Glucose Consumption Levels Research Articles

NREP, transcriptionally upregulated by HIF-1α, aggravates breast cancer cell growth and metastasis by promoting glycolysis

Breast cancer (BC) poses a great threat to women’s health. Neuronal regeneration related protein (NREP) is a multifunctional protein that is involved in embryonic development, regeneration, and human disease. However, the biological function of NREP in tumors is rarely reported and its role in BC remains unknown. Bioinformatics analysis showed that NREP is highly expressed and closely correlated with poor survival in BC patients. Under hypoxic conditions, NREP was upregulated in BC cells, and this promotion was reversed by hypoxia-inducible factor HIF-1α suppression. Luciferase reporter system and chromatin immunoprecipitation assays confirmed that HIF-1α directly binds to the promoter of NREP to increase the transcriptional activity of NREP. NREP suppression inhibited cell proliferation, arrested the cell cycle at the G1/S phase, and promoted apoptosis and caspase-3 activity in BC cells. Suppression of NREP decreased the tube formation ability of HUVECs. In addition, NREP downregulation showed an inhibition effect on cell migration, invasion, and EMT of BC cells. In NREP overexpressed cells, all these changes were reversed. In vivo, animal experiments also confirmed that NREP promotes BC tumor growth and metastasis. In addition, NREP promoted cellular glycolysis and enhanced the levels of glucose consumption, ATP, lactate production, and glucose transporters expression in NREP-overexpressed BC cells. In summary, our results demonstrated that NREP could be transcriptional activated by HIF-1α, which may aggravate BC tumor growth and metastasis by promoting cellular glycolysis. This result suggested that NREP may play an essential part in BC progression.

VEGF/Nrp1/HIF-1α promotes proliferation of hepatocellular carcinoma through a positive feedback loop.

To investigate the role of neuropilin1 (Nrp1) in glucose metabolism and proliferation of hepatocellular carcinoma (HCC) cells and to analyze its mechanism of action. The CRISPR gene knockout technique was used to knock out the Nrp1 gene in two HCC cell lines. The effect of Nrp1 on the proliferation of HCC cells was assessed in the CCK8 assay and plate cloning assay. The expression levels of glucose consumption, lactate production, and essential proteins of the glycolytic pathway were detected to explore the effect of Nrp1 on glucose metabolism in HCC cells. Using CoCl2 to revert the expression of hypoxia inducible factor-1α (HIF-1α), the role of HIF-1α in the pro-HCC cell metabolism of Nrp1 were demonstrated. The protein synthesis inhibitor CHX and proteasome inhibitor MG-132 was used to analyze the molecular mechanism of action of Nrp1 on HIF-1α. The Kaplan-Meier method was used to calculate survival rates and plot survival curves. Based on the CCK8 assay and plate cloning assay, we found that Nrp1 knockout significantly inhibited the proliferation of HCC cells. Nrp1 inhibitor suppressed lactate production and glucose consumption in HCC cells. Knockout of Nrp1 decreased the expression of glycolytic pathway-related proteins and HIF-1α protein. Furthermore, by joint use of CoCl2 and NRP1 knockout, we confirmed that reverting HIF-1α expression could reverse the effect of Nrp1 knockout on HCC cell metabolism in vitro. Mechanistically, Nrp1 showed a close correlation with the stability of HIF-1α protein in protein stability assay. Finally, we revealed that high expression of Nrp1 in HCC tissues was associated with poor overall survival and disease-free survival of the patients. Nrp1 accelerates glycolysis and promotes proliferation of HCC by regulating HIF-1α protein stability and through the VEGF/Nrp1/HIF-1α positive feedback loop.

Porphyromonas gingivalis msRNA P.G_45033 induces amyloid-β production by enhancing glycolysis and histone lactylation in macrophages.

High expression of amyloid-β (Aβ) in periodontal tissue could contribute to exacerbating the development of both periodontitis and Alzheimer's disease (AD). Porphyromonas gingivalis (P. gingivalis) as a periodontal pathogen expresses msRNAs, which can regulate gene transcription in host cells. The aim of this study is to reveal the mechanism of msRNA P.G_45033, a high copy msRNA in P. gingivalis, inducing Aβ expression in macrophages, and provide a new insight to explain the development of periodontitis, and also to explain the role of periodontal infection on AD. The levels of glucose consumption, pyruvate and lactate productions in macrophages after transfection with msRNA P.G_45033 were detected. Miranda, TargetScan, and RNAhybrid databases were used to predict the target gene of msRNA P.G_45033, and GO analysis was conducted to describe the functions of the overlapping ones. RT2 glucose-metabolism PCR Array was used to verify the relationship between msRNA P.G_45033 and the expression of genes related to glucose metabolism. The levels of histone Kla were detected using western blotting. The levels of Aβ in the macrophages and the culture medium were detected by immunofluorescence and ELISA, respectively. The levels of glucose consumption, pyruvate and lactate productions were increased after transfection of msRNA P.G_45033 in macrophages. GO analysis revealed that target genes were enriched in the metabolic process. RT2 glucose-metabolism PCR Array showed the expression of genes associated with glycolysis. The results of western blotting showed that the level of histone Kla was increased in macrophages. The results of immunofluorescence and ELISA showed that Aβ levels in macrophages and culture medium were increased after transfection. The present study revealed that msRNA P.G_45033 can induce Aβ production by enhancing glycolysis and histone Kla in macrophages.

