Non-small Cell Lung Cancer Tissues Research Articles

Novel circular RNA hsa_circ_0036683 suppresses proliferation and migration by mediating the miR-4664-3p/CDK2AP2 axis in non-small cell lung cancer.

The aim of the present study was to investigate the function of novel circular RNA hsa_circ_0036683 (circ-36683) in non-small cell lung cancer (NSCLC). RNA sequencing was used to screen out differentially expressed miRNAs. Expression levels of miR-4664-3p and circ-36683 were evaluated in lung carcinoma cells and tissues by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The effects of miR-4664-3p and circ-36683 on proliferation and migration were assessed using cell counting kit-8 (CCK-8), wound healing and transwell migration assays and xenograft experiments. The targeting relationship of circ-36683/miR-4664-3p/CDK2AP2 was assessed by luciferase reporter assays, western blot, qRT-PCR and argonaute2-RNA immunoprecipitation (AGO2 RIP). Co-immunoprecipitation (Co-IP), 5-ethynyl-2'-deoxyuridine (EdU) staining and CCK-8 were used to validate the indispensable role of CDK2AP2 in suppressing cell proliferation as a result of CDK2AP1 overexpression. By RNA sequencing, miR-4664-3p was screened out as an abnormally elevated miRNA in NSCLC tissues. Transfection of miR-4664-3p could promote cell proliferation, migration and xenograft tumor growth. As a target of miR-4664-3p, CDK2AP2 expression was downregulated by miR-4664-3p transfection and CDK2AP2 overexpression could abolish the proliferation promotion resulting from miR-4664-3p elevation. Circ-36683, derived from back splicing of ABHD2 pre-mRNA, was attenuated in NSCLC tissue and identified as a sponge of miR-4664-3p. The functional study revealed that circ-36683 overexpression suppressed cell proliferation, migration and resulted in G0/G1 phase arrest. More importantly, the antioncogenic function of circ-36683 was largely dependent on the miR-4664-3p/CDK2AP2 axis, through which circ-36683 could upregulate the expression of p53/p21/p27 and downregulate the expression of CDK2/cyclin E1. The present study revealed the antioncogenic role of circ-36683 in suppressing cell proliferation and migration and highlighted that targeting the circ-36683/miR-4664-3p/CDK2AP2 axis is a promising strategy for the intervention of NSCLC.

Identification of Key Genes and Signaling Pathways in Entrectinibresistant Non-small Cell Lung Cancer Using Bioinformatic Analysis and Experimental Verification.

Entrectinib, a ROS1 inhibitor, is effective in patients with ROS1-positive non-small-cell lung cancer (NSCLC). However, entrectinib resistance remains a challenge worldwide. The biomarkers of entrectinib resistance and molecular mechanisms have not been clarified based on the Gene Expression Omnibus (GEO) database. The aim of this study is to identify key genes and signaling pathways involved in the development of entrectinib-resistant NSCLC through bioinformatics analysis and experimental validation. Differentially expressed genes (DEGs) were screened between entrectinib resistant and parental human NSCLC cell lines of the GSE214715 dataset, lung adenocarcinoma (LUAD) and non-tumor adjacent tissues of the GSE75037 dataset, and NSCLC and non-tumor adjacent tissues of the GSE18842 dataset. Functional enrichment analyses were performed, including Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Overlapped DEGs among those three datasets were identified using the Venn diagram package. The transcriptional levels of key genes were investigated using the University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). The association between transcriptional levels of key genes and survival was analyzed using Kaplan-Meier Plotter (https://www.kmplot.com/analysis/). The correlations between hub genes and immune cell infiltration were investigated using the Tumor Immune Estimation Resource (TIMER) database. Specific signaling pathway enrichment analysis was performed using Gene Set Enrichment Analysis (GSEA) of LinkedOmics. Competitive endogenous RNA (ceRNA) networks, genome-wide association studies (GWAS), and drug sensitivity analyses of key genes were further investigated. The expression of ZEB2 was subsequently confirmed in both parental HCC78 cells and entrectinib-resistant HCC78 cells using real-time quantitative polymerase chain reaction (qRT-PCR). 708 DEGs were identified between entrectinib-resistant CUTO28 (CUTO28-ER) and parental CUTO28 cell lines in the GSE214715 dataset. One thousand three hundred and ninety-five DEGs were identified between entrectinib resistant (CUTO37-ER) and parental CUTO37 cell lines in the GSE214715 dataset. Eight hundred and forty-nine DEGs were identified between LUAD and non-tumor adjacent tissues in the GSE75037 dataset. Seven hundred and sevety-three DEGs were identified between NSCLC and non-tumor adjacent tissues in the GSE18842 dataset. Among these three datasets, seven overlapped DEGs were identified, including ZBED2, CHI3L2, CELF2, SEMA5A, ZEB2, S100A12, and PDK4. Among these seven overlapped DEGs, the expression levels of CHI3L2, ZEB2, and S100A12 were downregulated in those three datasets. The results of analysis using the UALCAN database showed that these three genes were significantly downregulated in LUAD and LUSC patients compared with the normal population. However, only the lower transcriptional level of ZEB2 was linked to worse survival in patients with lung cancer. GSEA analysis revealed that ZEB2 was significantly negatively correlated with nucleotide excision repair (NER) in LUAD, and homologous recombination (HR) and NER in LUSC, which were linked to drug resistance. A ceRNA network of THRB-AS1/ has-miR-1293/ ZEB2 in LUAD was established. We have identified core genes associated with non-small cell resistance to entrectinib, including CHI3L2, ZEB2, and S100A12. ZEB2 is a core gene associated with acquired resistance to entetinib in NSCLC.

