RNA-sequencing expression profiles and corresponding clinical information for lung carcinoma were downloaded from The Cancer Genome Atlas (TCGA) dataset (https://portal.gdc.com). The University of Alabama at Birmingham cancer data analysis portal (UALCAN; https://ualcan.path.uab.edu) was used for analysis.

Between July 2022 and May 2023, 80 specimens of lung carcinoma and their adjacent tissues (>5 cm away from the cancerous tissue) resected surgically were collected, all of which were from Department of Respiratory Medicine, Shanghai Xuhui Central Hospital, Fudan University (Shanghai, China). All specimens were frozen in liquid nitrogen for RNA extraction. All participating patients gave their written informed consent. The present study was approved by the Shanghai Xuhui Central Hospital's Ethics Committee (approval no. 2022021).

MRC-5, A549, HCC827, PC9 and HCC2935 were from Authenticated Cell Cultures. MRC-5 and HCC2935 were cultured using DMEM medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G, 100 U/ml streptomycin sulfate, and 2 mM L-glutamine. A549 cells were cultured using F12K medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G, 100 U/ml streptomycin sulfate, and 2 mM L-glutamine. PC9 and HCC827 were cultured using RPMI1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G, 100 U/ml streptomycin sulfate, and 2 mM L-glutamine. The cells were cultured in a 37˚C incubator containing 5% CO₂.

The siRNA targeting EGFR-AS1 and HDAC1, si-NC, miR-449a mimics, miR-449a inhibitor, miR-NC and inhibitor NC were all purchased from Shanghai Genechem Co., Ltd. For transfection, cells were seeded in 24 well plates at 5,000 cells per well or 800,000 per 15 cm dish and 10 nM siRNA, si-NC, miR-449a mimics, miR-449a inhibitor, miR-NC or inhibitor NC were transfected (at 37˚C) into cells, respectively, using Lipofectamine 3000^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. At 48 h after the transfection, cells were subjected to RNA isolation or western blotting. The siRNA, miRNA mimics and miRNA inhibitors sequences used were: si-EGFR-AS1-1, sense (SS): 5'-GCAAGTTGAGTGCAAATAACT-3', anti-sense (AS): 5'-TTATTTGCACTCAACTTGCTA-3'; si-EGFR-AS-2, SS: 5'-CCACAGTATTCACAAAGAATT-3', AS: 5'-TTCTTTGTGAATACTGTGGTG-3'; si-HDAC1-1, SS: 5'-CAGCGATGACTACATTAAATT-3', AS: 5'-TTTAATGTAGTCATCGCTGTG-3'; si-HDAC1-2, SS: 5'-GCTTCAATCTAACTATCAAAG-3', AS: 5'-TTGATAGTTAGATTGAAGCAA-3'; miR-449a mimics, SS:5'-TGGCAGTGTATTGTTAGCTGGT-3', AS: 5'-ACCAGCTAACAATACACTGCCA-3'; miR-449a inhibitor, SS: 5'-AGGCTCACATAATCAATCGACCA-3', AS: 5'-TGGCAGTGTATTGTTAGCTGGT-3'; miR-NC, SS: 5'-GCATCAAGGTGAACTTCAAGA-3', AS: 5'-TCTTGAAGTTCACCTTGATGC-3'; inhibitor-NC, SS: 5'-GCATCAAGGTGAACTTCAA-3', AS: 5'-TTGAAGTTCACCTTGATGC-3'.