PSME3 induces radioresistance and enhances aerobic glycolysis in cervical cancer by regulating PARP1

Cervical cancer (CC) ranks the fourth in gynecologic cancers. The incidence and mortality of CC has been decreased due to the cancer screening and early treatments in recent years, but the prognosis of CC patients at advanced stage is still sorrowful. Whether PSME3 exerted a role in the radioresistance of CC cells remains to be investigated. In this study, the expression of PSME3 in mRNA and protein levels was measured by RT-qPCR and western blot analysis, and increased expression of PSME3 in CC tissues and cells was observed. CCK-8 and colony formation assay revealed that the cell viability and proliferation of Hela and CaSki cells treated with different doses of X-ray was reduced due to the depletion of PSME3, indicating that silencing of PSME3 enhanced the radiosensitivity of CC cells. In addition, repair on DNA damage in CC cells was enhanced by PSME3 and the damage was attenuated by PSME3. Besides, the expression of glycolysis-related proteins (GLUT1, PGC-1α, LDHA and HK2) were enhanced by PSME3 but reduced by silencing PSME3 in CC cells. PSME3 restraint attenuated the levels of glucose consumption and lactate production, suggesting PSME3 depletion suppressed abnormal glycolysis of CC cells. Mechanically, PSME3 increased the PARP1 expression via elevating c-myc. Finally, we observed PSME3 attenuation inhibited CC growth in vivo. In conclusion, PSME3 enhanced radioresistance and aerobic glycolysis in CC by regulating PARP1, which might shed a light into the function of PSME3 in CC treatment.

Glucuronic acid metabolites of phenolic acids target AKT-PH domain to improve glucose metabolism

ObjectivePhenolic acids widely exist in the human diet and exert beneficial effects such as improving glucose metabolism. It is not clear whether phenolic acids or their metabolites play a major role in vivo. In this study, caffeic acid (CA) and ferulic acid (FA), the two most ingested phenolic acids, and their glucuronic acid metabolites, caffeic-4′-O-glucuronide (CA4G) and ferulic-4′-O-glucuronide (FA4G), were investigated. MethodsThree insulin resistance models in vitro were established by using TNF-α, insulin and palmitic acid (PA) in HepG2 cells, respectively. We compared the effects of FA, FA4G, CA and CA4G on glucose metabolism in these models by measuring the glucose consumption levels. The potential targets and related pathways were predicted by network pharmacology. Fluorescence quenching measurement was used to analyze the binding between the compounds and the predicted target. To investigate the binding mode, molecular docking was performed. Then, we performed membrane recruitment assays of the AKT pleckstrin homology (PH) domain with the help of the PH-GFP plasmid. AKT enzymatic activity was determined to compare the effects between the metabolites with their parent compounds. Finally, the downstream signaling pathway of AKT was investigated by Western blot analysis. ResultsThe results showed that CA4G and FA4G were more potent than their parent compounds in increasing glucose consumption. AKT was predicted to be the key target of CA4G and FA4G by network pharmacology analysis. The fluorescence quenching test confirmed the more potent binding to AKT of the two metabolites compared to their parent compounds. The molecular docking results indicated that the carbonyl group in the glucuronic acid structure of CA4G and FA4G might bind to the PH domain of AKT at the key Arg-25 site. CA4G and FA4G inhibited the translocation of the AKT PH domain to the membrane, while increasing the activity of AKT. Western blot analysis demonstrated that the metabolites could increase the phosphorylation of AKT and downstream glycogen synthase kinase 3β in the AKT signaling pathway to increase glucose consumption. ConclusionIn conclusion, our results suggested that the metabolites of phenolic acids, which contain glucuronic acid, are the key active substances and that they activate AKT by targeting the PH domain, thus improving glucose metabolism.

Upregulation of SOCS2 causes mitochondrial dysfunction and promotes ferroptosis in pancreatic cancer cells.

