Expression In Non-small Cell Lung Cancer Tissues Research Articles

Circular RNA circ_0004630 promotes malignancy and glycolysis of nonsmall cell lung cancer by sponging microRNA-1208 and regulating leucine-rich repeat kinase 2 expression.

Emerging evidence has discovered that circular RNAs play important regulators of nonsmall cell lung cancer (NSCLC), but the role and potential molecular mechanism of hsa_circ_100549 (circ_0004630) involved in NSCLC is poorly defined. In this study, circ_0004630, microRNA-1208 (miR-1208), and leucine-rich repeat kinase 2 (LRRK2) expression were detected using real-time quantitative polymerase chain reaction. Cell proliferation, colony formation, apoptosis, and invasion were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine, colony formation, flow cytometry, and transwell assays. Protein levels of glucose transporter 1, Hexokinase 2, and LRRK2 were detected using western blot assay. Glucose consumption, lactate production, and adenosine triphosphatecontent were assessed using the corresponding kits. After predicting via bioinformatics software Circinteractome and Targetscan, the binding between miR-1208 and circ_0004630 or LRRK2 was verified by a dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assay. The xenograft tumor model analyzed the biological role circ_000460 on tumor growth in vivo. It was found that circ_0004630 and LRRK2 were increased, and miR-1208 was low expression in NSCLC tissues and cells. Functionally, the downregulation of circ_0004630 inhibited NSCLC cell proliferation, invasion, glycolysis, and accelerated apoptosis in vitro. In mechanism, circ_0004630 might work as a sponge of miR-1208 to modulate LRRK2 expression. In addition, DUXAP8 deficiency cured neuroblastoma tumor growth in vivo. In conclusion, circ_0004630 knockdown might suppress NSCLC cell proliferation, metastasis, and glycolysis partly by the miR-1208/LRRK2 axis. Our findings hinted at an important theoretical basis for further elucidating the pathogenesis of NSCLC and targeted therapy.

O-GlcNAcylation of hexokinase 2 modulates mitochondrial dynamics and enhances the progression of lung cancer.

Non-small cell lung cancer (NSCLC) stands as the prevailing manifestation of lung cancer, with current therapeutic modalities linked to a dismal prognosis, necessitating further advancements. Hexokinase 2 (HK2), a critical enzyme positioned on the mitochondrial membrane, exerts control over diverse biological pathways, thereby regulating cancer. Nevertheless, the precise role and mechanism of HK2 in NSCLC remain inadequately elucidated, warranting comprehensive investigation. HK2 expression in NSCLC tissues and cell lines was detected through immunohistochemistry and western blot analysis. Concurrently, shRNA assays were applied to scrutinize the impact of HK2 on cell proliferation, apoptosis, migration, and invasion processes in NSCLC cell lines, utilizing CCK8, flow cytometry, wound-healing assay, and transwell techniques. The involvement of HK2 in mitochondrial dynamics was probed through western blot analysis, mitochondrial membrane potential assay, and assessment of ROS generation. Next, the functional role of HK2 was assessed by examining its influence on xenograft tumor growth in nude mice in vivo. Further research has demonstrated that HK2 played a role in NSCLC through its O-GlcNAcylation process. The results of the study revealed that HK2 O-GlcNAcylation promoted the proliferation, migration, and invasive characteristics of NSCLC cells, while alleviating mitochondrial damage, whereas O-GlcNAcylation inactivation yielded the opposite effect. Furthermore, in vivo experiments in nude mice illustrated that HK2 O-GlcNAcylation could stimulate tumor growth in NSCLC. These results suggested that HK2 may impact mitochondrial dynamics in NSCLC through its O-GlcNAcylation, thereby contributing to the progression of NSCLC.

Dysregulation of SIGLEC1 in non-small cell lung cancer: prognostic implications and immunomodulatory role-a multicenter cohort study

