Glycolysis In Non-small Cell Lung Cancer Cells Research Articles

EGCG enhances antitumor effect of apatinib in nonsmall cell lung cancer by targeting VEGF signaling to inhibit glycolysis.

Nonsmall cell lung cancer (NSCLC), one of the most aggressive malignancies globally, is characterized by poor prognosis and limited life expectancy. Epigallocatechin-3-gallate (EGCG), a natural polyphenol found in green tea, has emerged as a promising anticancer agent due to its potent antitumor properties. However, the role and the underlying mechanisms of EGCG in NSCLC remain poorly understood. Hence, this research aimed to explore the effect of EGCG on the antitumor effect of apatinib in NSCLC throughvascular endothelial growth factor(VEGF)-regulated glycolysis. Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine staining, wound healing, transwell, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and flow cytometry assays were carried out to evaluate the proliferation, migration, invasion, and apoptosis of H1299 cells, respectively. Furthermore, western blot analysis was used to detect the expressions of VEGF, p-vascular endothelial growth factor receptor-2, hypoxia-inducible factor 1α, neuropilin-1, phosphorylated-phosphatidylinositol 3-kinase, and phosphorylated-AKT. The transfection efficiency of H1299 cells with VEGF overexpression plasmid was also assessed by western blot analysis. Glycolysis was analyzed by estimating extracellular acidification rate, lactate concentration, glucose uptake, and the expressions of lactate dehydrogenase A, pyruvate kinase M2, and hexokinase 2. The results demonstrated that VEGF activated glycolysis in NSCLC cells. EGCG alone and apatinib alone or in combination inhibited cell viability, proliferation, invasion, migration, and glycolysis whereas promoted apoptosis in NSCLC cells. EGCG regulated glycolysis levels in NSCLC through VEGF overexpression, and enhanced the antitumor effect of apatinib in NSCLC through VEGF-regulated glycolysis. Taken together, EGCG strengthened the protective effects of apatinib in NSCLC through glycolysis mediated by VEGF.

Mechanisms of neural infiltration-mediated tumor metabolic reprogramming impacting immunotherapy efficacy in non-small cell lung cancer

BackgroundCurrent evidence underlines the active role of neural infiltration and axonogenesis within the tumor microenvironment (TME), with implications for tumor progression. Infiltrating nerves stimulate tumor growth and dissemination by secreting neurotransmitters, whereas tumor cells influence nerve growth and differentiation through complex interactions, promoting tumor progression. However, the role of neural infiltration in the progression of non-small cell lung cancer (NSCLC) remains unclear.MethodsThis study employs the techniques of immunohistochemistry, immunofluorescence, RNA sequencing, molecular biology experiments, and a murine orthotopic lung cancer model to deeply analyze the specific mechanisms behind the differential efficacy of NSCLC immunotherapy from the perspectives of neuro-tumor signal transduction, tumor metabolism, and tumor immunity.ResultsThis study demonstrates that nerve growth factor (NGF) drives neural infiltration in NSCLC, and 5-hydroxytryptamine (5-HT), which is secreted by nerves, is significantly elevated in tumors with extensive neural infiltration. Transcriptome sequencing revealed that 5-HT enhanced glycolysis in NSCLC cells. Pathway analysis indicated that 5-HT activated the PI3K/Akt/mTOR pathway, promoting tumor metabolic reprogramming. This reprogramming exacerbated immunosuppression in the TME. Neutralizing 5-HT-mediated metabolic reprogramming in tumor immunity enhanced the efficacy of PD-1 monoclonal antibody treatment in mice.ConclusionsThe findings of this study provide a novel perspective on the crosstalk between nerves and lung cancer cells and provide insights into further investigations into the role of nerve infiltration in NSCLC progression.

TRIM27 revealing by tumor educated platelet RNA-sequencing, as a potential biomarker for malignant ground-glass opacities diagnosis mediates glycolysis of non-small cell lung cancer cells partially through HOXM1.

