. Using qRT-PCR and Western blotting, ALKBH5, forkhead box F1 (FOXF1) and hexokinase 2 (HK-2) expressions in IH tissues and IH-derived endothelial cells XPTS-1 were assessed. The Me-RIP assay was used to analyze FOXF1 m6A level. CCK8, colony formation, flow cytometry and transwell assays were employed to determine IH cell viability, proliferation, apoptosis, migration and invasion. The interactions between YTH (YT521-B homology) domain 2 (YTHDF2), FOXF1 and HK-2 were analyzed by RIP, dual luciferase reporter gene assay and/or ChIP assay. The in vivo IH growth was evaluated in immunocompromised mice. FOXF1 was overexpressed in IH tissues, and its silencing inhibited IH cell proliferation, migration and invasion whereas promoting cell apoptosis in vitro. ALKBH5 upregulation facilitated FOXF1 mRNA stability and expression in IH cells in a m6A-YTHDF2-dependent manner. FOXF1 downregulation reversed the impact of ALKBH5 upregulation on IH cellular phenotypes. It also turned out that FOXF1 positively regulated HK-2 expression in IH cells through interacting with the HK-2 promoter. HK-2 upregulation abolished FOXF1 knockdown's inhibition on IH cell aggressive behaviors. ALKBH5 or FOXF1 silencing suppressed IH tumor development via HK-2 signaling in immunocompromised mice. ALKBH5 promoted FOXF1 expression m6A-YTHDF2 dependently, which in turn elevated HK-2 expression, thereby accelerating IH development.

Impact of glucose metabolism on PD-L1 expression in sorafenib-resistant hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer-related mortality worldwide. Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that binds to programmed cell death-1 (PD-1), which is expressed in activated T cells and other immune cells and has been employed in cancer therapy, including HCC. Recently, PD-L1 overexpression has been documented in treatment-resistant cancer cells. Sorafenib is a multikinase inhibitor and the only FDA-approved treatment for advanced HCC. However, several patients exhibit resistance to sorafenib during treatment. This study aimed to assess the effect of glucose deprivation on PD-L1 expression in HCC cells. We used PD-L1-overexpressing HepG2 cells and IFN-γ-treated SK-Hep1 cells to explore the impact of glycolysis on PD-L1 expression. To validate the correlation between PD-L1 expression and glycolysis, we analyzed data from The Cancer Genome Atlas (TCGA) and used immunostaining for HCC tissue analysis. Furthermore, to modulate PD-L1 expression, we treated HepG2, SK-Hep1, and sorafenib-resistant SK-Hep1R cells with rapamycin. Here, we found that glucose deprivation reduced PD-L1 expression in HCC cells. Additionally, TCGA data and immunostaining analyses confirmed a positive correlation between the expression of hexokinase II (HK2), which plays a key role in glucose metabolism, and PD-L1. Notably, rapamycin treatment decreased the expression of PD-L1 and HK2 in both high PD-L1-expressing HCC cells and sorafenib-resistant cells. Our results suggest that the modulation of PD-L1 expression by glucose deprivation may represent a strategy to overcome PD-L1 upregulation in patients with sorafenib-resistant HCC.

Diagnostic and prognostic significance of tissue lncRNA HOTAIR and hexokinase 2 mRNA expression in patients with pancreatic ductal adenocarcinoma