PurposeTo investigate the clinical significance and functional role of SIGLEC1-positive cells in non-small cell lungcancer (NSCLC) patients, focusing on their prognostic impact and therapeutic response.MethodsA multicenter retrospective cohort analysis was conducted, integrating data from multiple sources. Weanalyzed SIGLEC1 expression in NSCLC tissues, clinicopathological features, overall survival outcomes,chemotherapy responsiveness, and sensitivity to targeted therapies. We also developed a prognostic model basedon SIGLEC1 expression and clinical variables.ResultsSIGLEC1 expression was significantly downregulated in NSCLC tissues, and the density of SIGLEC1-positivecells was inversely correlated with various clinicopathological features. Notably, patients with high infiltration ofSIGLEC1-positive cells exhibited significantly better overall survival outcomes. Furthermore, elevated SIGLEC1expression was associated with improved responsiveness to chemotherapy and demonstrated distinct patterns ofsensitivity to targeted therapies. A robust prognostic model was developed by integrating SIGLEC1 expression andclinical variables.ConclusionsThis study highlighted the downregulation of SIGLEC1 in NSCLC tissues and its significant associationwith patient prognosis and therapeutic response. The findings suggested that SIGLEC1 played a critical role inmodulating the tumor immune microenvironment and has potential as both a prognostic biomarker and therapeutictarget in NSCLC.

The NEDD4/FLRT2 axis regulates NSCLC cell stemness.

Lung cancer is the leading cause of cancer-related death worldwide. The treatment for lung cancer, particularly for non-small cell lung cancer (NSCLC), remains a clinical challenge. Cancer stem cells are vital for lung cancer development. This study aimed to determine the influence of the neuronally expressed developmentally downregulated 4-fibronectin leucine-rich transmembrane 2 (NEDD4-FLRT2) axis on cancer cell stemness in NSCLC. FLRT2 expression in NSCLC tissues and stem cells was investigated using western blot and RT-qPCR. The sphere formation assay and the abundance of stemness markers were employed to confirm the stemness of NSCLC stem cells. The CCK-8, colony formation, and Trans-well assays, as well as flow cytometry, were used to determine NSCLC stem cell growth, metastasis, and apoptosis, respectively. The Co-IP assay was used to confirm the binding between NEDD4 and FLRT2. Xenograft tumor mouse models were used to investigate tumorigenesis in vivo. Here, we reported that FLRT2 expression was reduced in NSCLC tissues, cells, and NSCLC stem cells. FLRT2 upregulation inhibited NSCLC stem cell proliferation, sphere formation, and drug resistance and promoted drug-resistant cell apoptosis. Furthermore, FLRT2 overexpression demonstrated antitumor effects in a xenograft tumor mouse model. Mechanically, FLRT2 was ubiquitinated and degraded by E3 ligase NEDD4. NEDD4 overexpression significantly abolished the inhibitory effects of FLRT2 on NSCLC stemness, as evidenced by in vitro and in vivo experiments. This study revealed that FLRT2 acted as a tumor suppressor by inhibiting cancer cell stemness in NSCLC. NEDD4 promoted ubiquitination degradation of FLRT2 protein. NEDD4 counteracted the inhibitory effects of FLRT2 on NSCLC stem cell tumorigenesis.

Urolithin A promotes the degradation of TMSB10 to deformation F-actin in non-small-cell lung cancer

BackgroundLung cancer is one of the most frequently diagnosed cancers and non-small-cell lung cancer (NSCLC) poses major diagnoses. Urolithin A (UA) is a natural compound produced by the gut microbiota through the metabolism of polyphenol ellagitannins (ETs) and ellagic acid (EA), which has been found to inhibit epithelial-mesenchymal transition (EMT) in lung cancer cell lines. However, the mechanism of UA function in NSCLC remains elusive. ProposeThis study aimed to investigate the potential effectiveness of UA in NSCLC therapeutic and uncovering its underlying mechanisms. MethodsEffects of UA treatment, TMSB10 gene knockdown or overexpression on NSCLC cell phenotype were evaluated by availability, transwell assays. The downstream factors and pathways of UA were investigated by proteomics. TMSB10 expression in NSCLC tissues was detected by bioinformatics analysis as well as immunohistochemistry. Confocal imaging, GST pull-down and western blotting investigated the mechanism of UA induced TMSB10 degradation. ResultsIn the present study, we demonstrated that UA shows an inhibitory role in NSCLC cell proliferation, migration, and invasion. This inhibition is attributed to the accelerated degradation of TMSB10, a biomarker among various cancers, via the autophagy-lysosome pathway. Additionally, knocked down of TMSB10 showed a similar phenotype with UA treatment. The reduction of TMSB10 protein level following decreased ATP level inhibits the F-actin formation for cell migration, thereby disrupting the equilibrium between G-actin-TMSB10 and G-actin-ATP interactions in A549 cells. ConclusionOur results reveal that UA is potential for NSCLC therapeutics through reducing the protein level of TMSB10 to deformation the F-actin.

SLC12A8 promotes the migration and invasion of non-small cell lung cancer (NSCLC) cells.