Efficient ground-glass opacities (GGOs) diagnosis is challenging. A diagnostic method distinguishing malignant from benign GGOs is warranted. In this study, we sought to construct a noninvasive method based on tumor educated platelet (TEP) RNA profiles for malignant GGOs diagnosis and explore the molecular mechanism of the potential biomarker for the first time. Based on TEP RNA-sequencing (TEP RNA-seq) in benign and malignant GGOs, a classification model was constructed using differentially expressed genes (DEGs) and was used to evaluate diagnostic performance. High-throughput quantitative polymerase chain reaction (HT-qPCR) verified 23 genes selected from the top 60 DEGs between benign and malignant GGOs. The correlation between 17 verified DEGs and 22 key glycolytic genes was analyzed. Tripartite motif-containing 27 (TRIM27) overexpressing and knockdown (KD) cell models were constructed using A549 and PC-9 cells, respectively in which cell growth, apoptosis, migration and invasion were evaluated. The protein levels of HK-1/2, PKM1/2, LDHA and GLUT1 were evaluated by western blot. Glycolysis was evaluated through adenosine triphosphate (ATP), reactive oxygen species (ROS), lactate acid (LD) production, glucose uptake, and lactate dehydrogenase (LDH) activity assays. RNA-seq was performed in loss-of TRIM27-KD PC-9 cells to clarify the downstream factors of TRIM27 which was verified using western blot and immunofluorescence double staining. In 81 samples, the 1,647-DEG-based classification model exhibited area under the curve (AUC), sensitivity, and specificity values of 0.99 [95% confidence interval (CI): 0.972-1.000], 100%, and 91%, respectively, while the top 60-DEG-based classification model exhibited AUC, sensitivity, and specificity values of 0.986 (95% CI: 0.962-1.000), 98%, and 91%, respectively. TRIM27 achieved AUC of 0.87 in the diagnosis of malignant GGOs, with 83.93% sensitivity, 78.79% specificity, 81.15% accuracy, 77.05% positive predictive value (PPV) and 85.25% negative predictive value (NPV). TRIM27 was highly expressed in non-small cell lung cancer (NSCLC) cells, and accelerated cell migration and invasion. In addition, TRIM27 was found to promote glycolysis in NSCLC cells partially through HMOX1 which was negatively correlated with TRIM27. We constructed a novel TEP RNA-seq based classifier for malignant GGOs diagnosis. TRIM27, an important target discovered, could accelerate migration, invasion and regulate glycolysis partially through HMOX1 in NSCLC cells, thus providing scientific support for TRIM27 as a diagnostic biomarker for malignant GGO diagnosis.

PLA2G2D fosters angiogenesis in non-small cell lung cancer through aerobic glycolysis

Non-small cell lung cancer (NSCLC) stands prominent among the prevailing and formidable oncological entities. The immune and metabolic-related molecule Phospholipase A2, group IID (PLA2G2D) exerts promotional effects on tumor progression. However, its involvement in cancer angiogenesis remains elusive. Therefore, this investigation delved into the functional significance of PLA2G2D concerning angiogenesis in NSCLC. This study analyzed the expression and enriched pathways of PLA2G2D in NSCLC tissues through bioinformatics analysis, and measured the expression of PLA2G2D in NSCLC cells using qRT-PCR and western blot (WB). Subsequently, the viability and angiogenic potential of NSCLC cells were assessed employing CCK-8 and angiogenesis assays, respectively. The expression profile of angiogenic factors was analyzed through WB. Finally, the expression of glycolysis pathway-related genes, extracellular acidification rate and oxygen consumption rate, and the levels of pyruvate, lactate, citrate, and malate were analyzed in NSCLC cells using qRT-PCR, Seahorse XF 96, and related kits. Bioinformatics analysis revealed the upregulation of PLA2G2D in NSCLC tissues and its association with VEGF and glycolysis signaling pathways. Molecular and cellular experiments demonstrated that upregulated PLA2G2D promoted the viability, angiogenic ability, and glycolysis pathway of NSCLC cells. Rescue assays revealed that the effects of high expression of PLA2G2D on the viability, angiogenic ability, and glycolysis of NSCLC cells were weakened after the addition of the glycolysis inhibitor 2-DG. In summary, PLA2G2D plays a key role in NSCLC angiogenesis through aerobic glycolysis, displaying great potential as a target for anti-angiogenesis therapy.

Pirfenidone inhibits TGF-β1-induced metabolic reprogramming during epithelial-mesenchymal transition in non-small cell lung cancer.

Metastasis is an important contributor to increased mortality rates in non-small cell lung cancer (NSCLC). The TGF-β signalling pathway plays a crucial role in facilitating tumour metastasis through epithelial-mesenchymal transition (EMT). Glycolysis, a key metabolic process, is strongly correlated with NSCLC metastasis. Pirfenidone (PFD) has been shown to safely and effectively inhibit TGF-β1 in patients with lung diseases. Furthermore, TGF-β1 and glycolysis demonstrate an interdependent relationship within the tumour microenvironment. Our previous study demonstrated that PFD effectively inhibited glycolysis in NSCLC cells, prompting further investigation into its potential antitumour effects in this context. Therefore, the present study aims to investigate the potential antitumour effect of PFD in NSCLC and explore the relationship among TGF-β1, glycolysis and EMT through further experimentation. The antitumour effects of PFD were evaluated using five different NSCLC cell lines and a xenograft tumour model. Notably, PFD demonstrated a significant antitumour effect specifically in highly glycolytic H1299 cells. To elucidate the underlying mechanism, we compared the efficacy of PFD after pretreatment with either TGF-β1 or a TGF-β receptor inhibitor (LY2109761). The energy metabolomics analysis of tumour tissue demonstrated that PFD, a chemosensitizing agent, reduced lactate and ATP production, thereby inhibiting glycolysis and exerting synergistic antineoplastic effects. Additionally, PFD combined with cisplatin targeted TGF-β1 to inhibit glycolysis during EMT and enhanced the chemosensitization of A549 and H1299 cells. The magnitude of the anticancer effect exhibited by PFD was intricately linked to its metabolic properties.