METTL14-mediated Bim mRNA m6A modification augments osimertinib sensitivity in EGFR-mutant NSCLC cells.

Resistance to osimertinib represents a significant challenge for the successful treatment of non-small cell lung cancer (NSCLC) harboring activating mutations in epidermal growth factor receptor (EGFR). N6-methyladenosine (m6A) on mRNAs is critical for various biological processes, yet whether m6A regulates osimertinib resistance of NSCLC remains unknown. In this study, we demonstrated that developing osimertinib-resistant phenotypes depends on m6A reduction resulting from downexpression of m6A methyltransferase METTL14 in EGFR-mutant NSCLCs. Both in vitro and in vivo assay showed that specific knockdown of METTL14 was sufficient to confer osimertinib resistance and elevated expression of METTL14 rescued the efficacy of osimertinib in the resistant NSCLC cells. Mechanistically, METTL14 promoted m6A methylation of pro-apoptotic Bim mRNA and increased Bim mRNA stability and expression, resulting in activating the Bim-dependent pro-apoptotic signaling and thereby promoting osimertinib-induced cell apoptosis. Analysis of clinical samples revealed that decreased expression of METTL14 was observed in osimertinib-resistant NSCLC tissues and significantly associated with a poor prognosis. In conclusion, our study reveals a novel regulatory mechanism by which METTL14-mediated m6A methylation of Bim mRNA inhibited osimertinib resistance of NSCLC cells. It offers more evidences for the involvement of m6A modification in regulation of osimertinib resistance, and provides potential therapeutic targets for novel approaches to overcome the tolerance of osimertinib and other EGFR-TKIs. Implications: This study offers more evidences for the involvement of METTL14-mediated m6A modification in regulation of osimertinib resistance, and provides potential therapeutic targets for novel approaches to overcome the tolerance of osimertinib and other EGFR-TKIs.

RILPL2 as a potential biomarker for predicting enhanced Tcell 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.

CTSG may inhibit disease progression in HIV-related lung cancer patients by affecting immunosuppression

