The human normal lung epithelial cell line, BEAS-2B (cat. no. HTX2075), was obtained from Otwo Biotech (Shenzhen Haodihuatuo Biotechnology Co., Ltd.). The lung cancer cell line, A549 (cat. no. CCL-185), and the 293T cell line (cat. no. CRL-11268) were obtained from the American Type Culture Collection. The lung cancer cell line, H460 (cat. no. CL-0299), was obtained from Procell Life Science & Technology Co., Ltd. All cell lines were authenticated by their suppliers through STR profiling. The A549, BEAS-2B, and 293T cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 10% foetal bovine serum (Gemini Bio Products), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO₂. H460 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) with the same supplements and conditions.

A549 cells were treated in triplicate with 0.7% dimethyl sulfoxide (DMSO; solvent used to dissolve RSL3 and Fer-1), 2 μM RSL3 (28) (cat. no. HY-100218A; MedChemExpress), or 2 μM RSL3 and 5 μM Fer-1 (29) (cat. no. S7243; Selleck Chemicals) for 24 h. Total RNA was extracted using TRIzol reagent (cat. no. T9424; Sigma-Aldrich; Merck KGaA), and the quantity and purity of the extracted RNA were analysed using an RNA 6000 Nano LabChip Kit (cat. no. 5067-1511; Agilent Technologies, Inc.) by Bioanalyzer 2100 (Agilent Technologies, Inc.). The 2x 150 bp paired-end sequencing was performed using an Illumina Novaseq™ 6000 at LC-Bio Technology (Hangzhou) Co., Ltd.

Using genes from the aforementioned 9 samples with a mean fragments per kilobase of transcript per million mapped reads value >1, differentially expressed genes (DEGs) between the treatment and control samples were identified using DEseq2 software (version 1.40.2; https://github.com/mikelove/DESeq2), based on fold change >1.5 and P<0.05 as the cut-off. Biological Process (BP) of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the DEGs were performed using Metascape (https://metascape.org/gp) (30). Heatmaps, volcano plots and Venn diagrams were created using OmicStudio (https://www.omicstudio.cn). Protein-protein interaction (PPI) networks were examined using STRING (https://www.string-db.org/). The co-expression analysis of mRNA-lncRNA was performed using the online resource, weishengxin (https://www.bioinformatics.com.cn). GO analysis of LUCAT1 and the prediction of target genes of DElncRNAs was performed using LncACTdb 3.0 (http://bio-bigdata.hrbmu.edu.cn/LncACTdb) (31). Expression specificity analysis of mRNA and lncRNA was conducted using index τ, following the method described by Li et al (32). The location of LUCAT1 was predicted using lncLocator (http://www.csbio.sjtu.edu.cn/bioinf/lncLocator2/) (33). LUCAT1 data were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/) [accession nos. GSE104462 (34) and GSE158458 (35)] and were associated with liver cancer lines (including HepG2, Hep3B and Huh7). Potential miRNAs targeting LUCAT1 or the 3'-untranslated region (3'-UTR) of GCH1 were predicted using DIANA-LncBase v3 (https://diana.e-ce.uth.gr/lncbasev3/interactions) (36) or TargetScan (http://www.targetscan.org). miR-34a-5p and LUCAT1 or GCH1 binding sites were predicted using miRDB (http://www.mirdb.org) (37) or TargetScan. Pan-cancer data were downloaded from the UCSC Xena Pan-Cancer Atlas Hub (https://xenabrowser.net/datapages/), and the expression of lncRNAs were analysed using R software (version 4.2.2; https://cran.r-project.org/src/base/R-4/).