SOCS2 exerts oncogenic effects in a variety of tumors, but its role in pancreatic cancer has not been studied. The purpose of this study was to explore the role of SOCS2 in pancreatic cancer. The expression level of SOCS2 and the content of mitochondrial DNA (mtDNA) in the cells were detected by real-time PCR (qRT-PCR), and SOCS2 was overexpressed in PANC-1 and Capan-2 cells by transfection with pcDNA3.2-SOCS2. CCK-8, cell colony formation assay, and flow cytometry were used respectively to detect the cell proliferation rate, cell colony formation ability, and the level of ROS in the cells. The ATP level, glucose consumption level, and Fe2 + level in the cells were assessed by biochemical assays. And Western blot determined the protein expression levels of SOCS2 as well as ferroptosis-related proteins, namely, SLC7A11, DMT1, TFRC, and FTH. We found that SOCS2 was significantly down-regulated in pancreatic cancer cells. Overexpression of SOCS2 significantly decreased the viability of PANC-1 and Capan-2 cells, reduced the content of mtDNA and the level of ATP, and caused mitochondrial dysfunction with an accumulation of ROS. Aside from these effects, up-regulation of SOCS2 raised the levels of Fe2 +, DMT1 and TFRC, and decreased the level of SLC7A11 and FTH in PANC-1 and Capan-2 cells, thereby inducing the occurrence of ferroptosis. In conclusion, up-regulated SOCS2 may enhance mitochondrial dysfunction and ferroptosis in pancreatic cancer cells, which can be used as a molecular target for the diagnosis and treatment of pancreatic carcinoma.

Loss of MiR-155 Sensitizes FLT3-ITD+AML to Chemotherapy and FLT3 Inhibitors via Glycolysis Blocking by Targeting PIK3R1.

FLT3 tyrosine kinase inhibitors in combination with chemotherapy have shown some success in patients with FLT3 mutations. But a variety of mechanisms have led to the rapid resistance to the treatment. One of the most prominent is the metabolic alteration on aerobic glycolysis. We aim to explore the role of a high expressing microRNA, miR-155, in mediating resistance to chemotherapy and FLT3 inhibitor treatment. The deep sequencing data mining revealed the connection between glycolysis and drug resistance. MV411 cells with miR-155 knockout (KO) not only had increased sensitivity to FLT3 inhibitors but also Adriamycin (ADM) treatment. When combined with glycolysis inhibition the treatment response in MV411 cells further increased. Whereas in miR-155 KO cells, a lower glucose consumption level and lactic acid level were observed, and western blotting showed a decreased expression of key enzymes in glycolysis pathways. A negative correlation between PIK3R1 and miR-155 level can be observed in the sequencing data from FLT3-ITD+ AML patients. Moreover, luciferase reporter assay revealed that the 3'UTR of PIK3R1 mRNA can interact with the seed sequence of miR-155-5p. In conclusion, the loss of miR-155 increased treatment sensitivity to both chemotherapy and FLT3 inhibitors in FLT3-ITD+ AML cells via glycolysis blocking by targeting PIK3R1.

P-092: RED BLOOD CELL GLUCOSE CONSUMPTION AND METABOLISM IN SICKLE CELL DISEASE

Purpose: Glucose is the single most essential component of the red blood cells (RBCs), being its sole energy provider and therefore is implicated in the energy metabolomics of the RBCs. This in turn play a crucial role in RBC lifespan, which in Sickle Cell Disease (SCD) patients is reduced to about 20 days from the usual average 120 days. This motivated us to investigate the process of glucose consumption and metabolism in red blood cells of Sickle Cell Patient of Odisha. Materials and methods: Blood samples of homozygous SCD patients (HbSS) were collected randomly with informed consent from different parts of Odisha state depending on the availability and accessibility of the subjects. The samples were studied for their hematological and biochemical parameters including levels of glucose. The red blood cells of SCD patients aged and sex matched normal (HbAA) as well as sickle cell traits (HbAS) were separated and incubated for different time intervals to measure the glucose consumption and Pyruvate and Lactate formation. Along with this the membrane stability of the cells were estimated and time dependent glucose uptake was also carried out. Identification of the haemoglobin variant was done by performing sickling test, haemoglobin electrophoresis and HPLC Hb. Variant analysis. All the biochemical estimations were done following the standard methods. Results: In the red blood cells of SCD cases (HbSS), the levels of glucose consumption were noted to be the higher than the sickle cell trait (HbAS) cases and control/normal (HbAA) samples. Concurrently, formation of blood Pyruvate and Lactate showed higher rate in comparison to HBAA and HbAS red cells indicating raised metabolic turnover in the sickle RBCs. In the time dependent study, the red cells of HbSS patients showed higher rate of glucose consumption compared to trait and normal cells. This data also correlates with the higher hemolytic features of the RBCs showing lower membrane stability with increased metabolism. Conclusion: The sickle cell mutation causes much more damage to the life of RBCs than mere morphological sickling of cells. The energy consumption plays a vital role in the life span, shape, stability and overall biochemistry of the RBCs, thus being a critical factor of RBC metabolism, which in turn determines the overall blood-based health of the SCD patients. Understanding and pin-pointing the key factors of energy metabolism thus holds many potential and promising factors in controlling the sickling, and indeed overall health of patients in case of sickle cell disease. The authors do not declare any conflict of interest