This study aims to investigate the impacts of SLC12A8 on the invasion, migration, and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells. GEPIA database was employed to examine SLC12A8 expression pattern in lung cancer cells. Subsequently, qRT-PCR and Western blot analyses were conducted to assess SLC12A8 expression in NSCLC tissues and cell lines. The overall prognosis of NSCLC patients was evaluated using Kaplan-Meier plot and univariate and multivariate COX regression curves. The knockdown of SLC12A8 was established using lentivirus-mediated shRNA in A549 and H1299 cells. Cell proliferation, invasion, migration, and apoptosis were evaluated using CCK-8 assay, transwell, and flow cytometry techniques, respectively. Western blot analysis was performed to measure the expression levels of EMT-related proteins (E-cadherin and vimentin). The expression level of SLC12A8 was found to be significantly higher in both NSCLC cell lines and tissues. High SLC12A8 expression was correlated with a poor prognosis in NSCLC patients. Knocking down SLC12A8 led to a significant decrease in proliferation, migration, and invasion abilities, while promoting apoptosis in NSCLC cells. Additionally, SLC12A8 knockdown resulted in decreased levels of N-cadherin and vimentin, along with increased E-cadherin expression. The results indicate that reducing SLC12A8 expression may suppress the malignant phenotype of NSCLC cells, as well as the EMT. SLC12A8 may serve as a target for the clinical management of NSCLC progression.

RILPL2 as a potential biomarker for predicting enhanced T cell infiltration in non-small cell lung cancer

Our previous bioinformatics analysis has revealed that Rab-interacting lysosomal protein-like 2 (RILPL2) is associated with tumor immune microenvironment in non-small cell lung cancer (NSCLC). In our study, we collected 140 patients with primary NSCLC to verify the RILPL2 expression and its prognostic value, the relationship between RILPL2 expression and CD4+, CD8+T cell infiltration. A total of 140 patients who had been diagnosed with primary NSCLC (including 66 lung adenocarcinomas and 74 lung squamous cell carcinomas) were enrolled in our study. Immunohistochemical (IHC) staining was performed to analyze the expression of RILPL2, CD4, and CD8 in these patients. Compared with peri-cancer tissues, the RILPL2 expression in NSCLC tissues was significantly lower (P < 0.0001). RILPL2 expression was significantly related to clinical stage (P = 0.019), and low RILPL2 expression indicated higher stage. Low RILPL2 expression predicted worse overall survival (OS) in NSCLC patients (P = 0.017). Correlational analyses revealed that RILPL2 expression was significantly positively correlated with CD4+T cell infiltration in NSCLC (R = 0.294, P < 0.001), LUAD subgroup (R = 0.256, P = 0.038), and LUSC subgroup (R = 0.333, P = 0.004); RILPL2 expression was also significantly positively correlated with CD8+ T cell infiltration in NSCLC (R = 0.263, P = 0.002), LUAD subgroup (R = 0.280, P = 0.023), and LUSC subgroup (R = 0.250, P = 0.031). In conclusion, RILPL2 expression was downregulated in NSCLC; low RILPL2 expression was significantly related to higher stage and worse prognosis; RILPL2 expression was significantly positively correlated with CD4+, CD8+T cell infiltration.

HTR3A Promotes Non-small Cell Lung Cancer Through the FOXH1/Wnt3A Signaling Pathway.

5-Hydroxytryptamine receptors (5-HTRs) are strongly correlated with tumor progression in various types of cancer. Despite this, the underlying mechanisms responsible for the role of 5-HTRs in non-small cell lung cancer (NSCLC) remains unclear. This study aimed to investigate the relationship between 5-hydroxytryptamine receptor 3A (HTR3A) and NSCLC development. Our findings indicated a higher distribution of HTR3A expression in NSCLC tissues when compared with normal tissues, where patients with high HTR3A levels demonstrated shorter overall survival times. In vitro analyses revealed that overexpression of HTR3A facilitated the proliferation and migration of NSCLC cell lines (A549 and NCI-H3255). Similarly, a notable acceleration of tumor growth and enhanced pulmonary tumorigenic potential were observed in HTR3A-overexpressing tumor-bearing mice. Mechanistically, upregulation of Forkhead Box H1 (FOXH1) by HTR3A led to the activation of Wnt3A/β-catenin signaling pathways, thereby promoting the development of NSCLC. Our report thus highlights the significance of the HTR3A/FOXH1 axis during tumor progression in NSCLC, proposing HTR3A as a possible diagnostic indicator and candidate target for clinical therapy.