ObjectivesLung cancer is an independent risk factor for pulmonary complications following HIV infection. This study aimed to examine the expression and clinical significance of Cathepsin G (CTSG) protein in both non-HIV and HIV-related lung cancers.MethodsThe data related to lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) in the TCGA dataset and the data related to healthy individuals in the GTEx dataset, the GEPIA2 database was used to excavate the distinction in the expression of CTSG protein in non-small cell lung cancer (NSCLC) tissues versus normal non-cancerous tissues. The Ualcan database was used to compare the differences in CTSG expression at different stages of LUAD and LUSC. Immunohistochemistry (IHC) was used to detect the expression of CTSG proteins in the pathological tissues of patients with HIV-related lung cancer and patients with lung cancer without co-infection, the Kaplan-Meier method was used for survival analysis.ResultsWe observed that CTSG expression in NSCLC is lower compared to adjacent non-tumor tissues and correlates with NSCLC clinical stage. CTSG protein expression in HIV-related lung cancer tissues was lower than in adjacent tissues and lower than in lung cancer tissues without HIV infection, with a statistically significant difference (P < 0.05). It correlated with CD4 + T cell count and CD4+/CD8 + T cell ratio, as well as with the pathological type, distant metastasis, and clinical stage of HIV-related lung cancer, all with statistical significance (P < 0.05).ConclusionsCTSG could potentially mitigate disease advancement in HIV-related lung cancer patients by inhibiting immune depletion, serving as a prospective immunotherapeutic target for both non-HIV and HIV-associated lung cancers.

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.

Bone marrow mesenchymal stem cells-derived exosomal miR-145-5p reduced non-small cell lung cancer cell progression by targeting SOX9

BackgroundThe role of miR-145-5p in non-small cell lung cancer (NSCLC) has been studied, however, the regulation of hBMSCs-derived exosomes (Exo) transmitted miR-145-5p in NSCLC was still unknown. This study aimed to investigate the role of hBMSCs-derived exosomes (Exo) in the progression of NSCLC.MethodsThe Exo was extracted from hBMSCs and added to A549 and H1299 cell culture, followed by the detection of cell proliferation, migration, and invasion. The correlation between the expression of miR-145-5p and SOX9, as well as their binding relationship was determined by correlation analysis, luciferase gene reporter assay and RNA pull-down assays. The in vivo animal model was established to further verify the impact of hBMSCs-Exo.ResultsIt showed that miR-145-5p was downregulated and SOX9 was upregulated in NSCLC tissues. HBMSCs-derived Exo, and hBMSCs-Exo with overexpression of miR-145-5p could inhibit cell proliferation, migration, and invasion of both A549 and H1299 cells, and prevent against tumor progression in vivo. MiR-145-5p and SOX9 were found to be able to bind to each other, and a negative correlation were observed between the expression of them in NSCLC tissues. Furthermore, inhibition of SOX9 could reversed the suppressed role of miR-145-5p in vitro and in vivo.ConclusionTherefore, HBMSCs-Exo effectively transmitted miR-145-5p, leading to the suppression of malignant development in NSCLC through the regulation of SOX9.

Hypoxia-enhanced YAP1-EIF4A3 interaction drives circ_0007386 circularization by competing with CRIM1 pre-mRNA linear splicing and promotes non-small cell lung cancer progression

BackgroundThe progression of non-small cell lung cancer (NSCLC) is significantly influenced by circular RNAs (circRNAs), especially in tumor hypoxia microenvironment. However, the precise functions and underlying mechanisms of dysregulated circRNAs in NSCLC remain largely unexplored.MethodsDifferentially expressed circRNAs in NSCLC tissues were identified through high-throughput RNA sequencing. The characteristics of circ_0007386 were rigorously confirmed via Sanger sequencing, RNase R treatment and actinomycin D treatment. The effects of circ_0007386 on proliferation and apoptosis were investigated using CCK8, cloning formation assays, TUNEL staining, and flow cytometry assays in vitro. In vivo, xenograft tumor models were used to evaluate its impact on proliferation. Mechanistically, the regulatory relationships of circ_0007386, miR-383-5p and CIRBP were examined through dual luciferase reporter assays and rescue experiments. Additionally, we detected the binding of EIF4A3 to CRIM1 pre-mRNA by RNA immunoprecipitation and the interaction between YAP1 and EIF4A3 under hypoxic conditions by co-immunoprecipitation.ResultsOur investigation revealed a novel circRNA, designated as circ_0007386, that was upregulated in NSCLC tissues and cell lines. Circ_0007386 modulated proliferation and apoptosis in NSCLC both in vitro and in vivo. Functionally, circ_0007386 acted as a sponge for miR-383-5p, targeting CIRBP, which influenced NSCLC cell proliferation and apoptosis via the PI3K/AKT signaling pathway. Furthermore, under hypoxic conditions, the interaction between YAP1 and EIF4A3 was enhanced, leading to the displacement of EIF4A4 from binding to CRIM1 pre-mRNA. This facilitated the back-splicing of CRIM1 pre-mRNA, increasing the formation of circ_0007386. The circ_0007386/miR-383-5p/CIRBP axis was significantly associated with the clinical features and prognosis of NSCLC patients.ConclusionsCirc_0007386, regulated by YAP1-EIF4A3 interaction under hypoxia conditions, plays an oncogenic role in NSCLC progression via the miR-383-5p/CIRBP axis.

USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway

The regulatory significance of ubiquitin-specific peptidase 32 (USP32) in tumor is significant, nevertheless, the biological roles and regulatory mechanisms of USP32 in non-small cell lung cancer (NSCLC) remain unclear. According to our research, USP32 was strongly expressed in NSCLC cell lines and tissues and was linked to a bad prognosis for NSCLC patients. Interference with USP32 resulted in a significant inhibition of NSCLC cell proliferation, migration potential, and EMT development; on the other hand, USP32 overexpression had the opposite effect. To further elucidate the mechanism of action of USP32 in NSCLC, we screened H1299 cells for interacting proteins and found that USP32 interacts with BAG3 (Bcl2-associated athanogene 3) and deubiquitinates and stabilizes BAG3 in a deubiquitinating activity-dependent manner. Functionally, restoration of BAG3 expression abrogated the antitumor effects of USP32 silencing. Furthermore, USP32 increased the phosphorylation level of the RAF/MEK/ERK signaling pathway in NSCLC cells by stabilizing BAG3. In summary, these findings imply that USP32 is critical to the development of NSCLC and could offer a theoretical framework for the clinical diagnosis and management of NSCLC patients in the future.

The Advantage of Targeted Next-Generation Sequencing over qPCR in Testing for Druggable EGFR Variants in Non-Small-Cell Lung Cancer.

The emergence of targeted therapies in non-small-cell lung cancer (NSCLC), including inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, has increased the need for robust companion diagnostic tests. Nowadays, detection of actionable variants in exons 18-21 of the EGFR gene by qPCR and direct DNA sequencing is often replaced by next-generation sequencing (NGS). In this study, we evaluated the diagnostic usefulness of targeted NGS for druggable EGFR variants testing in clinical NSCLC material previously analyzed by the IVD-certified qPCR test with respect to DNA reference material. We tested 59 NSCLC tissue and cytology specimens for EGFR variants using the NGS 'TruSight Tumor 15' assay (Illumina) and the qPCR 'cobas EGFR mutation test v2' (Roche Diagnostics). The sensitivity and specificity of targeted NGS assay were evaluated using the biosynthetic and biological DNA reference material with known allelic frequencies (VAF) of EGFR variants. NGS demonstrated a sufficient lower detection limit for diagnostic applications (VAF < 5%) in DNA reference material; all EGFR variants were correctly identified. NGS showed high repeatability of VAF assessment between runs (CV% from 0.02 to 3.98). In clinical material, the overall concordance between NGS and qPCR was 76.14% (Cohen's Kappa = 0.5933). The majority of discordant results concerned false-positive detection of EGFR exon 20 insertions by qPCR. A total of 9 out of 59 (15%) clinical samples showed discordant results for one or more EGFR variants in both assays. Additionally, we observed TP53 to be a frequently co-mutated gene in EGFR-positive NSCLC patients. In conclusion, targeted NGS showed a number of superior features over qPCR in EGFR variant detection (exact identification of variants, calculation of allelic frequency, high analytical sensitivity), which might enhance the basic diagnostic report.

METTL5: A Potential Biomarker for Nonsmall Cell Lung Cancer That Promotes Cancer Cell Proliferation by Interacting with IGF2BP3.