BackgroundPancreatic adenocarcinoma, recognized for its aggressive behavior and frequent late-stage diagnosis, imposes significant challenges in early detection and prognosis. This study aimed to evaluate the diagnostic and prognostic potential by measuring the expression levels of long non-coding RNA HOTAIR and the glycolytic enzyme hexokinase 2 (HK2) mRNA in both tumorous and adjacent non-tumorous pancreatic tissue samples (n = 25 each) using RT-qPCR. ResultsBoth lncRNA HOTAIR and HK2 expression levels significantly increased in tumorous pancreatic tissues compared to non-tumorous tissue (P = 0.001). However, their levels in stage T2 and T3 showed no statistically significant difference (P = 0.01). lncRNA HOTAIR and HK2 expression levels positively correlated with each other (P = 0.001; r = 0.95); however, no significant associations were found with serum tumor markers CA19-9 and CEA (P = 0.01; r = 005; p = 0.1, r = 0.2). ROC analysis demonstrated the significant abilities of both lncRNA HOTAIR and HK2 expression levels to discriminate between tumorous and non-tumorous pancreatic tissues (AUC = 0.92 and 0.84, respectively) with 96% and 88% sensitivity, and 72% and 40% specificity, respectively, at optimal cut-off values of 1.12 and 0.84 relative expression units. Patients with elevated lncRNA HOTAIR and HK2 expression had shorter median survival (8 and 7 months, respectively), increasing the risk of adverse outcomes or recurrence 4–4.8 times (HR = 4.08, p = 0.07; HR = 4.8, p = 0.01), thus emphasizing their prognostic potential in pancreatic cancer.ConclusionlncRNA HOTAIR and HK2 expression levels exhibit diagnostic potential in pancreatic tumors. Elevated levels of both markers correlate strongly with adverse outcomes, underscoring their prognostic value.

TIMELESS promotes reprogramming of glucose metabolism in oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC), the predominant malignancy of the oral cavity, is characterized by high incidence and low survival rates. Emerging evidence suggests a link between circadian rhythm disruptions and cancer development. The circadian gene TIMELESS, known for its specific expression in various tumors, has not been extensively studied in the context of OSCC. This study aims to explore the influence of TIMELESS on OSCC, focusing on cell growth and metabolic alterations.MethodsWe analyzed TIMELESS expression in OSCC using western blot, immunohistochemistry, qRT-PCR, and data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). The role of TIMELESS in OSCC was examined through clone formation, MTS, cell cycle, and EdU assays, alongside subcutaneous tumor growth experiments in nude mice. We also assessed the metabolic impact of TIMELESS by measuring glucose uptake, lactate production, oxygen consumption, and medium pH, and investigated its effect on key metabolic proteins including silent information regulator 1 (SIRT1), hexokinase 2 (HK2), pyruvate kinase isozyme type M2 (PKM2), recombinant lactate dehydrogenase A (LDHA) and glucose transporter-1 (GLUT1).ResultsElevated TIMELESS expression in OSCC tissues and cell lines was observed, correlating with reduced patient survival. TIMELESS overexpression enhanced OSCC cell proliferation, increased glycolytic activity (glucose uptake and lactate production), and suppressed oxidative phosphorylation (evidenced by reduced oxygen consumption and altered pH levels). Conversely, TIMELESS knockdown inhibited these cellular and metabolic processes, an effect mirrored by manipulating SIRT1 levels. Additionally, SIRT1 was positively associated with TIMELESS expression. The expression of SIRT1, HK2, PKM2, LDHA and GLUT1 increased with the overexpression of TIMELESS levels and decreased with the knockdown of TIMELESS.ConclusionTIMELESS exacerbates OSCC progression by modulating cellular proliferation and metabolic pathways, specifically by enhancing glycolysis and reducing oxidative phosphorylation, largely mediated through the SIRT1 pathway. This highlights TIMELESS as a potential target for OSCC therapeutic strategies.

DNA methylation-regulated HK1 overexpression contributes to irradiation-resistance by promoting glycolysis in non-small cell lung cancer.

Irradiation-resistance presents a substantial challenge in the successful application of radiotherapy for non-small-cell lung cancer (NSCLC). However, the specific molecular mechanisms responsible for irradiation-resistance have yet to be completely understood. In this research, the DNA methylation and gene expression patterns resulting from irradiation treatment were produced using the DNA methylation BeadChip and RNA-Seq. An integrated analysis was carried out to identify the genes that are differentially expressed and regulated by DNA methylation. As results, the upregulation of gene expression and downregulation of DNA methylation of hexokinase 1 (HK1), a protein associated with glycolysis, were observed in irradiation-resistant NSCLC cells. Additionally, treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-Aza-dC) resulted in increased expression of HK1. Furthermore, it was found that overexpression of HK1 could enhance irradiation-resistance by impacting glycolysis. Collectively, our study indicate that irradiation-induced alterations in DNA methylation lead to the upregulation of HK1, which in turn promotes glycolysis and contributes to radiotherapy resistance in NSCLC. Therefore, targeting HK1 presents a potential novel strategy for addressing the issue of radiotherapy failure in NSCLC.

