Regulation of tumorigenesis and ferroptosis in non-small cell lung cancer by a novel BBOX1-AS1/miR-326/PROM2 axis.

Dysregulation of long non-coding RNAs (lncRNAs) is associated with the tumorigenesis and ferroptosis of non-small cell lung cancer (NSCLC). BBOX1 antisense RNA 1 (BBOX1-AS1) functions as an oncogenic driver in NSCLC. Here, we aim to investigate the regulation effect and underlying mechanism of BBOX1-AS1 in NSCLC progression and ferroptosis. RNA expression was detected by quantitative real-time PCR (qRT-PCR), and protein expression was measured by immunoblotting. Cell growth was assessed by CCK-8 and colony formation assays. Transwell assay was applied to evaluate cell invasion and migration. RNA pull-down and dual-luciferase reporter assays were applied to verify the relationship between miR-326 and BBOX1-AS1 or prominin 2 (PROM2). The role of BBOX1-AS1 in NSCLC tumorigenicity was also analyzed by xenograft assays. Silencing BBOX1-AS1 or PROM2 impeded NSCLC cell growth, migration, and invasion. Silencing BBOX1-AS1 induced cell apoptosis and ferroptosis. BBOX1-AS1 up-regulated PROM2 expression, and re-expression of PROM2 reversed the effects of BBOX1-AS1 depletion on cell malignant phenotypes and ferroptosis. BBOX1-AS1 post-transcriptionally modulated PROM2 expression by sponging miR-326. MiR-326 was validated as a mediator of BBOX1-AS1 in regulating NSCLC cell malignant phenotypes and ferroptosis. Additionally, BBOX1-AS1 deficiency in vivo resulted in the suppression of xenograft tumor growth. Together, our study defines a novel BBOX1-AS1/miR-326/PROM2 axis in regulating NSCLC malignant progression and ferroptosis, offering new evidence for the oncogenic role of BBOX1-AS1 in NSCLC. These findings may provide a basis for the future usage of targeting BBOX1-AS1 in NSCLC treatment.

Identification and Verification of Metabolism-related Immunotherapy Features and Prognosis in Lung Adenocarcinoma.

BACKGROUND: Lung cancer is a frequent malignancy with a poor prognosis. Extensive metabolic alterations are involved in carcinogenesis and could, therefore, serve as a reliable prognostic phenotype. AIMS: Our study aimed to develop a prognosis signature and explore the relationship between metabolic characteristic-related signature and immune infiltration in lung adenocarcinoma (LUAD). OBJECTIVE: TCGA-LUAD and GSE31210 datasets were used as a training set and a validation set, respectively. METHOD: A total of 513 LUAD samples collected from The Cancer Genome Atlas database (TCGA-LUAD) were used as a training dataset. Molecular subtypes were classified by consensus clustering, and prognostic genes related to metabolism were analyzed based on Differentially Expressed Genes (DEGs), Protein-Protein Interaction (PPI) network, the univariate/multivariate- and Lasso- Cox regression analysis. RESULTS: Two molecular subtypes with significant survival differences were divided by the metabolism gene sets. The DEGs between the two subtypes were identified by integrated analysis and then used to develop an 8-gene signature (TTK, TOP2A, KIF15, DLGAP5, PLK1, PTTG1, ECT2, and ANLN) for predicting LUAD prognosis. Overexpression of the 8 genes was significantly correlated with worse prognostic outcomes. RiskScore was an independent factor that could divide LUAD patients into low- and high-risk groups. Specifically, high-risk patients had poorer prognoses and higher immune escape. The Receiver Operating Characteristic (ROC) curve showed strong performance of the RiskScore model in estimating 1-, 3- and 5-year survival in both training and validation sets. Finally, an optimized nomogram model was developed and contributed the most to the prognostic prediction in LUAD. CONCLUSION: The current model could help effectively identify high-risk patients and suggest the most effective drug and treatment candidates for patients with LUAD.

Hsa_circ_0069244 acts as the sponge of miR-346 to inhibit non-small cell lung cancer progression by regulating XPC expression.