Circ_0006324 regulates cell proliferation, cell-cycle progression, apoptosis, and glycolysis of non-small cell lung cancer cells through miR-496/TRIM59 axis.

Increasing evidence suggests that circular RNA (circRNA) plays an important role in non-small cell lung cancer (NSCLC) progression. This study aimed to investigate the role and potential molecular mechanism of circ_0006324 in NSCLC. The expression levels of circ_0006324, miR-496, miR-488-5p, and tripartite motif-containing 59 (TRIM59) mRNA were determined by quantitative real-time polymerase chain reaction (PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, EdU assay, and flow cytometry were carried out to evaluate cell proliferation and apoptosis. The extracellular acidification rate and lactic acid production were examined to assess cell glycolysis. Western blot assay was used to detect protein levels. The target relationship of circ_0006324/miR-496/TRIM59 axis was validated by RNA pull-down assay, dual luciferase reporter assay, and radio immunoprecipitation assay. Xenograft tumor assay was performed to reveal the function of circ_0006324 in vivo. Circ_0006324 was upregulated in NSCLC and related to tumor node metastasis stage and distant metastasis. Knockdown of circ_00006324 impeded NSCLC cell proliferation, glycolysis, and promoted cell apoptosis. MiR-496 was verified as a target of circ_0006324 and circ_00006324 mediated the altering of cell proliferation, apoptosis, and glycolysis of NSCLC cells through targeting miR-496. TRIM59 was verified as a target of miR-496, and circ_0006324 positively regulated TRIM59 expression by targeting miR-496. Overexpression of TRIM59 could reverse the effects of circ_0006324 silencing on the proliferation, apoptosis, and glycolysis of NSCLC cells. Circ_0006324 knockdown impeded NSCLC tumor growth in vivo. Circ_0006324 functioned as a tumor promoter in NSCLC to promote cell proliferation, cell cycle progression, and glycolysis and inhibit cell apoptosis via miR-496/TRIM59 axis.

Circular RNA, circular RARS, promotes aerobic glycolysis of non-small-cell lung cancer by binding with LDHA.

Accumulating evidence has highlighted the critical roles of circular RNAs (circRNAs) in non-small-cell lung cancer (NSCLC). This study aims to unveil the roles of circRARS (circular RARS) (hsa_circ_0001551) in NSCLC. Quantitative real-time PCR was used to determine the expression of circRARS in NSCLC tissues and cells. Kaplan-Meier analysis was used to determine the prognostic value of circRARS expression. CCK8, transwell, and wound healing assays were used to assess the proliferation, invasion, and migration abilities of NSCLC cells. RNA pull-down, cell fraction, glucose consumption, lactate production, and lactate dehydrogenase activity assays were conducted to explore the potential mechanisms of circRARS in NSCLC. circRARS is upregulated in NSCLC tissues and positively correlated with smoking status, lymph node metastasis, and higher tumor stages. NSCLC patients with high expression of circRARS have poor overall survival. Functional assays demonstrated that circRARS accelerated the proliferation, invasion, and migration of NSCLC cells in vitro. The cell fraction suggested that circRARS mainly accumulated in cytoplasm and the RNA pull-down assay showed lactate dehydrogenase (LDHA) could bind with circRARS. Furthermore, circRARS positively regulates LDHA activity and LDHA expression at the transcription level. Moreover, downregulated circRARS decreases glucose consumption and lactate production and compromises aerobic glycolysis in NSCLC cells. Finally, rescue assays showed circRARS could promote NSCLC cell proliferation by regulating LDHA activity. This study shows that circRARS can promote glycolysis and tumor progression in NSCLC by regulating LDHA.