Transforming growth factor beta-1 upregulates glucose transporter 1 and glycolysis through canonical and noncanonical pathways in hepatic stellate cells.

BACKGROUNDHepatic stellate cells (HSCs) are the key effector cells mediating the occurrence and development of liver fibrosis, while aerobic glycolysis is an important metabolic characteristic of HSC activation. Transforming growth factor-β1 (TGF-β1) induces aerobic glycolysis and is a driving factor for metabolic reprogramming. The occurrence of glycolysis depends on a high glucose uptake level. Glucose transporter 1 (GLUT1) is the most widely distributed glucose transporter in the body and mainly participates in the regulation of carbohydrate metabolism, thus affecting cell proliferation and growth. However, little is known about the relationship between TGF-β1 and GLUT1 in the process of liver fibrosis and the molecular mechanism underlying the promotion of aerobic glycolysis in HSCs.AIMTo investigate the mechanisms of action of GLUT1, TGF-β1 and aerobic glycolysis in the process of HSC activation during liver fibrosis.METHODSImmunohistochemical staining and immunofluorescence assays were used to examine GLUT1 expression in fibrotic liver tissue. A Seahorse extracellular flux (XF) analyzer was used to examine changes in aerobic glycolytic flux, lactate production levels and glucose consumption levels in HSCs upon TGF-β1 stimulation. The mechanism by which TGF-β1 induces GLUT1 protein expression in HSCs was further explored by inhibiting/promoting the TGF-β1/mothers-against-decapentaplegic-homolog 2/3 (Smad2/3) signaling pathway and inhibiting the p38 and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. In addition, GLUT1 expression was silenced to observe changes in the growth and proliferation of HSCs. Finally, a GLUT1 inhibitor was used to verify the in vivo effects of GLUT1 on a mouse model of liver fibrosis.RESULTSGLUT1 protein expression was increased in both mouse and human fibrotic liver tissues. In addition, immunofluorescence staining revealed colocalization of GLUT1 and alpha-smooth muscle actin proteins, indicating that GLUT1 expression was related to the development of liver fibrosis. TGF-β1 caused an increase in aerobic glycolysis in HSCs and induced GLUT1 expression in HSCs by activating the Smad, p38 MAPK and P13K/AKT signaling pathways. The p38 MAPK and Smad pathways synergistically affected the induction of GLUT1 expression. GLUT1 inhibition eliminated the effect of TGF-β1 on HSC proliferation and migration. A GLUT1 inhibitor was administered in a mouse model of liver fibrosis, and GLUT1 inhibition reduced the degree of liver inflammation and liver fibrosis.CONCLUSIONTGF-β1 induces GLUT1 expression in HSCs, a process related to liver fibrosis progression. In vitro experiments revealed that TGF-β1-induced GLUT1 expression might be one of the mechanisms mediating the metabolic reprogramming of HSCs. In addition, in vivo experiments also indicated that the GLUT1 protein promotes the occurrence and development of liver fibrosis.

CircWHSC1 regulates malignancy and glycolysis by the miR-212-5p/AKT3 pathway in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive malignant tumor in breast cancer (BC). Circular RNA circWHSC1 (circWHSC1) is connected with the progression of tumors. However, the role and regulatory mechanism of circWHSC1 in TNBC are unclear. MethodsThe expression of circWHSC1, microRNA (miR)-212-5p, and protein kinase B-3 (AKT3) mRNA in BC tissues and/or cells was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The viability, colony formation, migration, invasion, and apoptosis of TNBC cells were determined by cell counting kit-8 (CCK-8), colony formation, transwell, or flow cytometry assays. The levels of glucose consumption and lactate production were assessed with commercial kits. The levels of hexokinase II (HK2) and AKT3 protein were detected by western blotting. The role of circWHSC1 in vivo was verified by tumor xenograft assay. The relationship between miR-212-5p and circWHSC1 or AKT3 was verified via dual-luciferase reporter and RNA pull-down assays. ResultsCircWHSC1 was upregulated in BC tissues and cells. Also, circWHSC1 could discriminate BC tissues and paracancerous normal tissues. TNBC patients with high circWHSC1 possessed a poor prognosis. CircWHSC1 silencing reduced TNBC cell growth in vivo and repressed proliferation, migration, invasion, glycolysis, and induced apoptosis of TNBC cells in vitro. CircWHSC1 regulated AKT3 expression by sponging miR-212-5p. Silencing of miR-212-5p overturned circWHSC1 knockdown-mediated impacts on malignancy and glycolysis of TNBC cells. AKT3 overexpression reversed the inhibitory effect of miR-212-5p mimic on malignancy and glycolysis of TNBC cells. ConclusionsCircWHSC1 accelerated malignancy and glycolysis of TNBC cells by the miR-212-5p/AKT3 axis. The research provided a potential prognostic biomarker and therapeutic target for TNBC.