The LUCAT1-expression vector was constructed using the PB-CMV-MCS-EF1α-RedPuro vector (cat. no. PB514B-2; System Biosciences, LLC) by YouBio. A549 cells in 6-well plates were co-transfected with 0.5 μg LUCAT1-expression vector (LUCAT1) or its control (Vector) and 0.2 μg Super PiggyBac Transposase Expression Vector (cat. no. PB210PA-1; System Biosciences, LLC) using the jetPRIME transfection reagent (cat. no. 101000046; Polyplus-transfection SA) at room temperature, and cells that stably expressed LUCAT1 were selected using 2 μg/ml puromycin (cat. no. A1113803; Gibco; Thermo Fisher Scientific, Inc.) and subsequently maintained with 1 μg/ml puromycin. LUCAT1 siRNA (siLUCAT1) and its control (siNC), mimics of miR-34a-5p (miR-34a-5p) and its control (miR-NC), and inhibitors of miR-34a-5p (anti-miR-34a-5p) and its control (anti-NC), synthesised by Genepharm, Inc., were transfected into H460 cells at a final concentration of 50 nM under the same conditions as aforementioned. The sequences used in these experiments are shown in Table SI. The gene RNA expression level was detected 48 h after transfection, and the protein expression level was detected 72 h after transfection.

The viability of cells was assessed using Cell Counting Kit-8 (CCK-8; cat. no. CK04; Dojindo Molecular Technologies, Inc.). To measure cell proliferation, cells were seeded at a density of 1×10³ cells/well into 96-well plates and cultured for 0, 24, 48 and 72 h. To measure cytotoxicity, cells were seeded at a density of 5×10³ cells/well into 96-well plates, cultured overnight and treated with RSL3 and Erastin (A549 cells were treated with 2 μM RSL3 or 20 μM Erastin and H460 cells were treated with 8 μM RSL3 or 20 μM Erastin) for 24 h. After adding the mixture (CCK-8 reagent: medium, 1:10) into the wells and incubating at 37°C for 1 h, a microplate reader (LabSystems Multiskan MS; Thermo Fisher Scientific, Inc.) was used to measure the absorbance at 450 nm.

Cells were seeded at a density of 1×10³ cells/well into 6-well plates, cultured overnight and treated with RSL3 or Erastin (A549 cells were treated with 0.1 μM RSL3 or 4 μM Erastin and H460 cells were treated with 2 μM RSL3 or 4 μM Erastin) for 10 days. After 20 min of methanol fixation at room temperature, 0.2% crystal violet was used to stain the cells for 5 min at room temperature. ImageJ software (version 1.53a; National Institutes of Health) was used to count colonies with >50 cells, and analysis was performed using GraphPad Prism (version 9.0; Dotmatics).

Lipid ROS was detected using a BODIPY 581/591 C11 Kit (cat. no. D3861; Invitrogen; Thermo Fisher Scientific, Inc.). Cells were seeded at a density of 1.5×10⁵ cells/well into 6-well plates, cultured overnight and treated with RSL3 or Erastin (A549 cells were treated with 2 μM RSL3 or 20 μM Erastin and H460 cells were treated with 8 μM RSL3 or 20 μM Erastin) at 37°C for 24 h. Subsequently, the cells were harvested and stained with 2 μM C11-BODIPY (581/591) probe at 37°C for 30 min in the dark. At least 1×10⁴ cells were collected to measure the fluorescence using the BD FACSAria™ II flow cytometer (BD Biosciences). FlowJo V10 software (FlowJo, LLC) was used to analyse the data.

TRIzol reagent was used to extract the total RNA, and the extracted RNA was reverse transcribed into cDNA using an ImProm-II^™ Reverse Transcriptase Reagent Kit (cat. no. A3803; Promega Corporation) according to the manufacturer's instructions. qPCR was performed in a Mx3000P instrument (Agilent Technologies, Inc.) using PowerUp™ SYBR™ Green Master Mix (cat. no. A24742; Applied Biosystems; Thermo Fisher Scientific, Inc.). For detecting miRNA expression, poly(A) tails were added before RT using E. coli poly(A) polymerase (cat. no. M0276S; New England BioLabs, Inc.). The qPCR conditions were as follows: i) 50°C for 2 min; ii) 95°C for 2 min; and iii) 40 cycles of 95°C for 5 sec and 60°C for 30 sec. To normalise the levels of miRNA expression and other genes, U6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA were utilised, and the relative gene expression levels were calculated using the 2^−ΔΔCq method (38). Table SII depicts the RT-qPCR primer sequences used in this assay.