Remodelin delays non-small cell lung cancer progression by inhibiting NAT10 via the EMT pathway.

Lung cancer remains the foremost reason of cancer-related mortality, with invasion and metastasis profoundly influencing patient prognosis. N-acetyltransferase 10 (NAT10) catalyzes the exclusive N (4)-acetylcytidine (ac4C) modification in eukaryotic RNA. NAT10 dysregulation is linked to various diseases, yet its role in non-small cell lung cancer (NSCLC) invasion and metastasis remains unclear. Our study delves into the clinical significance and functional aspects of NAT10 in NSCLC. We investigated NAT10's clinical relevance using The Cancer Genome Atlas (TCGA) and a group of 98 NSCLC patients. Employing WB, qRT-PCR, and IHC analyses, we assessed NAT10 expression in NSCLC tissues, bronchial epithelial cells (BECs), NSCLC cell lines, and mouse xenografts. Further, knockdown and overexpression techniques (siRNA, shRNA, and plasmid) were employed to evaluate NAT10's effects. A series of assays were carried out, including CCK-8, colony formation, wound healing, and transwell assays, to elucidate NAT10's role in proliferation, invasion, and metastasis. Additionally, we utilized lung cancer patient-derived 3D organoids, mouse xenograft models, and Remodelin (NAT10 inhibitor) to corroborate these findings. Our investigations revealed high NAT10 expression in NSCLC tissues, cell lines and mouse xenograft models. High NAT10 level correlated with advanced T stage, lymph node metastasis and poor overall survive. NAT10 knockdown curtailed proliferation, invasion, and migration, whereas NAT10 overexpression yielded contrary effects. Furthermore, diminished NAT10 levels correlated with increased E-cadherin level whereas decreased N-cadherin and vimentin expressions, while heightened NAT10 expression displayed contrasting results. Notably, Remodelin efficiently attenuated NSCLC proliferation, invasion, and migration by inhibiting NAT10 through the epithelial-mesenchymal transition (EMT) pathway. Our data underscore NAT10 as a potential therapeutic target for NSCLC, presenting avenues for targeted intervention against lung cancer through NAT10 inhibition.

Expression and potential molecular mechanism of TOP2A in metastasis of non-small cell lung cancer

DNA topoisomerase II alpha (TOP2A) expression, gene alterations, and enzyme activity have been studied in various malignant tumors. Abnormal elevation of TOP2A expression is considered to be related to the development of non-small cell lung cancer (NSCLC). However, its association with tumor metastasis and its mode of action remains unclear. Bioinformatics, real-time quantitative PCR, immunohistochemistry and immunoblotting were used to detect TOP2A expression in NSCLC tissues and cells. Cell migration and invasion assays as well as cytoskeletal staining were performed to analyze the effects of TOP2A on the motility, migration and invasion ability of NSCLC cells. Cell cycle and apoptosis assays were used to verify the effects of TOP2A on apoptosis as well as cycle distribution in NSCLC. TOP2A expression was considerably upregulated in NSCLC and significantly correlated with tumor metastasis and the occurrence of epithelial–mesenchymal transition (EMT) in NSCLC. Additionally, by interacting with the classical ligand Wnt3a, TOP2A may trigger the canonical Wnt signaling pathway in NSCLC. These observations suggest that TOP2A promotes EMT in NSCLC by activating the Wnt/β-catenin signaling pathway and positively regulates malignant events in NSCLC, in addition to its significant association with tumor metastasis. TOP2A promotes the metastasis of NSCLC by stimulating the canonical Wnt signaling pathway and inducing EMT. This study further elucidates the mechanism of action of TOP2A, suggesting that it might be a potential therapeutic target for anti-metastatic therapy.