Objective: To examine if METTL5 promotes the proliferation of nonsmall cell lung cancer (NSCLC) cells by interacting with IGF2BP3. Methods: The expression patterns of METTL5 and IGF2BP3 in NSCLC tissues, their relationship with survival rate, and their correlation were analyzed using bioinformatics and clinical sample analyses. The effects of METTL5 overexpression and IGF2BP3 knockdown, as well as those of METTL5 knockdown and IGF2BP3 overexpression, on the proliferation of NSCLC cells were analyzed by transfecting appropriate constructs. The interaction between METTL5 and IGF2BP3 was verified using the co-immunoprecipitation (Co-IP) assay. The invivo effects of METTL5 and IGF2BP3 on NSCLC growth were analyzed using the tumor-bearing nude mouse model. Results: METTL5 and IGF2BP3 expression levels were positively correlated and were associated with poor clinical prognosis. The METTL5 and IGF2BP3 expression levels were upregulated in the clinical NSCLC samples. IGF2BP3 expression did not affect METTL5 expression but was regulated by METTL5. IGF2BP3 overexpression mitigated the METTL5 knockdown-induced impaired cell proliferation. Meanwhile, IGF2BP3 knockdown suppressed METTL5-mediated NSCLC cell proliferation. The Co-IP assay results revealed the interaction between METTL5 and IGF2BP3 in NSCLC cells. IGF2BP3 knockdown suppressed tumor growth, whereas IGF2BP3 overexpression enhanced tumor volume and quality. Conclusion: METTL5 induces NSCLC cell proliferation by interacting with IGF2BP3. Thus, METTL5 is a potential biomarker and a therapeutic target for NSCLC.

METTL14 plays an oncogenic role in NSCLC by modulating ferroptosis and the m6A modification of GPX4

Context N6-methyladenosine (m6A) of RNA is involved in the progression of non-small cell lung cancer (NSCLC). Objective This study investigated the role of METTL14 in NSCLC and the mechanism. Materials and methods Expression levels were assessed by quantitative real-time PCR and ELISA assays. Cells viability was assessed by cell counting kit-8. M6A methylation was analysed by methylated RNA immunoprecipitation (MeRIP), RIP, luciferase assay, and mRNA stability assay. Results The results showed that METTL14 was highly expressed in NSCLC tissues and cell lines. Knockdown of METTL14 inhibited the cell viability while induced ferroptosis of NSCLC cells. Mechanistically, METTL14 interacts with GPX4, mediates m6A modification of GPX4, enhances its mRNA stability, and upregulates its expression. In addition, IGF2BP1 recognises the m6A-methylated GPX4 and mediates the elevated mRNA stability. Moreover, GPX4 reversed the effects of METTL14 depletion. Discussion and conclusion The METTL14/GPX4 axis promotes NSCLC progression by inhibiting cell ferroptosis through the recognition of m6A modification mediated by IGF2BP1.

YTH domain family protein 3 accelerates non-small cell lung cancer immune evasion through targeting CD8+ T lymphocytes

Immune evasion is one of the critical hallmarks of malignant tumors, especially non-small cell lung cancer (NSCLC). Emerging findings have illustrated the roles of N6-methyladenosine (m6A) on NSCLC immune evasion. Here, this study investigated the function and underlying mechanism of m6A reader YTH domain family protein 3 (YTHDF3) on NSCLC immune evasion. YTHDF3 was found to be highly expressed in NSCLC tissue and act as an independent prognostic factor for overall survival. Functionally, up-regulation of YTHDF3 impaired the CD8+ T antitumor activity to deteriorate NSCLC immune evasion, while YTHDF3 silencing recovered the CD8+ T antitumor activity to inhibit immune evasion. Besides, YTHDF3 up-regulation reduced the apoptosis of NSCLC cells. Mechanistically, PD-L1 acted as the downstream target for YTHDF3, and YTHDF3 could upregulate the transcription stability of PD-L1 mRNA. Overall, YTHDF3 targeted PD-L1 to promote NSCLC immune evasion partially through escaping effector cell cytotoxicity CD8+ T mediated killing and antitumor immunity. In summary, this study provides an essential insight for m6A modification on CD8+ T cell-mediated antitumor immunity in NSCLC, which might inspire an innovation for lung cancer tumor immunotherapy.