Circ-PGC increases the expression of FOXR2 by targeting miR-532-3p to promote the development of non-small cell lung cancer

ABSTRACT This study was to explore the function of circular progastricsin (circ-PGC) in NSCLC. The histological morphology of tumor tissues was observed by hematoxylin and eosin (HE) staining. The expression of circ-PGC, miR-532-3p and forkhead box R2 (FOXR2) mRNA was measured by real-time quantitative polymerase chain reaction (RT-qPCR). The protein level of FOXR2 was checked by western blot. In functional analyses, cell viability, colony formation, cell apoptosis, cell migration and cell invasion were investigated using cell counting kit-8 (CCK-8) assay, colony formation assay, flow cytometry assay, wound healing assay and transwell assay. Besides, glycolysis metabolism was assessed by the levels of glucose consumption, lactate production and adenosine triphosphate (ATP) production. The predicted relationship between miR-532-3p and circ-PGC and FOXR2 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The results showed that circ-PGC and FOXR2 were upregulated in NSCLC tissues and cells. Circ-PGC knockdown or FOXR2 knockdown inhibited NSCLC cell viability, colony formation, cell migration, invasion and glycolysis metabolism, and FOXR2 overexpression rescued these inhibitory effects caused by circ-PGC knockdown. MiR-532-3p harbored the same binding site with circ-PGC and FOXR2, and circ-PGC positively regulated FOXR2 expression by targeting miR-532-3p. The expression of β-catenin and c-Myc was decreased in cells after circ-PGC knockdown but recovered with miR-532-3p inhibition or FOXR2 overexpression. Circ-PGC downregulation also inhibited tumor growth in vivo. In conclusion, circ-PGC positively regulated FOXR2 expression by competitively binding to miR-532-3p, thereby promoting the development of NSCLC, and the Wnt/β-catenin signaling pathway might be activated by the circ-PGC/miR-532-3p/FOXR2 network.

A novel circ_0099999/miR-330-5p/FSCN1 ceRNA crosstalk in pancreatic cancer

Background Pancreatic cancer is a lethal malignancy in both sexes throughout the world. Circular RNAs (circRNAs) have been implicated in the development of pancreatic cancer by operating as competing endogenous RNAs (ceRNAs). Here, we explored circ_0099999-mediated ceRNA activity in regulating pancreatic tumorigenesis. Methods Ribonuclease R (RNase R) and subcellular localization assays were utilized to characterize circ_0099999. The levels of circ_0099999, microRNA (miR)-330-5p, and fascin actin-bundling protein 1 (FSCN1) were gauged by quantitative real-time PCR (qRT-PCR) and western blot. Cell proliferation, colony formation, apoptosis, migration, and invasion were evaluated by the Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, and transwell assays, respectively. The levels of glucose consumption and lactate production were determined using the assay kits. A direct relationship between miR-330-5p and circ_0099999 or FSCN1 was validated by dual-luciferase reporter assay. Tumour xenograft assays were used to analyse the role of circ_0099999 in vivo. Results Circ_0099999 was highly up-regulated in pancreatic cancer tissues and cells. Knockdown of circ_0099999 impeded cell proliferation, migration, invasion, glycolysis, and promoted apoptosis in vitro, as well as diminished tumour growth in vivo. Circ_0099999 targeted miR-330-5p, and miR-330-5p was a downstream mediator of circ_0099999 function. FSCN1 was a direct and functional target of miR-330-5p. Furthermore, circ_0099999 operated as a ceRNA for miR-330-5p to modulate FSCN1 expression. Conclusions Our findings established a novel causal mechanism, circ_0099999/miR-330-5p/FSCN1 ceRNA crosstalk, in regulating pancreatic carcinogenesis and provided that inhibition of circ_0099999 might have therapeutic benefits in pancreatic cancer.