P300 reduces TUBB4B expression to facilitate the biological process of migration and invasion of non-small cell lung cancer cells

This article explored the mechanism of E1A binding protein p300 (P300) and beta-tubulin 4B isotype-encoding gene (TUBB4B) in regulating the migration and invasion of non-small cell lung cancer (NSCLC) cells. TUBB4B and P300 expression in NSCLC tissues and cells was monitored by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. TUBB4B function on NSCLC cell migration, invasion and epithelial-mesenchymal transition (EMT) was monitored by wound healing assay, Transwell experiment and Western blot. The regulation of P300 on TUBB4B was monitored by qRT-PCR and Western blot. Mechanism of P300 and TUBB4B in regulating NSCLC cell migration and invasion was explored by rescue experiment. A xenograft tumor model was established by using nude mouse. As a result, low TUBB4B expression and high P300 expression was discovered in NSCLC tissues and cells. TUBB4B and P300 expression showed a negative correlation in NSCLC tissues. Lower TUBB4B but higher P300 was observed in tumor tissues of NSCLC patients with metastasis. TUBB4B overexpression suppressed NSCLC cell migration, invasion and EMT. TUBB4B silencing had opposite results. P300 overexpression inhibited TUBB4B expression, and P300 silencing facilitated TUBB4B overexpression in NSCLC cells. TUBB4B overexpression counteracted the promotion of P300 overexpression on NSCLC cell invasion and migration. TUBB4B silencing abrogated the inhibition of P300 knockdown on NSCLC cell invasion and migration. TUBB4B overexpression suppressed NSCLC cell in vivo growth. Thus, TUBB4B could be reduced by P300 in NSCLC. It exerted suppression role on NSCLC cell migration, invasion and EMT. TUBB4B may be a novel target for NSCLC treatment.

Knockdown of NCAPD3 inhibits the tumorigenesis of non-small cell lung cancer by regulation of the PI3K/Akt pathway

BackgroundAccumulating evidence indicates that aberrant non-SMC condensin II complex subunit D3 (NCAPD3) is associated with carcinogenesis of various cancers. Nevertheless, the biological role of NCAPD3 in the pathogenesis of non-small cell lung cancer (NSCLC) remains unclear.MethodsImmunohistochemistry and Western blot were performed to assess NCAPD3 expression in NSCLC tissues and cell lines. The ability of cell proliferation, invasion, and migration was evaluated by CCK-8 assays, EdU assays, Transwell assays, and scratch wound healing assays. Flow cytometry was performed to verify the cell cycle and apoptosis. RNA-sequence and rescue experiment were performed to reveal the underlying mechanisms.ResultsThe results showed that the expression of NCAPD3 was significantly elevated in NSCLC tissues. High NCAPD3 expression in NSCLC patients was substantially associated with a worse prognosis. Functionally, knockdown of NCAPD3 resulted in cell apoptosis and cell cycle arrest in NSCLC cells as well as a significant inhibition of proliferation, invasion, and migration. Furthermore, RNA-sequencing analysis suggested that NCAPD3 contributes to NSCLC carcinogenesis by regulating PI3K/Akt/FOXO4 pathway. Insulin-like growth factors-1 (IGF-1), an activator of PI3K/Akt signaling pathway, could reverse NCAPD3 silence-mediated proliferation inhibition and apoptosis in NSCLC cells.ConclusionNCAPD3 suppresses apoptosis and promotes cell proliferation via the PI3K/Akt/FOXO4 signaling pathway, suggesting a potential use for NCAPD3 inhibitors as NSCLC therapeutics.

Suppression of cyclin-dependent kinase 1 synergizes with checkpoint kinase 2 inhibitor to promote death of non-small cell lung cancer cells with radiotherapeutic resistance

ObjectiveRadiotherapy resistance poses a prevalent clinical challenge in non-small cell lung cancer (NSCLC), necessitating the development of ongoing treatment protocols following the emergence of radiotherapy resistance, as well as the identification of radiotherapy sensitization drugs. The objective of this study was to exclusively investigate therapeutic targets associated with radiotherapy resistance in NSCLC, thereby expanding the range of treatment alternatives available to patients grappling with radiotherapy resistance. MethodsVarious databases were employed to screen for therapeutic targets associated with radiotherapeutic resistance. The impact of administering inhibitors for cyclin-dependent kinase 1 (CDK1) and checkpoint kinase 2 (CHEK2) on cell proliferation and apoptosis in radioresistant NSCLC cell lines was assessed. ResultsThe potential of CDK1 and CHEK2 kinase as a novel biomarker and therapeutic target in radioresistant NSCLC cells is evident. The up-regulation of CDK1 and CHEK2 expression in NSCLC tissues, as well as their activation in NSCLC cells exposed to X-irradiation, leads to radiotherapeutic resistance. Additionally, the heightened expression of CDK1 and CHEK2 may contribute to an increased score of cancer stem cells (CSCs) in NSCLC patients. The suppression of CDK1 in combination with a CHEK2 inhibitor synergistically promotes the death of NSCLC cells with radiotherapeutic resistance. Notably, the sensitivity of radioresistant NSCLC cell lines to the CHEK2 inhibitor (PV-1019) is enhanced through the pharmacological or genetic inhibition of CDK1. ConclusionsThe current study provides new perspectives on the synergistic effects of CDK1 and CHEK2 inhibitors in inducing apoptosis in NSCLC cells that have acquired resistance to radiotherapy.