UBQLN4 promotes the proliferation and invasion of non-small cell lung cancer cell by regulating PI3K/AKT pathway

BackgroundUbiquilin-4 (UBQLN4), a member of the ubiquilin family, has received limited attention in cancer research to date. Here, we investigated for the first time the functional role and mechanism of UBQLN4 in non-small cell lung cancer (NSCLC).MethodsThe Cancer Genome Atlas (TCGA) database was employed to validate UBQLN4 as a differentially expressed gene. Expression differences of UBQLN4 in NSCLC cells and tissues were assessed using immunohistochemistry (IHC) experiment and western blotting (WB) experiment. Kaplan-Meier analysis was conducted to examine the association between UBQLN4 expression and NSCLC prognosis. Functional analyses of UBQLN4 were performed through cell counting kit-8 (CCK-8), colony formation, and transwell invasion assays. The impact of UBQLN4 on tumor-associated signaling pathways was assessed using the path scan intracellular signaling array. In vivo tumorigenesis experiments were conducted to further investigate the influence of UBQLN4 on tumor formation.ResultsUBQLN4 exhibited up-regulation in both NSCLC tissues and cells. Additionally, over-expression of UBQLN4 was associated with an unfavorable prognosis in NSCLC patients. Functional loss analyses demonstrated that inhibiting UBQLN4 could suppress the proliferation and invasion of NSCLC cells in both in vitro and in vivo settings. Conversely, functional gain experiments yielded opposite results. Path scan intracellular signaling array results suggested that the role of UBQLN4 is associated with the PI3K/AKT pathway, a correlation substantiated by in vitro and in vivo tumorigenesis experiments.ConclusionWe validated that UBQLN4 promotes proliferation and invasion of NSCLC cells by activating the PI3K/AKT pathway, thereby facilitating the progression of NSCLC. These findings underscore the potential of targeting UBQLN4 as a therapeutic strategy for NSCLC.

Low Expression of SCN4B Predicts Poor Prognosis in Non-Small Cell Lung Cancer.

Sodium voltage-gated channel beta subunit 4 (SCN4B) plays a suppressive role in various tumors. However, the role of SCN4B in non-small cell lung cancer (NSCLC) is not yet clear. This study aims to investigate the expression of SCN4B in NSCLC patients and its correlation with prognosis. Firstly, the expression of SCN4B in non-small cell lung cancer (NSCLC) was analyzed using The Cancer Genome Atlas (TCGA) database. Then, differential expression genes (DEGs) were identified using R software. Next, DEG enrichment pathways were analyzed using the R package clusterProfiler. Protein-protein interaction networks were revealed through STRING analysis. A heatmap showed significant differential expression of SCN4B. Further analysis included examining SCN4B expression in a pan-cancer context and its correlation with 24 types of immune cells in NSCLC. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR), Western Blot, immunohistochemistry, and clinical data were used to validate SCN4B expression and prognostic value in NSCLC patients. SCN4B mRNA expression in non-small cell lung cancer tissues was significantly lower than in adjacent normal tissues (p < 0.001). Clinical correlation analysis confirmed its association with clinical pathology. Gene set enrichment analysis (GSEA) and tumor immune-related analyses indicated that SCN4B is involved in NSCLC-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and participates in immune infiltration. qRT-PCR, Western Blot, and immunohistochemistry also confirmed that SCN4B is downregulated in NSCLC patients and is associated with poor prognosis. SCN4B is downregulated in tumor tissues of NSCLC patients and is associated with a poor prognosis.

Circ_0060060 Accelerates Metastasis of Non-Small-Cell Lung Carcinoma by Upregulating FUT8

We aimed to elucidate the diagnostic and prognostic values of Circ_0060060 in non-small-cell lung cancer (NSCLC), and its regulatory effect on FUT8. Relative levels of Circ_0060060 and FUT8 were examined in NSCLC and adjacent tissues. Clinical information of recruited NSCLC patients was collected for assessing the prognostic value of Circ_0060060. Regulatory effects of Circ_0060060 and FUT8 on in vitro proliferative and migratory abilities of NSCLC were examined by cell counting kit-8 (CCK-8) and transwell assay, respectively. Finally, the interaction between Circ_0060060 and FUT8 was determined via dual-luciferase reporter assay and Pearson correlation test. Circ_0060060 and FUT8 were upregulated in NSCLC tissues. High level of Circ_0060060 or FUT8 predicted higher incidences of distant metastasis and worse overall survival in NSCLC patients. in vitro evidences have shown the roles of Circ_0060060 and FUT8 in stimulating proliferative and migratory abilities of PC-9 and SPC-A1 cells. Circ_0060060 directly targeted FUT8 and positively regulated its level. Circ_0060060 and FUT8 are effective diagnostic markers of NSCLC. Circ_0060060 stimulates proliferative and migratory abilities of NSCLC by regulating FUT8.