Total RNA was obtained from the cells as instructed by the manufacturer with TRIzol^® reagent (Thermo Fisher Scientific, Inc.). The RNA of clean and concentrated samples was measured using a Spectrometer (Thermo Fisher Scientific, Inc.) and the absorbance was between 260-280 nm. The cDNA synthesis was carried out based on the instructions of the miScript II RT Kit (Qiagen). Briefly, 1 µg total RNA was added to 12 µl DEPC treated water, 2 µl miScript Reverse Transcriptase Mix (Qiagen GmbH), 2 µl miScript Nucleics Mix (Qiagen GmbH) and 4 µl 5x miScript HiSpec buffer (Qiagen GmbH). After incubating at 37˚C for 60 min the mixture was heated to 95˚C for 5 min to terminate the reaction. Using commercialized primers TaqMan Universal Mix II No UNG plus specific PCR primers (Thermo Fisher Scientific, Inc.) were used to detect the expression level of miR-449a according to the manufacturer's instructions. Amplification was performed on the ABI StepOne Plus system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Using U6 as the internal reference, the 2^-ΔΔCq method was used to calculate the relative expression level of miR-449a (32). The quantitative detection of EGFR-AS1 and HDAC1 mRNA was also performed on the ABI StepOne Plus system. Using GAPDH as an internal reference, the 2^-ΔΔCq method was used to calculate the relative expression level of mRNAs (32). The incubation conditions were as follows: 95˚C for 30 sec, followed by 40 cycles at 95˚C for 8 sec and 60˚C for 30 sec. All experiments were repeated three times. Sequences of primers for amplification are given in Table I.

Cell survival was measured by Cell Counting Kit-8 (Beyotime Institute of Biotechnology). The exponentially growing cells were seeded at a density of 2,000 cells per well in 96-well plates and incubated overnight at 37˚C. Then, the cells were treated with 10 µl CCK-8 for 4 h at 48 h after transfection. Absorbance density (OD, 450 nm) was measured using a microplate reader (Thermo Fisher Scientific, Inc.). Experimental study was conducted on six replicate wells per group.

The cells were subjected to trypsinization, dilution in serum free medium and seeded at 50,000 cells per Transwell chamber (8 µm pore size; Corning) with or without Matrigel (BD Biosciences). Matrigel was frozen and thawed overnight at 4˚C on ice, diluted with Opti-MEM medium (Thermo Fisher Scientific, Inc.) at a ratio of 1:3 and mixed with precooled pipette tips to form a homogenized Matrigel matrix. The Transwell chamber was placed on ice, the diluted Matrigel with a concentration of 50 µl/cm² growth area was added and it was left at 37˚C for 30 min before use. The culture medium with 10 percent of FBS was put into the wells and incubated for 12 h. The cells were fixed with 4% paraformaldehyde (MilliporeSigma) for 30 min at room temperature and stained with 0.1% crystal violet (MilliporeSigma) staining solution for 15 min at room temperature . Subsequently, three randomly selected fields of vision were analyzed and the number of cells that migrated through each insert was counted under a light microscope using a 20x objective. Each experiment was conducted in triplicate.

The pmirGLO plasmid (1 µg; Promega Corporation) was digested with DraI and XbaI (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Insert DNA containing a wild-type miR-449a binding site (ACTGCC) or a mutant miR-449a binding site (TGACGG) was purchased from Shanghai Genechem Co., Ltd. Oligonucleotides were hybridized at 90˚C, then cooled to 4˚C over 5 min, and finally kept at 4˚C for 60 min. Hybridized inserts were ligated with T4 ligase (200 U, 10 µl reaction, 1:10 vector/insert ratio; Thermo Fisher Scientific, Inc.) into a multiple cloning site of pmirGLO downstream of the firefly luciferase gene for construct wild-type (wt-pmirGLO pGL3-EGFR-AS1) and mutant (mut-pmirGLO pGL3-EGFR-AS1) luciferase reporter plasmid. To identify EGFR-AS1 and miR-449a targets, wt-pmirGLO EGFR-AS1 or mut-pmirGLO-EGFR-AS1 were co-transfected with miR-449a mimics into HEK293 cells Lipofectamine 3000^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. At 48 h after transfection, a dual luciferase assay kit (Promega Corporation) was used to lyse cells.