METTL3-mediated the m6A modification of SF3B4 facilitates the development of non-small cell lung cancer by enhancing LSM4 expression.

Splicing factor B subunit 4 (SF3B4) has been confirmed to participate in the progression of many cancers and is considered to be a potential target for non-small cell lung cancer (NSCLC). Thus, the role and molecular mechanism of SF3B4 in NSCLC progression deserves further study. Quantitative real-time PCR and western blot were employed to detect the mRNA and protein levels of SF3B4, Sm-like protein 4 (LSM4) and methyltransferase-like 3 (METTL3). Cell proliferation, apoptosis, invasion, migration and stemness were tested by cell counting kit-8, colony formation, flow cytometry, transwell, wound healing, and sphere formation assays. The interaction between SF3B4 and METTL3 or LSM4 was confirmed by MeRIP, RIP and Co-IP assays. Mice xenograft models were constructed to assess the effects of METTL3 and SF3B4 on NSCLC tumorigenesis. SF3B4 had high expression in NSCLC tissues and was associated with the shorter overall survival of NSCLC patients. Knockdown of SF3B4 suppressed NSCLC cell proliferation, invasion, migration and stemness, while inducing apoptosis. METTL3 promoted SF3B4 mRNA stability by m6A modification, and its knockdown inhibited NSCLC cell growth, metastasis and stemness by downregulating SF3B4. SF3B4 could interact with LSM4, and sh-SF3B4-mediated the inhibition on NSCLC cell functions could be reversed by LSM4 overexpression. In addition, reduced METTL3 expression restrained NSCLC tumor growth, and this effect was reversed by SF3B4 overexpression. METTL3-stablized SF3B4 promoted NSCLC cell growth, metastasis and stemness via positively regulating LSM4.

The SOX2/PDIA6 axis mediates aerobic glycolysis to promote stemness in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is an aggressive and rapidly expanding lung cancer. Abnormal upregulation or knockdown of PDIA6 expression can predict poor prognosis in various cancers. This study aimed to investigate the biological function of PDIA6 in NSCLC. SOX2 and PDIA6 expression in NSCLC tissues and regulatory relationship between them were analyzed using bioinformatics. GSEA was performed on the enrichment pathway of PDIA6. qRT-PCR was utilized to examine expression of SOX2 and PDIA6 in NSCLC tissues and cells, and dual-luciferase reporter assay and ChIP experiments were performed to validate their regulatory relationship. CCK-8 experiment was conducted to assess cell viability, western blot was to examine levels of stem cell markers and proteins related to aerobic glycolysis pathway in cells. Cell sphere formation assay was used to evaluate efficiency of cell sphere formation. Reagent kits were used to measure glycolysis levels and glycolysis products. High expression of PDIA6 in NSCLC was linked to aerobic glycolysis. Knockdown of PDIA6 reduced cell viability, expression of stem cell surface markers, and cell sphere formation efficiency in NSCLC. Overexpression of PDIA6 could enhance cell viability and promote aerobic glycolysis, but the addition of 2-DG could reverse this result. Bioinformatics predicted the existence of upstream transcription factor SOX2 for PDIA6, and SOX2 was significantly upregulated in NSCLC, and they had a binding relationship. Further experiments revealed that PDIA6 overexpression restored repressive effect of knocking down SOX2 on aerobic glycolysis and cell stemness. This work revealed that the SOX2/PDIA6 axis mediated aerobic glycolysis to promote NSCLC cell stemness, providing new therapeutic strategies for NSCLC.

Circ_0082374 Promotes the Tumorigenesis and Suppresses Ferroptosis in Non-small Cell Lung Cancer by Up-Regulating GPX4 Through Sequestering miR-491-5p.