Gankyrin modulated non-small cell lung cancer progression via glycolysis metabolism in a YAP1-dependent manner

Non-small cell lung cancer (NSCLC) is highly malignant and heterogeneous form of lung cancer and involves various oncogene alterations. Glycolysis, an important step in tumor metabolism, is closely related to cancer progression. In this study, we investigated the biological function and mechanism of action of Gankyrin in glycolysis and its association with NSCLC. Analyzed of data from The Cancer Genome Atlas as well as NSCLC specimens and adjacent tissues demonstrated that Gankyrin expression was upregulated in NSCLC tissues compared to adjacent normal tissues. Gankyrin was found to significantly aggravate cancer-related phenotypes, including cell viability, migration, invasion, and epithelial mesenchymal transition (EMT), whereas Gankyrin silencing alleviated the malignant phenotype of NSCLC cells. Our results reveal that Gankyrin exerted its function by regulating YAP1 expression and increasing its nuclear translocation. Importantly, YAP1 actuates glycolysis, which involves glucose uptake, lactic acid production, and ATP generation and thus might contribute to the tumorigenic effect of Gankyrin. Furthermore, the Gankyrin-accelerated glycolysis in NSCLC cells was reversed by YAP1 deficiency. Gankyrin knockdown reduced A549 cell tumorigenesis and EMT and decreased YAP1 expression in a subcutaneous xenograft nude mouse model. In conclusion, both Gankyrin and YAP1 play important roles in tumor metabolism, and Gankyrin-targeted inhibition may be a potential anti-cancer therapeutic strategy for NSCLC.

Tanshinone IIA inhibits cell growth by suppressing SIX1‑induced aerobic glycolysis in non‑small cell lung cancer cells

Aerobic glycolysis plays a key role in cancer cell metabolism and contributes to tumorigenesis, including that of non-small cell lung cancer (NSCLC). Tanshinone IIA (Tan IIA), an active compound of Salvia miltiorrhiza, exhibits antitumor properties. Multiple mechanisms are involved in the antitumor action of Tan IIA in lung cancer, such as inhibiting cell growth, promoting cell apoptosis and influencing cellular metabolism. However, the effects of Tan IIA on NSCLC cells and its mechanisms of action remain unclear. The present study shows Tan IIA dose-dependently attenuated the growth of NSCLC cells and in vitro in a dose-dependent manner. Moreover, Tan IIA markedly decreased the ATP level, glucose uptake and lactate production in the NSCLC cells in vitro. Tan IIA also inhibited tumor growth in a xenograft model in vivo. Mechanically, Tan IIA treatment decreased sine oculis homeobox homolog 1 (SIX1) mRNA and protein levels, thus leading to the downregulation of pyruvate kinase isozyme M2, hexokinase 2 and lactate dehydrogenase A (LDHA) expression in A549 cells. SIX1 knockdown with small interfering-RNA inhibited glycolysis in NSCLC cells, suggesting that SIX1 plays a role in the antitumor effect of Tan IIA on NSCLC cells. More importantly, it was demonstrated that SIX1 expression was stimulated in patients with NSCLC and was positively correlated with the LDH serum level. Finally, SIX1 low expression levels predicted the poor prognosis of patients with NSCLC. In conclusion, the present study showed that Tan IIA functioned as an anti-glycolysis agent in NSCLC cells by downregulating SIX1 expression and inhibiting cell proliferation.

Osmundacetone modulates mitochondrial metabolism in non-small cell lung cancer cells by hijacking the glutamine/glutamate/α-KG metabolic axis

BackgroundOsmundacetone (OSC) is a bioactive phenolic compound isolated from Phellinus igniarius and that was shown to exert cytotoxic effects on cancer cells in our previous work. The antiproliferative impact of OSC on non-small cell lung cancer (NSCLC) and the underlying mechanisms, however, have not been studied. PurposeThis study aimed to explore the antiproliferative effect of OSC on NSCLC cells and the mechanisms involved. MethodsCell viability, colony formation and cell cycle distribution were measured following exposure to OSC in vitro. The anticancer activity of OSC was also examined using a xenograft growth assay in vivo. Furthermore, serum metabolomics analysis by GC–MS was done to detect alterations in the metabolic profile. Next, expression of GLS1 and GLUD1, the key enzymes in glutamine metabolism, was evaluated using RT-PCR and western blot. α-KG and NADH metabolites were assessed by ELISA. Mitochondrial functions and morphology were evaluated using the JC-1 probe and transmission electron microscopy, respectively. The ATP production rate in mitochondria of cells with OSC treatment was determined using a Seahorse XFe24 Analyzer. ResultsOSC selectively reduced the proliferation of A549 and H460 cells. OSC triggered G2/M cell cycle arrest and decreased the cell clone formation. A mouse xenograft model revealed that OSC inhibited tumor growth in vivo. Findings of serum metabolomics analyses indicated that the anticancer function of OSC was related to disorders of glutamine metabolism. Such a speculation was further verified by the expression level of GLUD1, which was downregulated by OSC treatment. Concentrations of the related metabolites α-KG and NADH were reduced in response to OSC treatment. Moreover, OSC led to disorganization of the mitochondrial ultrastructure and a decrease in mitochondrial membrane potential. OSC also decreased ATP production via oxidative phosphorylation (OXPHOS) but did not affect glycolysis in NSCLC cells. ConclusionThe key role of OSC in mitochondrial energy metabolism in NSCLC cells is to suppress tumor development and cell proliferation downregulating GLUD1 to inhibit the glutamine/glutamate/α-KG metabolic axis and OXPHOS. It indicats that OSC might be a potential natural agent for personalized medicine and an anticancer metabolic modulator in NSCLC chemotherapy.