MRPL35 Induces Proliferation, Invasion, and Glutamine Metabolism in NSCLC Cells by Upregulating SLC7A5 Expression.

Mitochondrial ribosomal protein L35 (MRPL35) has been reported to contribute to the growth of non-small cell lung cancer (NSCLC) cells. However, the functions and mechanisms of MRPL35 on glutamine metabolism in NSCLC remain unclear. The detection of mRNA and protein of MRPL35, ubiquitin-specific protease 39 (USP39), and solute carrier family 7 member 5 (SLC7A5) was conducted using qRT-PCR and western blotting. Cell proliferation, apoptosis, and invasion were evaluated using the MTT assay, EdU assay, flow cytometry, and transwell assay, respectively. Glutamine metabolism was analyzed by detecting glutamine consumption, α-ketoglutarate level, and glutamate production. Cellular ubiquitination analyzed the deubiquitination effect of USP39 on MRPL35. An animal experiment was conducted for invivo analysis. MRPL35 was highly expressed in NSCLC tissues and cell lines, and high MRPL35 expression predicted poor outcome in NSCLC patients. Invitro analyses suggested that MRPL35 knockdown suppressed NSCLC cell proliferation, invasion, and glutamine metabolism. Moreover, MRPL35 silencing hindered tumor growth invivo. Mechanistically, USP39 stabilized MRPL35 expression by deubiquitination and then promoted NSCLC cell proliferation, invasion, and glutamine metabolism. In addition, MRPL35 positively affected SLC7A5 expression in NSCLC cells invitro and invivo. Moreover, the anticancer effects of MRPL35 silencing could be rescued by SLC7A5 overexpression in NSCLC cells. MRPL35 expression was stabilized by USP39-induced deubiquitination in NSCLC cells, and knockdown of MRPL35 suppressed NSCLC cell proliferation, invasion, and glutamine metabolism invitro and impeded tumor growth invivo by upregulating SLC7A5, providing a promising therapeutic target for NSCLC.

WITHDRAWN: Circular RNA CircRNA-0000520 promotes non-small cell lung cancer progression through sponging microRNA-521

The non-small cell lung cancer (NSCLC) accounts for 85 % of all lung cancers. This study investigated how the circular RNA (circRNA)-0000520 affected NSCLC and the underlying mechanisms, providing potential target for developing effective treatments of NSCLC. Firstly, circRNA-0000520 expression in tissues and cells were explored by the GEO2R online tool analyzing the public dataset obtained from the Gene Expression Omnibus (GEO) database and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). CircRNA-0000520 was upregulated in NSCLC (P < 0.05). Knockdown of circRNA-0000520 decreased the proliferation, migration and invasion of NSCLC cells, while increased the apoptosis and G0/G1 arrest of NSCLC cells (P < 0.05). Next, the downstream miRNAs of circRNA-0000520 were detected. By using RT-qPCR, the miR-521 was found to be downregulated in both NSCLC tissues and cell lines. And circRNA-0000520 knockdown obviously led to an increase of miR-521 in NSCLC cells. Circinteractome database was used to predict the binding sites of miR-521 in circRNA-0000520. And the interaction between circRNA-0000520 and miR-521 were verified by dual-luciferase reporter assay and RNA immunoprecipitation verified the interaction between circRNA-0000520 and miR-521. Furthermore, miR-521 depletion reversed the decreased levels of proliferation, migration and invasion of NSCLC cells, and the increased levels of apoptosis and G0/G1 arrest of NSCLC cells (P < 0.05) induced by circRNA-0000520 knockdown. In general, this study further verified that circRNA-0000520 played oncogenic roles in NSCLC and promoted the malignant behavior of NSCLC, and firstly proved that circRNA-0000520 acted as a sponge of miR-521, suggesting the potential of circRNA-0000520/miR-521 axis as the promising therapeutic targets for patients with NSCLC.