RIPA Lysis Buffer (cat. no. ZE122-101S; Genstar Biosolutions Co., Ltd.) containing Protease Inhibitor Cocktail (cat. no. B14001; Bimake) was used to extract the total proteins from cells. The Pierce™ BCA Protein Assay Kit (cat. no. 23227; Thermo Fisher Scientific, Inc.) was utilised to quantify protein concentrations in the samples. Then, samples with 15 μg of protein were separated via 12% SDS-PAGE gels and transferred to nitrocellulose membranes (cat. no. 66485; Pall Life Sciences). After blocking the membrane with 5% non-fat milk at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: GAPDH (1:20,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.); GCH1 (1:2,000; cat. no. 28501-1-AP; ProteinTech Group, Inc.); GPX4 (1:5,000; cat. no. ab125066; Abcam) and FSP1 (1:2,000; cat. no. 20886-1-AP; ProteinTech Group, Inc.). Then, the membranes were incubated with secondary antibodies at room temperature for 1 h. The secondary antibodies used were as follows: Goat anti-mouse (1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc.) and goat anti-rabbit (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.). The signals were visualised using HRP Substrate Luminol Reagent (cat. no. WBKLS0500; MilliporeSigma) and imaged using a chemiluminescence imaging system (Tanon-4600SF; Tanon Science and Technology Co., Ltd.). ImageJ software was used for the semi-quantitation of protein.

Full-length LUCAT1 was inserted into the pGL3-basic vector (Promega Corporation) using XbaI and NdeI (cat. nos. R0145S and R0111S; New England BioLabs, Inc.) to create the LUCAT1 reporter vector. The GCH1 3'-UTR reporter vector was also constructed by insertion into the pGL3-basic vector. Using the QuickMutation™ Plus Kit (cat. no. D0208S; Beyotime Institute of Biotechnology), the binding sites 'CACUGCC' (position 869-875 in LUCAT1 or position 255-261 in the GCH1 3'-UTR) were changed to 'GCGACGG' to create mutated variants of the LUCAT1 and the GCH1 3'-UTR reporter vector. Dual-luciferase reporter assays were conducted by co-transfecting 293T cells with the Renilla Luciferase Expression Vector (Promega Corporation), the LUCAT1 or GCH1 3'-UTR reporter vector, and miR-NC or miR-34a-5p mimics using jetPRIME transfection reagent. After 48 h of transfection, the cells were lysed by the Dual-Luciferase Reporter Assay System (cat. no. E1910; Promega Corporation), and the luciferase activities were measured by GloMax^® 20/20 Luminometer (Promega Corporation). Transfection efficiencies were standardised using Renilla luciferase. Table SIII shows the sequences of the primers used in this assay.

The data are presented as the mean ± SD. Unpaired Student's t-test (two-tailed) and one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey's test were used to analyse the differences between the different groups, using GraphPad Prism software. P<0.05 was used to indicate a statistically significant difference.