An NF-\u03baB/OVOL2 circuit regulates glucose import and cell survival in non-small cell lung cancer

BackgroundTumor cells tend to utilize glycolysis rather than aerobic respiration even under aerobic conditions. OVOL2, an inhibitory C2H2 zinc finger transcription factor, is a potential tumor suppressor in cancers. However, the association between OVOL2 and tumor energy metabolism is unknown.MethodsWestern blotting was used to determine the expression of OVOL2 in different non-small cell lung cancer (NSCLC) cell lines and mouse models. The metabolic parameters in NSCLC cells following overexpression or knockdown OVOL2 were examined. To define the mechanism by which OVOL2 regulates aerobic glycolysis, interacting protein of OVOl2 and downstream molecular events were identified by luciferase assay and co-immunoprecipitation. We documented the regulatory mechanism in mouse xenograft models. Finally, clinical relevance of OVOL2, NF-κB signaling and GLUT1 was measured by immunostaining.ResultsOVOL2 is downregulated in NSCLC and overexpression of OVOL2 inhibits the survival of cancer cells. Moreover, OVOL2 directly binds to P65 and inhibits the recruitment of P300 but facilitates the binding of HDAC1 to P65, which in turn negatively regulates NF-κB signaling to suppress GLUT1 translocation and glucose import. In contrast, OVOL2 expression is negatively regulated by NF-κB signaling in NSCLC cells via the ubiquitin–proteasome pathway. Re-expression of OVOL2 significantly compromise NF-κB signaling-induced GLUT1 translocation, aerobic glycolysis in NSCLC cells and mouse models. Immunostaining revealed inverse correlations between the OVOL2 and phosphorylated P65 levels and between the OVOL2 and membrane GLUT1 levels, and a strong correlation between the phosphorylated P65 and membrane GLUT1 levels.ConclusionsThese results suggest a regulatory circuit linking NF-κB and OVOL2, which highlights the role of NF-κB signaling and OVOL2 in the modulation of glucose metabolism in NSCLC.5S647uSA4FfDHArbVFhiZ4Video

Circ_0078767 Inhibits the Progression of Non-Small-Cell Lung Cancer by Regulating the GPX3 Expression by Adsorbing miR-665.

Non-small-cell lung cancer (NSCLC) is one of the most serious cancers. The circular RNA_0078767 (circ_0078767) expression was decreased in NSCLC tissues. However, the molecular mechanism of circ_0078767 remains unknown. The expression of circ_0078767, microRNA-665 (miR-665), and glutathione peroxidase 3 (GPX3) was detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR). Cell proliferation, migration, and invasion were detected by colony formation assay and transwell assay, respectively. The lactate production and glucose consumption were tested by glycolysis. Western blot examined the protein levels of hexokinase-2 (HK2), matrix metalloproteinase-9 (MMP9), and GPX3 cells. Circinteractome predicted the relationship between miR-665 and circ_0078767 or GPX3 and was verified by dual luciferase reporter assays. The xenotransplantation model was established to study the role of circ_0078767 in vivo. The expression of circ_0078767 and GPX3 was decreased in NSCLC tissues, while the expression of miR-665 was increased. Circ_0078767 can sponge miR-665, and GPX3 is the target of miR-665. In vitro complement experiments showed that knockdown of circ_0078767 significantly promoted malignant behavior of NSCLC, while cotransfection of miR-665 inhibitor partially reduced this change. In addition, the GPX3 overexpression decreased the promoting effects of miR-665 upregulation on proliferation, migration, and invasion of NSCLC cells. Mechanically, circ_0078767 regulates the GPX3 expression in NSCLC cells by spongy miR-665. In addition, in vivo studies have shown that downregulation of circ_0078767 promotes tumor growth. Circ_0078767 silencing promotes proliferation, migration, invasion, and glycolysis of NSCLC cells by regulating the miR-665/GPX3 axis, suggesting that circ_0078767/miR-665/GPX3 axis may be a potential regulatory mechanism for the treatment of NSCLC.