Circ_0005962 functions as an oncogene to aggravate NSCLC progression.

BackgroundNon-small cell lung cancer (NSCLC) is a leading threat to human lives with high incidence and mortality. Circular RNAs were reported to play important roles in human cancers. The purpose of this study was to investigate the role of circ_0005962 and explore the underlying functional mechanisms.MethodsThe protein levels of Beclin 1, light chain3 (LC3-II/LC3-I), Pyruvate dehydrogenase kinase 4 (PDK4), Cleaved Caspase 3 (C-caspase 3), and proliferating cell nuclear antigen were examined using western blot analysis. Glycolysis was determined according to the levels of glucose consumption and lactate production. Xenograft model was constructed to investigate the role of circ_0005962 in vivo.Resultcirc_0005962 expressed with a high level in NSCLC tissues and cells. circ_0005962 knockdown inhibited proliferation, autophagy, and glycolysis but promoted apoptosis in NSCLC cells. miR-382-5p was targeted by circ_0005962, and its inhibition reversed the role of circ_0005962 knockdown. Besides, PDK4, a target of miR-382-5p, was regulated by circ_0005962 through miR-382-5p, and its overexpression abolished the effects of miR-382-5p reintroduction. circ_0005962 knockdown suppressed tumor growth in vivo.Conclusioncirc_0005962 knockdown restrained cell proliferation, autophagy, and glycolysis but stimulated apoptosis through modulating the circ_0005962/miR-382-5p/PDK4 axis. Our study broadened the insights into understanding the mechanism of NSCLC progression.

PFKFB3 Promotes Liver Fibrosis by Regulating Aerobic Glycolysis of Hepatic Stellate Cells

Background: Hepatic stellate cells (HSCs) are the key effector cells in the occurrence and development of liver fibrosis, while aerobic glycolysis is one of the important metabolic characteristics of HSC activation. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a homodimeric bifunctional enzyme, which is a rate-limiting enzyme in glycolysis. This metabolite is important for the dynamic regulation of glycolytic flux. However, little is known about the role of PFKFB3 in liver fibrosis. Objectives: In this study, we aimed to explore the effects of PFKFB3 on aerobic glycolysis in the process of HSC trans-differentiation and liver fibrosis. Methods: Immunohistochemical (IHC) staining and immunofluorescence assays were used to examine PFKFB3 expression in mice fibrotic liver tissue. The determination of extracellular acidification rate was used to examine changes in aerobic glycolytic flux, lactate production levels, and glucose consumption levels in HSCs upon TGF-β1 stimulation. Western blot analysis of the expression of PFKFB3, α-SMA protein, and type I collagen was done. Liver histopathology was also examined. Besides, glycolytic inhibition by pharmacologic approaches was used to demonstrate the critical role of glycolysis in liver fibrosis. Results: The PFKFB3 protein expression was increased in mouse fibrotic liver tissue. In addition, immunofluorescence revealed the colocalization of PFKFB3 and alpha-smooth muscle actin (α-SMA) protein. In vitro experiments showed that PFKFB3 could promote glycolysis flux, lactic acid production, and glucose consumption of hepatic stellate cells. The PFKFB3 inhibitor was used in a mouse model of liver fibrosis, and the inhibition of PFKFB3 reduced the degree of liver inflammation and liver fibrosis. Conclusions: PFKFB3 can promote HSC aerobic glycolysis, which, in turn, promotes HSC activation and liver fibrosis.

Aspirin modulates 2-hydroxyisobutyrylation of ENO1K281 to attenuate the glycolysis and proliferation of hepatoma cells

Aspirin can efficiently inhibit the glycolysis and proliferation of cancer cells, however, the underlying mechanism is poorly understood. Here, we report that aspirin attenuates the glycolysis and proliferation of hepatoma cells through modulating the levels of lysine 2-hydroxyisobutyrylation (Khib) of enolase 1 (ENO1). We found that aspirin decreased the levels of glucose consumption and lactate production in hepatoma cells. Moreover, 4 mM aspirin reduced the activities of ENO1, a key enzyme of glycolysis, and decreased the levels of ENO1 Khib in the cells. Interestingly, we identified that 4 mM aspirin could decrease the levels of Khib on many proteins by using pan Khib antibody in the cells. Interestingly, the activities of ENO1 could be rescued by the transient overexpression of ENO1, but not by ENO1 mutant (K281R). Moreover, we identified that the C646, an inhibitor of p300 which is a writer of Khib, could reduce the levels of ENO1 Khib, resulting in the decrease of ENO1 activities. The treatment with PDTC, an inhibitor of NF-κB which is a target of aspirin, could work well as C646 in the cells. Both of aspirin and C646 (or PDTC) displayed a stronger effect than the single treatment in the system. Functionally, ENO1, but not ENO1 mutant (K281R), could rescue the aspirin-induced inhibition of proliferation of liver cancer cells in vitro, suggesting that ENO1K281 is involved in the aspirin-mediated inhibition of liver cancer. Our finding provides new insights into the mechanism by which aspirin attenuates the glycolysis and proliferation of hepatoma cells.