Knockdown of circ_0076305 decreases the paclitaxel resistance of non-small cell lung cancer cells by regulating TMPRSS4 via miR-936.

Paclitaxel (PTX) is a commonly used as a chemotherapeutic drug for non-small cell lung cancer (NSCLC). Exploring the underlying mechanism of PTX resistance is of great significance for NSCLC treatment. The expression levels of RNA and protein were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assays. The targeted relationship was confirmed by dual-luciferase reporter assay and RNA-pull down assay. The PTX resistance and cell proliferation were assessed by cell counting kit-8 (CCK-8) assay and 5-Ethynyl-2'-deoxyuridine (EDU) assay, respectively. Cell migration and invasion were analyzed by transwell assays. Cell apoptosis was analyzed by flow cytometry, and cell glycolysis was analyzed using the commercial kits. The role of circular RNA_0076305 (circ_0076305) in regulating the PTX sensitivity in vivo was explored in xenograft tumor model. Circ_0076305 was up-regulated in PTX-resistant NSCLC tissues and cells. Mechanically, circ_0076305 bound to microRNA-936 (miR-936), and miR-936 targeted transmembrane serine protease 4 (TMPRSS4). Circ_0076305 could up-regulate TMPRSS4 expression by sponging miR-936 in NSCLC cells. miR-936 knockdown or TMPRSS4 overexpression reversed the anti-tumor effects of circ_0076305 knockdown in NSCLC cells with PTX treatment. Circ_0076305 silencing increased the PTX sensitivity of xenograft tumors in vivo. Circ_0076305 silencing promoted PTX sensitivity by targeting miR-936/TMPRSS4 axis in NSCLC cells.

EIF4A3-Induced CircDHTKD1 regulates glycolysis in non-small cell lung cancer via stabilizing PFKL.

Lung cancer (LC) is one of the malignancies with the highest incidence and mortality in the world, approximately 85% of which is non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) exert multiple roles in NSCLC occurrence and development. The sequencing results in previous literature have illustrated that multiple circRNAs exhibit upregulation in NSCLC. We attempted to figure out which circRNA exerts an oncogenic role in NSLCL progression. RT-qPCR evaluated circDHTKD1 level in NSCLC tissue specimens and cells. Reverse transcription as well as RNase R digestion assay evaluated circDHTKD1 circular characterization in NSCLC cells. FISH determined circDHTKD1 subcellular distribution in NSCLC cells. Loss- and gain-of-function assays clarified circDHTKD1 role in NSCLC cell growth, tumour growth and glycolysis. Bioinformatics and RIP and RNA pull-down assessed association of circDHTKD1 with upstream molecule Eukaryotic initiation factor 4A-III (EIF4A3) or downstream molecule phosphofructokinase-1 liver type (PFKL) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) in NSCLC cells. Rescue assays assessed regulatory function of PFKL in circDHTKD1-meidated NSCLC cellular phenotypes. CircDHTKD1 exhibited upregulation and stable circular nature in NSCLC cells. EIF4A3 upregulated circDHTKD1 in NSCLC cells. CircDHTKD1 exerted a promoting influence on NSCLC cell malignant phenotypes and tumour growth. CircDHTKD1 exerted a promoting influence on NSCLC glucose metabolism. CircDHTKD1 exerts a promoting influence on NSCLC glucose metabolism through PFKL upregulation. RIP and RNA pull-down showed that circDHTKD1 could bind to IGF2BP, PFKL could bind to IGF2BP2, and circDHTKD1 promoted the binding of PFKL to IGF2BP2. In addition, RT-qPCR showed that IGF2BP2 knockdown promoted PFKL mRNA degradation, suggesting that IGF2BP2 stabilized PFKL in NSCLC cells. CircDHTKD1 exhibits upregulation in NSCLC. We innovatively validate that EIF4A3-triggered circDHTKD1 upregulation facilitates NSCLC glycolysis through recruiting m6A reader IGF2BP2 to stabilize PFKL, which may provide a new direction for seeking targeted therapy plans of NSCLC.