A comprehensive metabolite fingerprint of fibrostenosis in patients with Crohn’s disease

Intestinal fibrostenosis in patients with Crohn’s disease (CD) is a common and untreatable comorbidity that is notoriously difficult to monitor. We aimed to find metabolites associated with the presence of fibrostenosis in patients with CD using targeted and untargeted metabolomics analyses of serum and primary cell cultures using hyphenated ultra-high performance liquid chromatography high-resolution mass spectrometry. Targeted metabolomics revealed 11 discriminating metabolites in serum, which were enriched within the arginine and proline metabolism pathway. Based on untargeted metabolomics and discriminant analysis, 166 components showed a high predictive value. In addition, human intestinal fibroblasts isolated from stenotic tissue were characterized by differential levels of medium-chain dicarboxylic acids, which are proposed as an energy source through beta-oxidation, when oxidative phosphorylation is insufficient. Another energy providing pathway in such situations is anaerobic glycolysis, a theory supported by increased expression of hexokinase 2 and solute carrier family 16 member 1 in stenotic fibroblasts. Of interest, four (unannotated) metabolic components showed a negative correlation with hexokinase 2 gene expression. Together, this study provides a discriminative metabolic fingerprint in the serum and in intestinal fibroblasts of stenotic and non-stenotic patients with CD suggestive for increased production of building blocks for collagen synthesis and increased glycolysis.

Changes in carbohydrate metabolism and soil microorganisms under the stress of polyamide and polyethylene nanoplastics during rice (Oryza sativa L.) growth

Nanoplastics (NPs) presence in agricultural soils can affect plant growth and impact the quality of agricultural products. To investigate the effect of polyamide (PA) NPs and polyethylene (PE) NPs on carbohydrate metabolism and soil microorganisms during rice growth, rice seedlings were exposed to soil containing 2 g/kg of 100 nm PA or 100 nm PE powder for 33 d. The results revealed that 100 nm PE reduced shoot length and dry weight of rice by 4.14 % and 15.68 %, respectively. Analyzing the expression of hexokinase-2 (HXK), phosphofructokinase-1 (PFK), pyruvate kinase (PK) and isocitrate dehydrogenase (IDH), which are four genes related to carbohydrate metabolism, 100 nm PA decreased the expression of PFK and increased the expression of PK and IDH. 100 nm PE increased the expression of HXK, PFK, PK, and IDH. The results of soil microorganisms showed that 100 nm PA significantly effects on 3 bacterial phyla (Bacteroidota, Deinococcota, and Desulfobacterota), whereas 100 nm PE significantly effects on phylum Rozellomycota, class Umbelopsidomycetes, and an unclassified Firmicutes. Our study provides direct evidence of the negative effects of PA and PE on rice, which may be important for assessing the risk of NPs on agroecosystems.

The Aurora kinase inhibitor AT9283 inhibits Burkitt lymphoma growth by regulating Warburg effect

Objective To investigate the effect of the kinase inhibitor AT9283 on Burkitt lymphoma (BL) cells and elucidate the underlying mechanisms. Methods The effect of AT9283 on the proliferation of BL cell lines was tested using the MTT assay. Apoptosis and cell cycle were measured by flow cytometry. The proteins associated with the cell cycle, apoptosis, and the Warburg effect were detected using Western blotting. Alterations in glycolytic metabolism in terms of glucose intake and lactate concentrations were determined by glucose and lactate assays. Results The current study utilized the GEPIA, the Human Protein Atlas (HAP) database and immunohistochemistry to conduct analyses, which revealed a high expression of Aurora kinases and Warburg effect-related proteins in malignant B-cell lymphoma tissues. AT9283 significantly inhibited the cell proliferation of BL cells and induced G2/M arrest. Additionally, AT9283 induced apoptosis in BL cells and reversed the Warburg effect by increasing glucose uptake and reducing lactate production. Moreover, the protein expression of hexokinase 2, pyruvate kinase M2, and lactate dehydrogenase A was significantly suppressed by AT9283, possibly through the inhibition of c-Myc and HIF-1α protein expression. Conclusion The reversal of the Warburg effect in BL cells and the subsequent inhibition of cell proliferation and induction of apoptosis were observed by targeting Aurora A and Aurora B with AT9283. This finding may present new therapeutic options and targets for BL.