Circular RNAs (circRNAs) have been identified to be dysregulated in non-small cell lung cancer (NSCLC) and implicated in the progression of this cancer. Here, this work aimed to investigate the role and mechanism of circ_0082374 on NSCLC progression. Levels of circ_0082374, miR-491-5p, GPX4 (glutathione peroxidase 4) and epithelial-mesenchymal transition (EMT)-related proteins were examined by quantitative real-time PCR or western blotting, respectively. Cell proliferation and metastasis were detected using cell counting kit-8, colony formation, EdU, transwell, and Scratch assays. Cell ferroptosis was evaluated by measuring cell survival after the treatment of different ferroptosis inducers or inhibitors, as well as the accumulation of intracellular reactive oxygen species (ROS), ferrous iron (Fe2+) and malondialdehyde (MDA). The binding between miR-491-5p and circ_0082374 or GPX4 was confirmed using dual-luciferase reporter and RNA pull-down assays. In vivo experiments were conducted using murine xenograft assay and immunohistochemistry. Circ_0082374 was a stable circRNA with high expression in NSCLC tissues and cells. Functionally, circ_0082374 silencing suppressed NSCLC cell proliferation and metastasis. Moreover, its down-regulation enhanced ferroptosis by decreasing iron and lipid peroxidation accumulation. Mechanistically, circ_0082374 could indirectly up-regulate GPX4 expression via miR-491-5p, indicating the circ_0082374/miR-491-5p/GPX4 competitive endogenous RNAs (ceRNA) network. Rescue experiments demonstrated that the miR-491-5p/GPX4 axis mediated the regulatory effects of circ_0082374 exerted on NSCLC cells. Moreover, knockdown of circ_0082374 impeded NSCLC growth and EMT via regulating miR-491-5p and GPX4. Circ_0082374 silencing could suppress NSCLC cell proliferation, metastasis and induce ferroptosis through miR-491-5p/GPX4 axis, suggesting a novel therapeutic approach for NSCLC patients.

Kinetochore scaffold 1 downregulation suppressed the development of non-small cell lung cancer by inactivating the phosphatidylinositol 3 kinase/protein kinase B (AKT)/nuclear factor-kappa B pathway.

Kinetochore scaffold 1 (KNL1) is indispensable for generating motile micro-tubule attachments and isolating chromosomes. KNL1 is highly expressed in multiple middle-route tissues and promotes tumor development. However, how it functions in non-small cell lung cancer (NSCLC) is unclear. Real-time quantitative PCR (RT-qPCR) and Western blotting (WB) were used to determine KNL1 expression in NSCLC tissues and cells. The sh-KNL1 or oe-KNL1 was transfected into NSCLC cells. The colony formation assay, cell counting kit-8 (CCK-8) assay, and flow cytometry were used to evaluate cell proliferation and apoptosis. A transwell assay was used to monitor invasion and migration. The CCK-8 assay was used to measure NSCLC cell sensitivity to chemotherapy drugs. WB confirmed the protein levels of apoptosis-related proteins, cell cycle-associated proteins, and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappaB (NF-κB) pathway. A PI3K/AKT/NF-κB pathway inhibitor was used to intervene in NSCLC cell transfection along with oe-KNL1, thus revealing the function of the pathway in carcinogenicity mediated by KNL1. In result KNL1 expression was substantially increased in NSCLC tissues and cells. High-level KNL1 expression is related to the poor prognosis of NSCLC patients. KNL1 silencing bolstered promoted NSCLC cell apoptosis and inhibited proliferation, cell cycle progression, invasion, and EMT, whereas KNL1 silencing had the opposite effect. KNL1 knockdown increased NSCLC cell sensitivity to chemical drugs. KNL1 promoted PI3K/AKT/NF-κB pathway activation, while PI3K/AKT/NF-κB pathway inhibition weakened the procancer effect mediated by KNL1 overexpression but had little influence on KNL1 levels. We conclude that KNL1 activates the PI3K/AKT/NF-κB pathway to increase NSCLC progression and attenuate NSCLC sensitivity to chemotherapy drugs.

Promotion of non-small cell lung cancer tumor growth by FHL2 via inducing angiogenesis and vascular permeability.