Functions of lncRNA DUXAP8 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) poses a serious threat to public health due to its significant morbidity and mortality rates. The processes of NSCLC formation and development are quite complex and involve numerous regulatory biomolecules. Long non-coding RNAs (lncRNAs) have attracted attention since they have been found to play critical roles in the tumorigenesis of various human malignancies. Recently, double homeobox A pseudogene 8 (DUXAP8) was identified as an oncogenic lncRNA that is overexpressed in different tumor types. In NSCLC, high expression of DUXAP8 is associated with poor prognosis in patients. The regulatory mechanism underlying the oncogenic effects of DUXAP8 can be divided into transcriptional level and post-transcriptional level. DUXAP8 promotes proliferation, epithelial-mesenchymal transition, and aerobic glycolysis in NSCLC cells. Moreover, DUXAP8 shows potential for the diagnosis and treatment of NSCLC. Herein, we review the molecular mechanisms underlying the DUXAP8-mediated phenotypes of NSCLC as well as its potential clinical applications.

Circ_0020123 regulates autophagy, glycolysis, and malignancy by upregulating IRF4 through eliminating miR-193a-3p-mediated suppression of IRF4 in non-small cell lung cancer.

Circular RNA is related to the tumorigenesis of various cancers. Circular RNA hsa_circ_0020123 (circ_0020123) has been uncovered to promote non-small cell lung cancer (NSCLC) progression. However, the regulatory mechanism of circ_0020123 in NSCLC is unclear. The quantitative real-time polymerase chain reaction was employed to detect the levels of circ_0020123, microRNA (miR)-193a-3p, and IRF4 interferon regulatory factor 4 (IRF4) in NSCLC tissues and cells. Loss-of-function experiments were performed to analyze the impacts of circ_0020123 silencing on NSCLC cell malignancy, autophagy, and glycolysis. Protein levels were detected using western blotting. The regulatory mechanism of circ_0020123 was analyzed by bioinformatics analysis and validated by the dual-luciferase reporter, RNA immunoprecipitation assay, and RNA pull-down assay. Xenograft assay was performed to verify the biological function of circ_0020123. We observed an overt elevation in circ_0020123 expression in NSCLC samples and cells, and NSCLC patients with high circ_0020123 expression had a poor prognosis. Circ_0020123 knockdown constrained xenograft tumor growth in vivo and curbed cell proliferation, migration, and glycolysis, and accelerated cell apoptosis and autophagy in NSCLC cells in vitro. Circ_0020123 could absorb miR-193a-3p to regulate IRF4 expression. miR-193a-3p silencing overturned circ_0020123 knockdown-mediated impacts on NSCLC cell malignancy, autophagy, and glycolysis. And IRF4 overexpression reversed miR-193a-3p mimic-mediated effects on NSCLC cell malignancy, autophagy, and glycolysis. Circ_0020123 promoted glycolysis and tumor growth by upregulating IRF4 through sequestering miR-193a-3p in NSCLC, offering a novel mechanism by which circ_0020123 is responsible for the malignancy, autophagy, and glycolysis of NSCLC cells.

Silencing of ISLR inhibits tumour progression and glycolysis by inactivating the IL‑6/JAK/STAT3 pathway in non‑small cell lung cancer.

Lung cancer is the second most frequent cancer type in both men and women, and it is considered to be one of the major causes of cancer-related mortality worldwide. However, few biomarkers are currently available for the diagnosis of lung cancer. The aim of the present study was to investigate the function of the immunoglobulin superfamily containing leucine-rich repeat (ISLR) gene in non-small cell lung cancer (NSCLC) cells, and to elucidate the underlying molecular mechanism of its action. The current study analysed ISLR expression in NSCLC tumour and normal tissues using The Cancer Genome Atlas cohort datasets. ISLR expression in NSCLC cell lines was determined using reverse transcription-quantitative PCR. Cell Counting Kit-8, soft agar colony formation, wound healing, Transwell, flow cytometry and glycolysis assays were performed to determine the effects of ISLR silencing or overexpression on cells. The expression levels of the genes involved in epithelial-mesenchymal transition (EMT), apoptosis and glycolysis were evaluated via western blotting. Transfected cells were exposed to the pathway activator, IL-6, to validate the regulatory pathway. ISLR was overexpressed in NSCLC tissues and cell lines. Overall, patients with high ISLR expression had lower survival rates. In addition, small interfering RNA-ISLR inhibited the proliferation, EMT, migration, invasion and glycolysis of NSCLC cells, and promoted their apoptosis. ISLR overexpression had the opposite effect on tumour progression and glycolysis in NSCLC cells. Gene set enrichment analysis and western blotting results indicated that the IL-6/Janus kinase (JAK)/STAT3 pathway was enriched in ISLR-related NSCLC. Knockdown of ISLR inhibited IL-6-induced proliferation, invasion, migration and glycolysis in human NSCLC cells. In summary, ISLR silencing can inhibit tumour progression and glycolysis in NSCLC cells by activating the IL-6/JAK/STAT3 signalling pathway, which is a potential molecular target for NSCLC diagnosis and treatment.