Tetraarsenic tetrasulfide triggers ROS-induced apoptosis and ferroptosis in B-cell acute lymphoblastic leukaemia by targeting HK2

PurposeAcute lymphoblastic leukemia (ALL) is the most common type of cancer diagnosed in children. Despite cure rates of higher than 85 %, refractory or relapsed ALL still exhibits a bleak prognosis indicative of the dearth of treatment modalities specific for relapsed or refractory ALL. Prior research has implicated metabolic alterations in leukemia pathogenesis, and literature on the therapeutic efficacy of arsenic compounds targeting metabolic pathways in B-cell acute lymphoblastic leukemia (B-ALL) cells is scarce. MethodsA compound extracted from realgar, tetraarsenic tetrasulfide (As4S4), and its antitumor effects on B-ALL were experimentally examined in vitro and in vivo. ResultsAs4S4 apparently targets B-ALL cells by inducing specific cellular responses, including apoptosis, G2/M arrest, and ferroptosis. Interestingly, these effects are attributed to reactive oxygen species (ROS) accumulation, and increased ROS levels have been linked to both the mitochondria-dependent caspase cascade and the activation of p53 signaling. The ROS scavenger N-acetylcysteine (NAC) can counteract the effects of As4S4 treatment on Nalm-6 and RS4;11 cells. Specifically, by targeting Hexokinase-2 (HK2), As4S4 induces alterations in mitochondrial membrane potential and disrupts glucose metabolism, leading to ROS accumulation, and was shown to inhibit B-ALL cell proliferation in vitro and in vivo. Intriguingly, overexpression of HK2 can partially desensitize B-ALL cells to As4S4 treatment. ConclusionTetraarsenic tetrasulfide can regulate the Warburg effect by controlling HK2 expression, a finding that provides both new mechanistic insight into metabolic alterations and pharmacological evidence for the clinical treatment of B-ALL.

HIF-1α promotes virus replication and cytokine storm in H1N1 virus-induced severe pneumonia through cellular metabolic reprogramming

The mortality of patients with severe pneumonia caused by H1N1 infection is closely related to viral replication and cytokine storm. However, the specific mechanisms triggering virus replication and cytokine storm are still not fully elucidated. Here, we identified hypoxia inducible factor-1α (HIF-1α) as one of the major host molecules that facilitates H1N1 virus replication followed by cytokine storm in alveolar epithelial cells. Specifically, HIF-1α protein expression is upregulated after H1N1 infection. Deficiency of HIF-1α attenuates pulmonary injury, viral replication and cytokine storm in vivo. In addition, viral replication and cytokine storm were inhibited after HIF-1α knockdown in vitro. Mechanistically, the invasion of H1N1 virus into alveolar epithelial cells leads to a shift in glucose metabolism to glycolysis, with rapid production of ATP and lactate. Inhibition of glycolysis significantly suppresses viral replication and inflammatory responses. Further analysis revealed that H1N1-induced HIF-1α can promote the expression of hexokinase 2 (HK2), the key enzyme of glycolysis, and then not only provide energy for the rapid replication of H1N1 virus but also produce lactate, which reduces the accumulation of the MAVS/RIG-I complex and inhibits IFN-α/β production. In conclusion, this study demonstrated that the upregulation of HIF-1α by H1N1 infection augments viral replication and cytokine storm by cellular metabolic reprogramming toward glycolysis mainly through upregulation of HK2, providing a theoretical basis for finding potential targets for the treatment of severe pneumonia caused by H1N1 infection.