Antiangiogenetic therapy is one of the effective strategies for non-small cell lung cancer (NSCLC) treatment. Four-and-a-half LIM-domain protein 2 (FHL2) serves as a key function in cell growth and metastasis of multiple cancers, but the role of FHL2 in NSCLC angiogenesis has not been intensely examined. FHL2 expression in NSCLC tissues and cell lines and its correlation with patients prognosis were investigated by using The Cancer Genome Atlas (TCGA) database and quantitative polymerase chain reaction (qPCR). Cell Counting Kit-8 (CCK-8) assay, EdU (5-ethynyl-2'-deoxyuridine) assay, and a xenograft model were used to investigate the effects of FHL2 on NSCLC progression in vitro and in vivo. CCK-8, wound-healing, Transwell invasion, tube formation, and permeability assays were performed to determine the roles of FHL2 in angiogenesis and vascular permeability. Vascular endothelial growth factor A (VEGFA) enzyme-linked immunosorbent assay (ELISA) assay, Western blot analysis, and MK-2206 were used to investigate the specific mechanism mediated by FHL2. We demonstrated that FHL2 was significantly upregulated in NSCLC tissues and cell lines and was associated with poor prognosis. FHL2 overexpression enhanced the cell viability of NSCLC cells, as well as the proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). In addition, we determined that FHL2 activated the AKT-mTOR signaling pathway in HUVECs by promoting VEGFA secretion from NSCLC cells, thereby inducing angiogenesis and vascular leakiness. We further confirmed that FHL2 also promoted NSCLC tumor growth in vivo. Our study revealed the role of FHL2 in NSCLC and the mechanism by which FHL2 promotes NSCLC tumorigenesis, providing novel insights into targeted therapy for NSCLC.

FUS-stabilized USP35 promotes growth, invasion and angiogenesis in NSCLC through deubiquitinating VEGFA.

Vascular endothelial growth factor A (VEGFA) is an important regulator for non-small cell lung cancer (NSCLC). Our study aimed to reveal its upstream pathway to provide new ideas for developing the therapeutic targets of NSCLC. The mRNA and protein levels of VEGFA, ubiquitin-specific peptidase 35 (USP35), and FUS were determined by quantitative real-time PCR and Western blot. Cell proliferation, apoptosis, invasion and angiogenesis were detected using CCK8 assay, EdU assay, flow cytometry, transwell assay and tube formation assay. The interaction between USP35 and VEGFA was assessed by Co-IP assay and ubiquitination assay. Animal experiments were performed to assess USP35 and VEGFA roles in vivo. VEGFA had elevated expression in NSCLC tissues and cells. Interferences of VEGFA inhibited NSCLC cell proliferation, invasion, angiogenesis, and increased apoptosis. USP35 could stabilize VEGFA protein level by deubiquitination, and USP35 knockdown suppressed NSCLC cell growth, invasion and angiogenesis via reducing VEGFA expression. FUS interacted with USP35 to promote its mRNA stability, thereby positively regulating VEGFA expression. Also, USP35 silencing could reduce NSCLC tumorigenesis by downregulating VEGFA. FUS-stabilized USP35 facilitated NSCLC cell growth, invasion and angiogenesis through deubiquitinating VEGFA, providing a novel idea for NSCLC treatment.

Expression of RSK4 protein in non-small cell lung cancer tissues, adjacent tissues and its correlation with clinicopathological features.

To evaluate the expression of Ribosomal S6 kinase 4 (RSK4) protein in non-small cell lung cancer (NSCLC) tissues and adjacent non-tumor tissues, and to elucidate its correlation with clinicopathological features of NSCLC. We analyzed 100 NSCLC patients treated at the Second Affiliated Hospital of Zhejiang University School of Medicine from June 2020 to June 2022. Patient demographics and clinical data, including gender, age, history of diabetes, tumor location, degree of tumor differentiation, lymph node metastasis, and clinical stage, were collected. RSK4 protein expression was assessed in tissue samples via immunohistochemical staining. RSK4 protein was positively expressed in 35.00% of cancerous tissues, significantly lower than the 69.00% observed in adjacent non-tumor tissues (P < 0.05). Patients with lower tumor differentiation, advanced Tumor Node Metastasis (TNM) stages, and lymph node metastases showed significantly reduced RSK4 expression compared to those with higher differentiation, earlier TNM stages, and no lymph node metastases (all P < 0.05). Cox regression analysis indicated that TNM stage, low differentiation, and lymph node metastases significantly influenced RSK4 expression (all P < 0.05). Survival analysis revealed a higher positive prognosis survival rate of 74.29% (26/35) among patients with positive RSK4 expression, versus 53.85% (35/65) in those with negative expression (P < 0.05). Spearman correlation analysis demonstrated a significant positive correlation of RSK4 expression with TNM stage, lymph node metastasis, and patient prognosis (all P < 0.05). Positive RSK4 expression in NSCLC tissues is significantly correlated with advanced cancer stage, poor differentiation, and presence of lymph node metastasis, suggesting a potential tumor suppressor role for RSK4 in NSCLC. This association underscores its prognostic relevance in NSCLC patients.