To study the interaction between HDAC1 3'-UTR and miR-449a targets, the pmirGLO plasmid (1 µg; Promega Corporation) was digested with DraI and XbaI (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Insert DNA containing a wild-type miR-449a binding site (CACTGCC) or a mutant miR-449a binding site (CTGACGG) was purchased from Shanghai Genechem Co., Ltd. Oligonucleotides were hybridized at 90˚C, then cooled to 4˚C over 5 min, and finally kept at 4˚C for 60 min. Hybridized inserts were ligated with T4 ligase (200 U, 10 µl reaction, 1:10 vector/insert ratio; Thermo Scientific) into a multiple cloning site of pmirGLO downstream of the firefly luciferase gene for construct HDAC1 3'-UTR wild-type (wt-pmirGLO-HDAC1 3'-UTR) and mutant (mut-pmirGLO-HDAC1 3'-UTR) luciferase reporter plasmid. Wild-type (wt-pmirGLO-HDAC1 3'-UTR) and or EGFR-AS1 (mut-pmirGLO-HDAC1 3'-UTR) luciferase reporters were co-transfected with miR-449a mimics into 293 cells using Lipofectamine 3000^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. At 48 h after transfection, a dual luciferase assay kit (Promega Corporation) was used to lyse cells. The test was carried out on the basis of the activity of Renilla luciferase.

A Panomis Luminometer (Affymetrix; Thermo Fisher Scientific, Inc.) was used with a standard method to measure the activity of Renilla luciferase.

The RIP was conducted according to manufacturer's instructions of EZMagna RIP kit (cat. no. 17-701; MilliporeSigma). The A549 cells were cultured to 80-80% confluence, then lysed with RIP lysis buffer (Millipore Sigma). The protein A/G magnetic beads underwent incubation with 5 µg antibodies specific for argonaute-(Ago)2 (cat. no. SAB4200085; MilliporeSigma) or normal murine IgG (cat. no. A7031; Beyotime Institute of Biotechnology) for 30 min at room temperature. The beads underwent incubation at 4˚C overnight with cell lysates after being washed three times with RIP wash buffer. The RNA purity and the concentration RNA was measured with a Nextwave TT 1000 spectrophotometer (Thermo Fisher Scientific, Inc.), determined at a 260-280 nm absorption. The RNeasy Micro Kit (Qiagen GmbH) was used for purification of RNA and quantification with qPCR. RT-qPCR was used to detect the co-precipitated RNA. The input % was utilized for calculating enrichment level of RNA.

The cells were lysed using Cell Lysis Solution (MilliporeSigma) at 4˚C with gentle agitation for 5 min at 1,000 rpm. Equal amounts (20 µg) of protein were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.) and separated on 10% SDS-PAGE gels, and transferred onto PVDF membranes (Cytiva). After blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated overnight at 4˚C with anti-HDAC1 antibody (cat. no. ab68436; Abcam; 1:1,000) and anti-β-actin antibody (cat. no. ab8226; Abcam; 1:2,000). After washing TBST buffer (0.1% Tween-20; Beyotime Institute of Biotechnology), the membranes were incubated with HRP-labeled goat anti-mouse IgG (cat. no. ab205719, Abcam; 1:50,000) at room temperature for 1 h. After washing the membranes three times, ECL luminescent solution (Thermo Fisher Scientific Inc.) was applied to the membranes and the results were observed on an Imagequant LAS4000 (Cytiva). The density of each band was quantified using ImageJ software (National Institutes of Health).

All analyses were performed using SPSS 20.0 (IBM Corp.). Data are presented as mean ± SD. A paired two-tailed t-test was used to compare EGFR-AS1, miR-449a and HDAC1 expression in lung carcinoma and their paired adjacent tissues. The unpaired Student's t-test was used to compare the significance between two groups, and one-way ANOVA and Tukey's post hoc test were used for multiple comparisons. The relationship of the EGFR-AS1 and miR-449a, HDAC1 and miR-449a, EGFR-AS1 and HDAC1 was measured with Pearson's correlative test. P<0.05 was considered to indicate a statistically significant difference.