The Long non-coding RNA MALAT1 functions as a competing endogenous RNA to regulate vascular remodeling by sponging miR-145-5p/HK2 in hypertension

ABSTRACT Long non-coding RNAs (LncRNAs) have been found to play a regulatory role in the pathophysiology of vascular remodeling-associated illnesses through the lncRNA-microRNA (miRNA) regulation axis. LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is thought to be involved in proliferation, migration, apoptosis, and calcification of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the regulatory role of MALAT1 on vascular remodeling in hypertension. Our data indicate that the expression of MALAT1 is significantly upregulated in hypertensive aortic smooth muscle. Knockdown of MALAT1 inhibited the proliferation, migration, and phenotypic transition of VSMCs induced by Ang II. Bioinformatics analysis was used to predict the complementary binding of miR-145-5p to the 3’-untranslated region of MALAT1. Besides, the expressions of MALAT1 and miR-145-5p were negatively correlated, while luciferase reporter assays and RNA immunoprecipitation assay validated the interaction between miR-145-5p and MALAT1. The proliferation, migration and phenotypic transformation of VSMCs induced by overexpression of MALAT1 were reversed in the presence of miR-145-5p. Furthermore, we verified that miR-145-5p could directly target and bind to hexokinase 2 (HK2) mRNA, and that HK2 expression was negatively correlated with miR-145-5p in VSMCs. Knockdown of HK2 significantly inhibited the effects of overexpression of MALAT1 on Ang II-induced VSMCs proliferation, migration and phenotypic transformation. Taken together, the MALAT1/miR-145-5p/HK2 axis may play a critical regulatory role in the vascular remodeling of VSMCs in hypertension.

HK2 in microglia and macrophages contribute to the development of neuropathic pain.

Neuropathic pain is a complex pain condition accompanied by prominent neuroinflammation involving activation of both central and peripheral immune cells. Metabolic switch to glycolysis is an important feature of activated immune cells. Hexokinase 2 (HK2), a key glycolytic enzyme enriched in microglia, has recently been shown important in regulating microglial functions. Whether and how HK2 is involved in neuropathic pain-related neuroinflammation remains unknown. Using a HK2-tdTomato reporter line, we found that HK2 was prominently elevated in spinal microglia. Pharmacological inhibition of HK2 effectively alleviated nerve injury-induced acute mechanical pain. However, selective ablation of Hk2 in microglia reduced microgliosis in the spinal dorsal horn (SDH) with little analgesic effects. Further analyses showed that nerve injury also significantly induced HK2 expression in dorsal root ganglion (DRG) macrophages. Deletion of Hk2 in myeloid cells, including both DRG macrophages and spinal microglia, led to the alleviation of mechanical pain during the first week after injury, along with attenuated microgliosis in the ipsilateral SDH, macrophage proliferation in DRGs, and suppressed inflammatory responses in DRGs. These data suggest that HK2 plays an important role in regulating neuropathic pain-related immune cell responses at acute phase and that HK2 contributes to neuropathic pain onset primarily through peripheral monocytes and DRG macrophages rather than spinal microglia.

Phlorizin ameliorates myocardial fibrosis by inhibiting pyroptosis through restraining HK1-mediated NLRP3 inflammasome activation

The specific role of phlorizin (PHL), which has antioxidant, anti-inflammatory, hypoglycemic, antiarrhythmic and antiaging effects, on myocardial fibrosis (MF) and the related pharmacological mechanisms remain unknown. The objective of this study was to determine the protective actions of PHL on isoprenaline (ISO)-induced MF and its molecular mechanisms in mice. PHL was administered at 100 and 200 mg/kg for 15 consecutive days with a subcutaneous injection of ISO (10 mg/kg). MF was induced by ISO and alleviated by treatment with PHL, as shown by reduced fibrin accumulation in the myocardial interstitium and decreased levels of myocardial enzymes, such as creatinine kinase-MB, lactate dehydrogenase, and aspartate transaminase. In addition, PHL significantly decreased the expression of the fibrosis-related factors alpha smooth muscle actin, collagen I, and collagen III induced by ISO. The generation of intracellular reactive oxygen species induced by ISO was attenuated after PHL treatment. The malondialdehyde level was reduced, whereas the levels of superoxide dismutase, catalase, and glutathione were elevated with PHL administration. Moreover, compared to ISO, the level of Bcl-2 was increased and the level of Bax protein was decreased in the PHL groups. PHL relieved elevated TNF-α, IL-1β, and IL-18 levels as well as cardiac mitochondrial damage resulting from ISO. Further studies showed that PHL downregulated the high expression of hexokinase 1 (HK1), NLRP3, ASC, Caspase-1, and GSDMD-N caused by ISO. In conclusion, our findings suggest that PHL protects against ISO-induced MF due to its antioxidant, anti-apoptotic, and anti-inflammatory activities and via inhibition of pyroptosis mediated by the HK1/NLRP3 signaling pathway in vivo.