Circ_0002346 Suppresses Non-Small-Cell Lung Cancer Progression Depending on the Regulation of the miR-582-3p/STXBP6 Axis.

Background Accumulating articles have reported the pivotal regulatory roles of circular RNAs (circRNAs) in non-small-cell lung cancer (NSCLC) tumorigenesis. Here, our purpose was to explore the role of circ_0002346 in NSCLC progression and its associated mechanism. Methods Cell proliferation ability was assessed by a 5-ethynyl-2′-deoxyuridine (EDU) assay and a colony formation assay. Transwell assays were conducted to analyze cell migration and invasion abilities. Cell apoptosis was analyzed by flow cytometry and by using a caspase3 activity assay kit. The glycolysis of NSCLC cells was analyzed using a fluorescence-based glucose/lactate assay kit. A dual-luciferase reporter assay and an RNA pull-down assay were performed to verify the binding relationship between microRNA-582-3p (miR-582-3p) and circ_0002346 or syntaxin-binding protein 6 (STXBP6). Results circ_0002346 level was prominently downregulated in NSCLC tissues and cell lines. circ_0002346 overexpression significantly suppressed the proliferation, migration, invasion, and glycolysis and triggered the apoptosis of NSCLC cells. circ_0002346 directly interacted with miR-582-3p, and circ_0002346 overexpression-mediated antitumor effects in NSCLC cells were partly reversed by miR-582-3p overexpression. miR-582-3p directly interacted with the 3′ untranslated region (3′UTR) of STXBP6, and STXBP6 silencing partly counteracted circ_0002346 overexpression-mediated antitumor influences in NSCLC cells. circ_0002346 can upregulate the expression of STXBP6 by acting as a miR-582-3p sponge in NSCLC cells. circ_0002346 overexpression suppressed xenograft tumor growth in vivo. Conclusion circ_0002346 overexpression suppressed the malignant properties of NSCLC cells by binding to miR-582-3p to induce the expression of STXBP6.

Circular RNA 0006349 Augments Glycolysis and Malignance of Non-small Cell Lung Cancer Cells Through the microRNA-98/MKP1 Axis.

Background: The involvement of dysregulated circular RNAs (circRNAs) in human diseases has been increasingly recognized. In this study, we focused on the function of a newly screened circRNA, circ_0006349, in the progression of non-small-cell lung cancer (NSCLC) and the molecules of action.Methods: The NSCLC circRNA dataset GSE101684, microRNA (miRNA) dataset GSE29250, and mRNA dataset GSE51852 obtained from the GEO database were used to identify the differentially expressed genes in NSCLC samples. Tumor and normal tissues were collected from 59 patients with NSCLC. The expression of circ_0006349, miR-98, and MAP kinase phosphatase 1 (MKP1) in collected tissue samples and in acquired cells was determined. The binding relationships between miR-98 and circ_0006349/MKP1 were predicted and validated. Altered expression of circ_0006349, miR-98, and MKP1 was introduced in NSCLC cells to examine their roles in cell growth, apoptosis, and glycolysis.Results: Circ_0006349 and MKP1 were upregulated, and miR-98 was poorly expressed in the collected tumor tissues and the acquired NSCLC cell lines. Circ_0006349 was identified as a sponge for miR-98 to elevate MKP1 expression. Silencing of circ_0006349 suppressed proliferation and increased apoptosis of Calu-3 and H1299 cells, and it reduced glycolysis, glucose uptake, and the production of lactate in cells. Upon circ_0006349 knockdown, further downregulation of miR-98 or upregulation of MKP1 restored the malignant behaviors of cells.Conclusion: This research demonstrated that circ_0006349 derepressed MKP1 expression by absorbing miR-98, which augmented the proliferation and glycolysis of NSCLC cells and promoted cancer development.

Circ_0001944 Contributes to Glycolysis and Tumor Growth by Upregulating NFAT5 Through Acting as a Decoy for miR-142-5p in Non-Small Cell Lung Cancer.