To identify genes involved in ferroptosis, A549 cells were treated with DMSO, RSL3 or RSL3 + Fer-1. RSL3 (2 μM) inhibited approximately half of the cell viability of A549 cells, which was prevented by co-incubation with 5 μM Fer-1 (Fig. S1). Subsequently, RNA-Seq was performed. When comparing the RSL3 treatment group with the DMSO group, 291 DEmRNAs were identified, including the ferroptosis-related genes, solute carrier family 7 member 11, prostaglandin-endoperoxide synthase 2, FTH1, heme oxygenase 1 and TP53 (1,39-42) (Fig. 1A and B), of which 82 were rescued (expression level was restored) by co-incubation with Fer-1, indicating that these 82 DEmRNAs may be important for ferroptosis (Fig. 1C). The DEmRNAs were enriched in the BP terms, 'response to oxygen levels' and 'positive control of programmed cell death' (Fig. 1D). KEGG pathway analysis revealed that the ferroptosis-related, 'HIF-1 signaling pathway' (43), and 'TNF signaling pathway' (44) were also enriched during ferroptosis (Fig. 1E). Among the top 10 hub mRNAs in the PPI network displayed using STRING, jun proto-oncogene, C-X-C motif chemokine ligand 8, epiregulin, cyclin-dependent kinase inhibitor 1A, serpin family E member 1 and DNA damage-inducible transcript 3 were previously reported to be involved in ferroptosis (45-50), suggesting that the remaining four genes, growth arrest and DNA damage inducible α (GADD45A), epithelial mitogen (EPGN), heparin-binding EGF-like growth factor (HBEGF) and transforming growth factor α (TGFA), may also have roles in ferroptosis (Fig. 1F and G). These results therefore identified novel genes that may be involved in ferroptosis caused by alkoxyl radicals and other rearrangement products generated by ferrous iron from lipid hydroperoxides.

In addition to the 82 DEmRNAs, 18 DElncRNAs were either upregulated or downregulated in the RSL3 group compared with the DMSO group, which was prevented by co-incubation with Fer-1 (Fig. 2A-D). The genes targeted by DElncRNAs were enriched in the BP terms 'regulation of growth' and 'response to oxygen levels' (Fig. 2E). Additionally, KEGG pathway analysis revealed that the 'MAPK signaling pathway' (51,52), 'Hippo signaling pathway' (53) and 'Wnt signaling pathway' (54) were enriched (Fig. 2F), suggesting that DElncRNAs are involved in ferroptosis.

Compared with mRNA expression levels, the global expression level of lncRNA was much lower (Fig. 2G). However, the lncRNAs displayed a greater degree of expression specificity compared with that of the mRNAs (Fig. 2H). To identify lncRNAs that were more important in ferroptosis, their correlations with DEmRNAs were examined and mRNA-lncRNA co-expression networks were constructed. Using a robust correlation (|r|>0.9, P<0.05) as the cut-off for mRNA-lncRNA pairs, 93 nodes (16 DElncRNAs and 77 DEmRNAs) and 589 mRNA-lncRNA pairs (544 with a positive correlation and 45 with a negative correlation) were identified. The mRNA-lncRNA pairs with the top 100 correlation coefficients (|r|) were used for network construction (Fig. 2I).

Among the 18 DElncRNAs, LUCAT1 was the most highly upregulated, ranking first among the candidates (Fig. 2J), and it had an important role in the constructed mRNA-lncRNA co-expression network (Fig. 2I). Therefore, it was next confirmed that LUCAT1 was upregulated following treatment with RSL3 and prevented by co-incubation with Fer-1 (Fig. 2K and L). After treating A549 cells with RSL3, LUCAT1 expression increased in a dose-dependent manner and maximum LUCAT1 expression occurred after 12 h of treatment with 4 μM RSL3 (Fig. 2M and N). To obtain more evidence for the connection between LUCAT1 and ferroptosis, the GEO database was utilised to explore the relationship between LUCAT1 and other ferroptosis inducers in cancer. It was found that LUCAT1 was elevated in HepG2 liver cancer cells treated with Erastin and in two sorafenib-resistant liver cancer lines (Hep3B and Huh7) by bioinformatic analysis (Fig. S2A-C), suggesting that ferroptosis inducer-induced upregulation of LUCAT1 may be common in different types of cancer. Furthermore, the top three GO terms for LUCAT1 predicted by the LncACTdb were 'superoxide metabolic process' (55), 'MAPK activity' (51,52) and 'oxygen and reactive oxygen species metabolic process' (56), which all have important roles in ferroptosis (Fig. 2O). These results reflected a possible close relationship between ferroptosis and LUCAT1.