Activation of Glycolysis by MCM10 Increases Stemness and Paclitaxel Resistance in Gastric Cancer Cells.

Chemotherapy is an essential avenue for curing malignancies; however, tumor cells acquire resistance to chemotherapeutic agents, eventually leading to chemotherapy failure. At present, paclitaxel (PTX) resistance seriously hinders the therapeutic efficacy of gastric cancer (GC). Investigating the molecular mechanism of PTX resistance in GC is critical. This study attempted to delineate the impact of MCM10 on GC resistance to PTX and its mechanism in GC. The expression of minichromosome maintenance complex component 10 (MCM10) in GC tissues, its enrichment pathways, and its correlation with glycolysis marker genes and stemness index (mRNAsi) were analyzed in a bioinformatics effort. Real-time quantitative polymerase chain reaction was used to assay the expression of MCM10 in cells. Cell counting kit-8 (CCK-8) was used to analyze cell viability and calculate the 50% inhibitor concentration (IC50) value. Western blot was used to measure the expression of MCM10, Hexokinase 2 (HK2) and stemness-related factors in cells. Sphere-forming assay was performed to study cell sphere-forming ability. Seahorse XF 96 was utilized to measure cell extracellular acidification and oxygen consumption rates. The content of glycolysisrelated products was tested with corresponding kits. MCM10 was significantly upregulated in GC and enriched in the glycolysis pathway, and it was positively correlated with both glycolysis-related genes and stemness index. High expression of MCM10 increased sphere-forming ability of drug-resistant cells and GC resistance to PTX. The stimulation of PTX resistance and drug-resistant cell stemness in GC by high MCM10 expression was mediated by the glycolysis pathway. MCM10 was upregulated in GC and drove stemness and PTX resistance in GC cells by activating glycolysis. These findings generated new insights into the development of PTX resistance in GC, implicating that targeting MCM10 may be a novel approach to improve GC sensitivity to PTX chemotherapy.

HK2 contributes to the proliferation, migration, and invasion of diffuse large B-cell lymphoma cells by enhancing the ERK1/2 signaling pathway.

Hexokinase 2 (HK2) has been associated with carcinogenic growth in numerous kinds of malignancies as essential regulators during the processing of glucose. This study aimed to explore the effects of HK2 on diffuse large B-cell lymphoma (DLBCL) cells via the ERK1/2 signaling. Expressions of HK2 and ERK1/2 were examined in DLBCL cell lines using quantitative reverse transcription polymerase chain reaction and western blotting. HK2 and ERK1/2 were attenuated through HK2 small-interfering RNA (siRNA) and ERK inhibitor FR180204, respectively, in U2932 and SU-DHL-4 cells. Cell Counting Kit-8, clone formation, transwell, and flow cytometry assays were used in evaluating the effects of HK2 and ERK1/2 on cell proliferation, migration, and apoptosis. Moreover, a xenograft model was created to assess the roles of HK2 in vivo. HK2 and ERK1/2 were evidently up-regulated in DLBCL cell lines. HK2 knockdown and FR180204 markedly suppressed the proliferation and clonogenesis of U2932 and SU-DHL-4 cells and promoted cell apoptosis in vitro. We also found that HK2 silencing suppressed tumor growth in vivo. Notably, HK2 knockdown inactivated the ERK1/2 signaling pathway both in vitro and in vivo. These data indicate that inhibition of HK2 may suppress the proliferation, migration, and invasion of DLBCL cells, partly via inhibiting the ERK1/2 signaling pathway.