Long non‑coding RNA OIP5‑AS1 facilitates the progression of ovarian cancer via the miR‑128‑3p/CCNG1 axis.

Long non-coding RNA (LncRNA) o-phthalaldehyde-interacting protein 5 antisense transcript 1 (OIP5-AS1) serves major roles in the progression of various types of cancer. The present study investigated its biological function in ovarian cancer (OC) and its mechanisms. The levels of OIP5-AS1, microRNA-128-3p (miR-128-3p) and cyclin G1 (CCNG1) were examined by reverse transcription-quantitative PCR. Cell viability, apoptosis, migration and invasion were detected to analyze cellular progression. Glycolytic metabolism was assessed by detecting the levels of glucose consumption and lactate production. CCNG1 and hexokinase 2 protein levels were measured by western blotting. Dual-luciferase reporter assay, RNA immunoprecipitation and RNA pull-down assays were performed to affirm the interaction between two molecules. OIP5-AS1 was found to be upregulated in OC tissues and cells. Knockdown of OIP5-AS1 suppressed cell viability, migration, invasion and glycolysis while promoting apoptosis in OC cells. OIP5-AS1 interacted with miR-128-3p and functioned as an oncogene by sequestering miR-128-3p. In addition, CCNG1 was a target gene for miR-128-3p and miR-128-3p regulated the CCNG1-induced effects on OC cells by downregulating CCNG1. OIP5-AS1 upregulated the expression of CCNG1 via targeting miR-128-3p. OIP5-AS1 knockdown also inhibited tumor growth of OC in vivo by modulating the expression of miR-128-3p and CCNG1. Collectively, these data illustrated that the oncogenic role of OIP5-AS1 in OC was associated with the miR-128-3p/CCNG1 axis at least in part. OIP5-AS1 might be a probable diagnostic and therapeutic biomarker for the treatment of OC patients.

Hypoxia in the microenvironment promotes glycolysis to aggravate tumor progression via modulating the lincRNA-p21 and its downstream genes in HCC

IntroductionLincRNA-p21 was found to inhibit hepatic stellate cell (HSC) activation and liver fibrosis via a signaling cascade of lincRNA-p21-miR-181b-PTEN. Hypoxia was also previously proved to regulate hepatocellular carcinoma (HCC) glycolysis by targeting HK2.Material and methodsLuciferase assay was carried out to examine the regulatory role of miR-181b in lincRNA-p21 and HK2 expression. Quantitative real-time PCR was performed to measure the expression of lincRNA-p21, miR-181b and HK2 mRNA. Western blot and immunohistochemistry were used to analyze the expression of HK2 protein.ResultsThe expression of lincRNA-p21 and HK2 was effectively suppressed by miR-181b in Hep3B and HepG2 cells. Besides, the luciferase activities of wild type lincRNA-p21 and HK2 were remarkably suppressed by miR-181b in Hep3B and HepG2 cells. Activation and suppression of lincRNA-p21 expression using pcDNA and shRNA revealed a negative correlation between miR-181b and lincRNA-p21 expression as well as a positive correlation between HK2 and lincRNA-p21 expression. Moreover, lincRNA-p21 shRNA could effectively reverse the effect of hypoxia-induced dysregulation in miR-181b and HK2 expression, as well as the altered levels of glucose consumption and lactate production in Hep3B and HepG2 cells. Furthermore, lincRNA-p21 was capable of altering the growth and miR-181b/HK2 expression of HepG2 xenograft tumors in nude mice.ConclusionsOur study investigated the molecular relationship between lincRNA-p21, miR-181b and HK2 in cellular and animal models, and validated that hypoxia could up-regulate the expression level of lincRNA-p21 in the microenvironment of solid hepatocellular carcinoma tumor, which accordingly led to aggravated glycolysis via elevated HK2 expression, thus inhibiting the apoptosis of HCC.