Circular RNAs (circRNAs) play a significant role in the progression of diverse malignancies. Here, we aimed to probe the function and mechanism of circ_0069244 in non-small cell lung cancer (NSCLC). In the present study, circ_0069244 was selected from the circRNA microarray datasets (GSE112214). Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to examine circ_0069244, miR-346 and XPC complex subunit, DNA damage recognition and repair factor (XPC) expression levels. Kaplan-Meier curve was employed to analyze the association between circ_0069244 expression and overall survival of NSCLC patients. Cell counting kit-8 (CCK-8) and 5-Bromo-2'-deoxyuridine (BrdU) experiments were utilized to examine the proliferation of NSCLC cells. Scratch healing and Transwell experiments were executed to examine the migration of NSCLC cells. Western blot was conducted to detect XPC expression at protein level in NSCLC cells. Bioinformatics analysis, dual-luciferase reporter gene and RNA immunoprecipitation (RIP) experiments predicted and validated the targeting relationships of circ_0069244 and miR-346, as well as miR-346 and 3'untranslated region (UTR) of XPC mRNA, respectively. We reported that circ_0069244 was remarkably down modulated in NSCLC and was linked to shorter survival and poor tumor histological grade in NSCLC patients. Functionally, circ_0069244 repressed NSCLC cell proliferation and migration. Furthermore, miR-346-5p was unveiled to be a downstream target of circ_0069244, and miR-346-5p specifically modulated XPC expression. Rescue experiments indicated that the inhibitory effect of circ_0069244 was abolished by co-expression of miR-346-5p mimics. Taken together, circ_0069244 restrained NSCLC progression by modulating the miR-346-5p/XPC axis.

Circ_0006988 promotes the proliferation, metastasis and angiogenesis of non-small cell lung cancer cells by modulating miR-491-5p/MAP3K3 axis.

Circular RNAs (circRNAs) are related to the progression of non-small cell lung cancer (NSCLC). However, the roles and mechanism of circ_0006988 are largely unknown. The levels of circ_0006988, Low-Density Lipoprotein Receptor Class A Domain Containing 3 (LDLRAD3), microRNA-491-5p (miR-491-5p), Mitogen-Activated Protein Kinase Kinase Kinase 3 (MAP3K3) were measured using quantitative real-time polymerase-chain reaction (qRT-PCR) and western blot assay. The characteristic of circ_0006988 was analyzed by RNase R assay and Actinomycin D assay. Functional analyses were processed by Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, flow cytometry analysis, transwell assay, wound-healing assay and tube formation assay. The interactions between circ_0006988 and miR-491-5p as well as miR-491-5p and MAP3K3 were analyzed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Murine xenograft model assay was processed to verify the function of circ_0006988 in vivo. Immunohistochemistry (IHC) assay was conducted to examine the level of Ki67. Circ_0006988 abundance was increased in NSCLC tissues and cells. Circ_0006988 silencing restrained NSCLC cell proliferation, migration, invasion and angiogenesis, and induced apoptosis. Circ_0006988 sponged miR-491-5p, which directly targeted MAP3K3. MiR-491-5p overexpression repressed NSCLC cell malignant behaviors. MiR-491-5p downregulation or MAP3K3 overexpression reversed the effect of circ_0006988 silencing on NSCLC cell progression. In addition, circ_0006988 knockdown reduced xenograft tumor growth. ssCirc_0006988 contributed to the development of NSCLC by miR-491-5p/MAP3K3 axis.

KLF5-induced BBOX1-AS1 contributes to cell malignant phenotypes in non-small cell lung cancer via sponging miR-27a-5p to up-regulate MELK and activate FAK signaling pathway.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a major histological subtype of lung cancer with high mortality and morbidity. A substantial amount of evidence demonstrates long non-coding RNAs (lncRNA) as critical regulators in tumorigeneis and malignant progression of human cancers. The oncogenic role of BBOX1 anti-sense RNA 1 (BBOX1-AS1) has been reported in several tumors. As yet, the potential functions and mechanisms of BBOX1-AS1 in NSCLC are obscure. METHODS: The gene and protein expression was detected by qRT-PCR and western blot. Cell function was determined by CCK-8, colony forming, would healing and transwell assays. Bioinformatics tools, ChIP assays, dual luciferase reporters system and RNA pull-down experiments were used to examine the interaction between molecules. Subcutaneous tumor models in nude mice were established to investigate in vivo NSCLC cell behavior. RESULTS: BBOX1-AS1 was highly expressed in NSCLC tissues and cells. High BBOX1-AS1 expression was associated with worse clinical parameters and poor prognosis. BBOX1-AS1 up-regulation was induced by transcription factor KLF5. BBOX1-AS1 deficiency resulted in an inhibition of cell proliferation, migration, invasion and EMT in vitro. Also, knockdown of BBOX1-AS1 suppressed NSCLC xenograft tumor growth in mice in vivo. Mechanistically, BBOX1-AS1 acted act as a competetive "sponge" of miR-27a-5p to promote maternal embryonic leucine zipper kinase (MELK) expression and activate FAK signaling. miR-27a-5p was confirmed as a tumor suppressor in NSCLC. Moreover, BBOX1-AS1-induced increase of cell proliferation, migration, invasion and EMT was greatly reversed due to the overexpression of miR-27a-5p. In addition, the suppressive effect of NSCLC progression owing to BBOX1-AS1 depletion was abated by the up-regulation of MELK. Consistently, BBOX1-AS1-mediated carcinogenicity was attenuated in NSCLC after treatment with a specific MELK inhibitor OTSSP167. CONCLUSIONS: KLF5-induced BBOX1-AS1 exerts tumor-promotive roles in NSCLC via sponging miR-27a-5p to activate MELK/FAK signaling, providing the possibility of employing BBOX1-AS1 as a therapeutic target for NSCLC patients.