5'-tRF-19-Q1Q89PJZ Suppresses the Proliferation and Metastasis of Pancreatic Cancer Cells via Regulating Hexokinase 1-Mediated Glycolysis.

tRNA-derived small RNAs (tDRs) are dysregulated in several diseases, including pancreatic cancer (PC). However, only a limited number of tDRs involved in PC progression are known. Herein, a novel tDR, 5'-tRF-19-Q1Q89PJZ (tRF-19-Q1Q89PJZ), was verified in PC plasma using RNA and Sanger sequencing. tRF-19-Q1Q89PJZ was downregulated in PC tissues and plasma, which was related to advanced clinical characteristics and poor prognosis. tRF-19-Q1Q89PJZ overexpression inhibited the malignant activity of PC cells in vitro, while tRF-19-Q1Q89PJZ inhibition produced an opposite effect. The differentially expressed genes induced by tRF-19-Q1Q89PJZ overexpression were enriched in "pathways in cancer" and "glycolysis". Mechanistically, tRF-19-Q1Q89PJZ directly sponged hexokinase 1 (HK1) mRNA and inhibited its expression, thereby suppressing glycolysis in PC cells. HK1 restoration relieved the inhibitory effect of tRF-19-Q1Q89PJZ on glycolysis in PC cells and on their proliferation and mobility in vitro. tRF-19-Q1Q89PJZ upregulation inhibited PC cell proliferation and metastasis in vivo and suppressed HK1 expression in tumor tissues. Furthermore, tRF-19-Q1Q89PJZ expression was attenuated under hypoxia. Collectively, these findings indicate that tRF-19-Q1Q89PJZ suppresses the malignant activity of PC cells by regulating HK1-mediated glycolysis. Thus, tRF-19-Q1Q89PJZ may serve as a key target for PC therapy.

The knockdown of lncRNA DLGAP1-AS2 suppresses osteosarcoma progression by inhibiting aerobic glycolysis via the miR-451a/HK2 axis.

Osteosarcoma (OS) is one of the most aggressive bone tumors worldwide. Emerging documents have shown that long noncoding RNAs (lncRNAs) elicit crucial regulatory functions in the process of tumorigenesis. LncRNA DLGAP1-AS2 is recognized as a regulator in several types of cancers, but its biological functions and molecular mechanisms in OS remain to be elucidated. RT-qPCR and In situ hybridization (ISH) were used to evaluate DLGAP1-AS2 expression in OS samples. Western blotting was used for the measurement of the protein levels of hexokinase 2 (HK2) and epithelial-mesenchymal transition (EMT)-related markers. The proliferation of OS cells was determined using a CCK-8 assay and EdU assay. TUNEL assay and flow cytometry were performed to assess OS cell apoptosis. Glucose metabolism in vitro assays were used. The binding relations among miR-451a, HK2, and DLGAP1-AS2 were validated by luciferase reporter assay. The cellular distribution of DLGAP1-AS2 in OS cells was determined by FISH and subcellular fractionation assays. Mouse xenograft models were established to perform the experiments in vivo. We found that DLGAP1-AS2 expression was upregulated in OS tissues and cells. Downregulation of DLGAP1-AS2 expression suppressed the malignancy of OS cells by restraining cell proliferation, the EMT process, invasiveness, migration, and aerobic glycolysis and accelerating apoptotic behaviors. Of note, silenced DLGAP1-AS2 restrained tumor growth and metastasis in vivo. However, DLGAP1-AS2 overexpression accelerated the progression of OS. We further found that DLGAP1-AS2 upregulation was induced by hypoxia and low glucose. Additionally, DLGAP1-AS2 bound to miR-451a to upregulate HK2 expression. Rescue assays revealed that the DLGAP1-AS2/miR-451a/HK2 axis contributed to OS cell malignancy by promoting aerobic glucose metabolism. Overall, these findings revealed a new regulatory pathway where DLGAP1-AS2 upregulated HK2 expression by sponging miR-451a to accelerate OS development.