Ex Vivo Perfusion With Methylprednisolone Attenuates Brain Death-induced Lung Injury in Rats

Background.The onset of brain death (BD) leads to the deterioration of potential donor lungs. Methylprednisolone is considered to increase lung oxygenation capacity and enhance the procurement yield of donor lungs, when applied in situ, during donor management. However, whether BD-induced lung damage is ameliorated upon treatment with methylprednisolone during acellular ex vivo lung perfusion (EVLP), remains unknown. We aimed to investigate whether the quality of lungs from brain-dead donors improves upon methylprednisolone treatment during EVLP.Methods.Rat lungs were randomly assigned to 1 of 3 experimental groups (n = 8/group): (1) healthy, directly procured lungs subjected to EVLP; (2) lungs from brain-dead rats subjected to cold storage and EVLP; and (3) lungs from brain-dead rats subjected to cold storage and EVLP with 40 mg methylprednisolone added to the perfusate. Ventilation and perfusion parameters, histology, edema formation, metabolic profile, and inflammatory status of lungs were investigated.Results.Methylprednisolone treated lungs from brain-dead donors improved positive inspiratory pressures needed to maintain tidal volumes of 7 mL/kg of body weight, which was 25.6 ± 5.8 cm H2O in untreated lungs and 18.0 ± 3.0 cm H2O in methylprednisolone treated lungs, after 6 h EVLP. Furthermore, dynamic lung compliance increased upon methylprednisolone treatment, with values of 0.11 ± 0.05 mL/cm H2O versus 0.18 ± 0.04 mL/cm H2O after 6 h of EVLP. Methylprednisolone treatment ameliorated the amount of lung edema, as corroborated by a reduction of 0.7 in the wet/dry ratio. Although glucose consumption levels were comparable, the BD-induced cumulative lactate production decreased from 0.44 ± 0.26 to 0.11 ± 0.16 mmol/L upon methylprednisolone treatment. Finally, BD-induced inflammatory status was reduced upon methylprednisolone treatment compared to untreated lungs from brain-dead donors, as reflected by lower proinflammatory gene expression levels of IL-1β, IL-6 and MCP-1, and IL-6 perfusate levels.Conclusions.We showed that methylprednisolone treatment during EVLP attenuates BD-induced lung injury.

Silencing of circular RNA circPDE5A suppresses neuroblastoma progression by targeting the miR-362-5p/NOL4L axis

Objective Neuroblastoma (NB) is the most common extra-cranial solid tumour in early childhood. Circular RNAs (circRNAs) have been implicated in the development of NB. The purpose of the current study was to explore the molecular action of circRNA phosphodiesterase 5 A (circPDE5A) in NB malignant progression. Materials and methods The expression levels of circPDE5A, miR-362-5p and nucleolar protein 4 like (NOL4L) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTS) assay. Cell migration and invasion were evaluated by transwell assay. The levels of glucose consumption and lactate production were measured using the commercial assay kits. Targeted correlations among circPDE5A, miR-362-5p and NOL4L were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. In vivo assays were performed to examine the role of circPDE5A in tumour growth in vivo. Results Our results revealed that circPDE5A was up-regulated in NB tissues and cells. The silencing of circPDE5A suppressed NB cell proliferation, migration, invasion, and glycolysis in vitro and diminished tumour growth in vivo. Moreover, circPDE5A directly targeted miR-362-5p by binding to miR-362-5p. CircPDE5A silencing impeded NB malignant progression in vitro through up-regulating miR-362-5p. Furthermore, NOL4L was a direct target of miR-362-5p, and NOL4L mediated the regulation of miR-362-5p on NB malignant progression in vitro. Additionally, circPDE5A functioned as a regulator of NOL4L expression via targeting miR-362-5p. Conclusions Our current findings identified that the knockdown of circPDE5A suppressed NB malignant progression at least in part by the regulation of the miR-362-5p/NOL4L axis, providing a novel rationale for developing circPDE5A as a potential target for NB management.

Flavonoids from Sophora alopecuroides L. improve palmitate-induced insulin resistance by inhibiting PTP1B activity in vitro

Seventeen flavonoids (1–17) were isolated from Sophora alopecuroides L.. Compounds 1 and 2 were new compounds, and compounds 5, 8, 11, 12, and 17 were isolated from S. alopecuroides for the first time. The sources of compounds 1 and 2 were determined from the seeds of S. alopecuroides by UPLC-QE-Orbitrap-MS, and compounds 1, 2, 7, 13, 14, 15, 16, and 17 were proven to improve the insulin resistance of C2C12 myotubes and significantly increase glucose consumption levels. Among them, compounds 1, 2, 13, 14, 16, and 17 could bind to protein tyrosine phosphatase 1B (PTP1B), thereby significantly inhibiting the enzyme activity of PTP1B. Compound 2 had the strongest inhibitory effect, with an inhibition rate of 95.22% at 0.1 μg mL−1.