Genes that are expressed at either high or low levels in tumours influence the growth and treatment of the tumours (57). LUCAT1 expression was higher in 18 different types of cancer tissues than in the corresponding normal tissues (Fig. S3A). Compared with normal tissues, LUCAT1 expression was higher in lung adenocarcinoma and lung squamous cell carcinoma (Fig. S3B and C). Similarly, two lung cancer cell lines, A549 and H460, expressed higher levels of LUCAT1 compared with the BEAS-2B normal lung epithelial cell line (Fig. S3D). These findings suggested that LUCAT1 functions as an oncogene in lung cancer.

The expression of LUCAT1 was lower in A549 cells than in H460 cells (Fig. S3D); therefore, the effect of overexpressing LUCAT1 in A549 cells was examined (Fig. 3A). As shown in Fig. 3B, LUCAT1 overexpression significantly enhanced A549 cell proliferation. When ferroptosis was induced by RSL3 and Erastin, overexpression of LUCAT1 enhanced the survival rate and colony formation of A549 cells (Fig. 3C and D). Similarly, lipid peroxidation levels were elevated by RSL3 and Erastin treatment; however, LUCAT1 overexpression reduced these levels (Fig. 3E). Collectively, these data suggested that LUCAT1 was a ferroptosis suppressor in A549 cells treated with RSL3 and Erastin.

As LUCAT1 was expressed at higher levels in H460 cells than in A549 cells (Fig. S3D), the effect of knocking down LUCAT1 expression in H460 cells was next explored (Fig. 4A). Knocking down LUCAT1 expression significantly inhibited the proliferation of H460 cells (Fig. 4B). Knocking down LUCAT1 expression also significantly reduced the survival rate and colony formation capacity of H460 cells following ferroptosis induction (Fig. 4C and D). Moreover, knocking down LUCAT1 expression increased the level of lipid peroxidation upon ferroptosis induction (Fig. 4E). These data suggested that knocking down LUCAT1 expression promoted RSL3- and Erastin-induced ferroptosis.

To investigate the mechanism by which LUCAT1 influences ferroptosis, the expression of the main ferroptosis resistance genes were detected. FSP1 and GCH1 were downregulated in LUCAT1-knock down cells, while GPX4 expression was not affected (Fig. 5A). Owing to the greater downregulation of GCH1, we considered that LUCAT1 primarily exerted its effects through GCH1. LncLocator analysis indicated that LUCAT1 is predominantly localised in the cytoplasm (Fig. 5B); thus, it may act as a miRNA sponge in regulating GCH1 expression. Next, the shared miRNAs targeting the 3'-UTR of GCH1 (predicted by TargetScan) and LUCAT1 (predicted by DIANA-LncBase v3) were analysed and miR-34a-5p was identified as a potential mediator between the two (Fig. 5C). As illustrated in Fig. 5D, the expression level of miR-34a-5p was elevated upon LUCAT1 knockdown. Moreover, transfection with miR-34a-5p mimics downregulated GCH1 expression (Fig. 5E and F). Additionally, miR-34a-5p mimics enhanced the RSL3-induced ferroptosis sensitivity as determined via cell viability and lipid ROS assays (Fig. 5G and H). Conversely, inhibition of miR-34a-5p with anti-miR-34a-5p increased the resistance of ferroptosis mediated by LUCAT1 inhibition (Fig. 5I and J).

Using miRDB and TargetScan, the potential miR-34a-5p binding sites with LUCAT1 and the 3'-UTR of GCH1 were identified (Fig. 5K). The potential binding sites between miR-34a-5p and LUCAT1 or the 3'-UTR of GCH1 were then validated using dual-luciferase reporter assays. The luciferase activity was markedly lower in 293T cells transfected with miR-34a-5p mimics than with miR-NC; however, no changes in luciferase activity were observed in the LUCAT1-mutated (MUT) or GCH1-MUT groups (Fig. 5L). In conclusion, these findings suggested that LUCAT1 may promote ferroptosis via the miR-34a-5p/GCH1 axis.