MicroRNA-130a targeting hypoxia-inducible factor 1 alpha suppresses cell metastasis and Warburg effect of NSCLC cells under hypoxia.

MicroRNAs have been demonstrated to play critical role in the development of non-small cell lung cancer (NSCLC) and hypoxia is a common hallmark of NSCLC. MiRNA-130a-3p (miR-130a) is a well-known tumor suppressor, and we intended to explore the role and mechanism of miR-130a in NSCLC cells under hypoxia. We used real-time quantitative polymerase chain reaction method to measure miR-130a expression, and found that miR-130a was downregulated in human NSCLC tumors and cell lines (A549 and H1299), accompanied with upregulation of hypoxia-inducible factor 1 alpha (HIF1A), a marker of hypoxia. Besides, miR-130a low expression was associated with tumor burden and poor overall survival. Moreover, miR-130a expression was even downregulated in hypoxia-treated A549 and H1299 cells. Ectopic expression of miR-130a suppressed Warburg effect, migration and invasion in hypoxic A549 and H1299 cells, as evidenced by decreased glucose consumption, lactate production, hexokinase 2 expression, and numbers of migration cells and invasion cells analyzed by commercial glucose and lactate assay kits, western blotting and transwell assays. Furthermore, overexpression of miR-130a restrained xenograft tumor growth of A549 cells in mice. However, recovery of HIF1A could reverse the suppressive effect of miR-130a overexpression on cell migration, invasion and Warburg effect in hypoxic A549 and H1299 cells. Mechanically, dual-luciferase reporter assay, RNA immunoprecipitation and RNA pull-down assay confirmed a target relationship between miR-130a and HIF1A. Collectively, we demonstrated an anti-tumor role of miR-130a in NSCLC cells under hypoxia through targeting HIF1A, suggesting a potential target for the interfering of NSCLC.

Antioxidant coumarin and pyrone derivatives from the insect-associated fungus Aspergillus Versicolor.

Two new compounds, versicolones A and B (1 and 2), and three known pyrone derivatives (3-5) were isolated from the insect-associated fungus Aspergillus versicolor. Their structures were elucidated through a combination of HRESIMS and NMR spectroscopic analysis. Versicolone A (1) was revealed as a coumarin derivative with the rare 5-alkyl side chain substitution. Compound 5 exhibited significant antioxidant activity with EC50 value of 8.0 µM in the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assay, which was more than 2-fold potency of the positive control trolox.

Long non-coding RNA HOTTIP promotes hypoxia-induced glycolysis through targeting miR-615-3p/HMGB3 axis in non-small cell lung cancer cells.

Increased glycolysis under hypoxic stress is a fundamentally important feature of non-small cell lung cancer (NSCLC) cells, but molecular mechanisms of hypoxia on glycolysis remain elusive. Herein, we aimed to explore whether lncRNAs and miRNAs are involved in the glycolytic reprogramming under hypoxic conditions. The levels of HOXA transcript at the distal tip (HOTTIP), miR-615-3p and high mobility group box 3 (HMGB3) mRNA were assessed by qRT-PCR. Western blot was performed to determine the protein expression of hexokinase 2 (HK-2) and HMGB3. Glucose consumption and lactate production were analyzed using a respective assay kit. The targeted correlation between miR-615-3p and HOTTIP or HMGB3 was verified using dual-luciferase reporter and RNA immunoprecipition assays. Our data revealed that HOTTIP was upregulated and miR-615-3p was downregulated in NSCLC tissues and cells. Hypoxia induced glycolysis, increased HOTTIP and HMGB3 mRNA levels and repressed miR-615-3p expression in NSCLC cells. HOTTIP deficiency or miR-615-3p expression restoration repressed hypoxia-induced glycolysis. Moreover, HOTTIP acted as a molecular sponge for miR-615-3p and HMGB3 was a direct target of miR-615-3p. The inhibitory effect of HOTTIP deficiency on glycolysis under hypoxic exposure was reversed by miR-615-3p restoration. Additionally, HOTTIP regulated HMGB3 expression by acting as a molecular sponge of miR-615-3p in NSCLC cells. In conclusion, our study suggested that HOTTIP might promote glycolysis under hypoxic conditions at least partly through regulating miR-615-3p/HMGB3 axis in NSCLC cells. Targeting HOTTIP might be a promising therapeutic strategy for NSCLC treatment.