BackgroundCircular RNAs (circRNAs) participate in the tumorigenesis of various cancers. CircRNA hsa_circ_0001944 (circ_0001944), derived from the TCONS_l2_00030860 gene, has been uncovered to be upregulated in NSCLC (non-small cell lung cancer). Nevertheless, the influence of circ_0001944 on glycolysis and tumor growth in NSCLC is unclear.MethodsExpression trend of circ_0001944 in NSCLC tissues and cells were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to assess the influence of circ_0001944 knockdown on proliferation, migration, invasion, and glycolysis of NSCLC cells. Protein levels were assessed by Western blotting. The regulatory mechanism of circ_0001944 was analyzed by bioinformatics analysis, dual-luciferase reporter assay, and/or RNA pull-down assay. The tumorigenicity of circ_0001944 was confirmed by xenograft assay.ResultsCirc_0001944 was highly expressed in NSCLC, and NSCLC patients with high expression of circ_0001944 had a worse prognosis. Circ_0001944 silencing decreased xenograft tumor growth in vivo and repressed proliferation, migration, invasion, and glycolysis of NSCLC cells in vitro. Circ_0001944 was verified as a decoy for microRNA (miR)-142-5p, which targeted NFAT5 (nuclear factor of activated T cells 5). MiR-142-5p was downregulated while NFAT5 was upregulated in NSCLC. Both miR-142-5p inhibition and NFAT5 overexpression offset the suppressive impact of circ_0001944 silencing on proliferation, migration, invasion, and glycolysis of NSCLC cells. Circ_0001944 adsorbed miR-142-5p to elevate NFAT5 expression in NSCLC cells.ConclusionCirc_0001944 promotes proliferation, migration, invasion, and glycolysis of NSCLC cells by upregulating NFAT5 through adsorbing miR-142-5p, offering a novel mechanism for understanding the advancement of NSCLC.

Circular RNA circ_0001421 contributes to colony formation, migration, invasion and glycolysis of non-small cell lung cancer via the miR-409-3p/TMEM14A axis

IntroductionAccumulating evidence testifies to the considerably significant roles of circular RNAs (circRNAs) in non-small cell lung cancer (NSCLC). This report describes the exploration of the molecular mechanism of circRNA_0001421 (circ_0001421) in NSCLC.Material and methodsThe relative levels of circ_0001421, microRNA-409-3p (miR-409-3p) and transmembrane protein 14A (TMEM14A) were assayed through quantitative real-time polymerase chain reaction (qRT-PCR). Cell colony formation ability was detected by colony formation assay. Transwell assay was exploited for assessing cell migration and invasion. Glycolysis was evaluated via ECAR measurement, glucose consumption, lactate production and protein detection. The protein levels were examined using Western blot. The target combination between miR-409-3p and circ_0001421 or TMEM14A was analyzed by dual-luciferase reporter assay. Xenotransplantation assay was applied for estimating the effect of circ_0001421 on NSCLC in 10 mice.ResultsCirc_0001421 was up-regulated in NSCLC tissues and cells. Down-regulation of circ_0001421 suppressed colony formation, migration, invasion and glycolysis of NSCLC cells. Circ_0001421 could sponge microRNA-409-3p (miR-409-3p) and miR-409-3p inhibition relieved the effects of circ_0001421 knockdown on NSCLC cells. MiR-409-3p targeted transmembrane protein 14A (TMEM14A) and circ_0001421 modulated TMEM14A expression via targeting miR-409-3p. Overexpression of miR-409-3p suppressed NSCLC progression by inhibiting TMEM14A. Circ_0001421 depression restrained tumor growth of NSCLC by the miR-409-3p/TMEM14A axis in vivo.ConclusionsCirc_0001421 facilitated the development of NSCLC via the regulation of the miR-409-3p/TMEM14A axis. Circ_0001421 may be a promising therapeutic target in NSCLC.

Targeting B7-H3 via chimeric antigen receptor T cells and bispecific killer cell engagers augments antitumor response of cytotoxic lymphocytes

BackgroundB7-H3, an immune-checkpoint molecule and a transmembrane protein, is overexpressed in non-small cell lung cancer (NSCLC), making it an attractive therapeutic target. Here, we aimed to systematically evaluate the value of B7-H3 as a target in NSCLC via T cells expressing B7-H3-specific chimeric antigen receptors (CARs) and bispecific killer cell engager (BiKE)-redirected natural killer (NK) cells.MethodsWe generated B7-H3 CAR and B7-H3/CD16 BiKE derived from an anti-B7-H3 antibody omburtamab that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. Antitumor efficacy and induced-immune response of CAR and BiKE were evaluated in vitro and in vivo. The effects of B7-H3 on aerobic glycolysis in NSCLC cells were further investigated.ResultsB7-H3 CAR-T cells effectively inhibited NSCLC tumorigenesis in vitro and in vivo. B7-H3 redirection promoted highly specific T-cell infiltration into tumors. Additionally, NK cell activity could be specially triggered by B7-H3/CD16 BiKE through direct CD16 signaling, resulting in significant increase in NK cell activation and target cell death. BiKE improved antitumor efficacy mediated by NK cells in vitro and in vivo, regardless of the cell surface target antigen density on tumor tissues. Furthermore, we found that anti-B7-H3 blockade might alter tumor glucose metabolism via the reactive oxygen species-mediated pathway.ConclusionsTogether, our results suggest that B7-H3 may serve as a target for NSCLC therapy and support the further development of two therapeutic agents in the preclinical and clinical studies.