UALCAN analysis of TCGA NSCLC showed a significant increase of EGFR-AS1 and HDAC1 in NSCLC (Fig. 1A and C), while the levels of miR-449a were lower (Fig. 1B) compared with adjacent tissues. Furthermore, qPCR findings indicated that EGFR-AS1 and HDAC1 were more highly expressed in A549, HCC827, PC9 and HCC2935 cells (NSCLC) compared with MRC-5 cells (healthy lung fibroblast), with the greatest expression in A549 and PC9 (Fig. 1D and F). A contrary tendency was seen with miR-449a (Fig. 1E). Thus, A549 and PC9 were chosen for the next experiment.

To investigate the role of EGFR-AS1 in NSCLC, siRNAs were used to inhibit EGFR-AS1 expression in A549 and PC9 cells (Fig. 2A and B). The CCK-8 assay showed that EGFR-AS1 markedly reduced A549 and PC9 proliferation (P<0.01; Fig. 2C and D). Furthermore, invasion and migration assays demonstrated that EGFR-AS1 could markedly inhibit A549 and PC9 in tumor cells (Fig. 2E-J).

In order to investigate the influence of miR-449a on A549 and PC9 cells, the influence of miR-449a on tumor growth, invasion and metastasis in NSCLC was investigated (Fig. 3A-D). The results showed that A549 and PC9 were significantly affected (P<0.01) and were also aggressive and metastatic (Fig. 3E and F).

HDAC1 siRNA was used to transfect A549 and PC9 cells and investigate the downregulation of HDAC1 on proliferation, invasion and metastasis (Fig. 4A and B). CCK-8 demonstrated that HDAC1 could significantly inhibit invasion and metastasis of A549, PC9 and A549 cells (P<0.01; Fig. 4C-J).

Using siEGFR-AS1, miR-449a, A549 and PC9 cells were cotransfected to study their HDAC1 expression. Western blotting and qPCR demonstrated partial restoration of HDAC1 in the siEGFR-AS1 + miR-449a inhibitors versus siEGFR-AS1 (Fig. 5 and Fig. S1).

Using ENCORI and UALCAN (30) prediction, it was shown that there were binding sites between EGFR-AS1 and miR-449a, and between miR-449ap and HDAC1 3'-UTR (Fig. 6A). The results of luciferase reporter gene analysis showed that there were targeted regulatory effects between EGFR-AS1 and miR-449a (Fig. 6B) and between miR-449a and HDAC1 3'-UTR (Fig. 6C). miRNAs, which occur in the cytoplasm, form part of RISC (RNA-induced silencing complex). It has been demonstrated that Ago2 is a component of RISC, that is involved in the silencing of the miRNA-mediated gene (33). Subsequently, the SNRNP70 antibody was used as a positive control (Fig. 6E) and RNA immunoprecipitation (RIP) was performed using the Ago2 antibody to analyze whether EGFR-AS1 and miR-449ap were present in RISC. The results showed that Ago2 antibody could enrich EGFR-AS1 and miR-449ap compared to the control (IgG) (Fig. 6D). These results suggest that EGFR-AS1 regulates the miR-449a/HDAC1 pathway through ceRNA in NSCLC.

The results of qPCR showed that among 80 patients with NSCLC, EGFR-AS1 and HDAC1 mRNA were highly expressed in tumor tissues and lowly expressed in paracancerous tissues (Fig. 6A and B). miR-449a was lowly expressed in tumor tissues and highly expressed in paracancerous tissues (Fig. 7C). Furthermore, it was found that the expression levels of EGFR-AS1 and HDAC1 mRNA were negatively correlated with miR-449a (Fig. 7D and E), while the expression levels of EGFR-AS1 was positively correlated with HDAC1 mRNA (Fig. 7F).