LINC01314 suppresses proliferation and invasion via epithelial-to-mesenchymal transition regulation in lung adenocarcinoma

Introduction

Lung cancer remains one of the most prevalent malignancies worldwide, exhibiting poor 5-year survival rates (1). In recent decades, it has emerged as the leading cause of cancer-related mortality, contributing to 18.7% of cancer deaths and accounting for 12.4% of new cases globally (2). Approximately 80–85% of newly diagnosed lung cancers are classified as non-small cell lung cancer (NSCLC), of which adenocarcinoma constitutes 50–60% of cases (3). Despite significant advances in early detection and comprehensive therapeutic strategies, the prognosis for patients with pulmonary adenocarcinoma remains dismal, with a 5year survival rate of only 10–20% in most countries (2,4). Among all causes of death that are associated with pulmonary adenocarcinoma, metastasis is critical.

Long non-coding RNAs (lncRNAs) are RNA molecules exceeding 200 nucleotides in length that lack significant open reading frames and, consequently, do not encode proteins (5). Beyond exhibiting temporal and spatial expression specificity, lncRNAs regulate gene expression at multiple levels, thereby influencing the onset and progression of various diseases, including cancer (6,7). Advances in tumor transcriptome profiling and growing evidence of lncRNA functionality have identified several differentially expressed lncRNAs associated with malignancies. These molecules contribute significantly to cancer development by modulating diverse biological processes (BP), such as genomic imprinting and transcriptional regulation (5,8,9). Accordingly, developing early detection strategies that employ prognostic lncRNA markers, along with novel therapeutic approaches, is imperative.

In our previous study, we employed microarray analysis and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to examine differential lncRNA expression in pulmonary adenocarcinoma tissues relative to adjacent non-cancerous tissues. Our results demonstrated that long intergenic non-protein coding RNA 1314 (LINC01314) was upregulated in 30 paired specimens and that its expression was lower in poorly differentiated adenocarcinoma tissues compared to well- or moderately differentiated ones in Chinese population (10). In the present study, microarray data from the Gene Expression Omnibus (GEO) database and sequencing data from The Cancer Genome Atlas (TCGA) project were downloaded and employed to assess LINC01314 expression levels in patients with pulmonary adenocarcinoma. To evaluate the putative anti-tumor properties of LINC01314, we conducted a series of in vitro assays. Additionally, to elucidate the mechanisms underlying LINC01314’s function, we performed a functional enrichment analysis, and the results were subsequently validated by western blotting. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-867/rc).

Methods

Cell lines and culture

Human pulmonary adenocarcinoma cell lines A549 and H1299 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) at 37 °C in a humidified atmosphere with 5% CO₂.

Cell transfection

The A549 and H1299 cell lines were seeded in 6-well plates at approximately 70% confluence one day prior to transfection. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the culture medium was replaced with RPMI-1640 containing 10% FBS 5 hours post-transfection. The sequences of the LINC01314 small interfering RNA (siRNA) were as follows: sense, 5'-GGACGAAAUGAGAGAACAUTT-3'; anti-sense, 5'-AUGUUCUCUCAUUUCGUCCTT-3'. A negative control (NC) RNA, with no sequence homology to any human gene, was employed; its sequences were: sense, 5'-UUCUCCGAACGUGUCACGUdTdT-3' and anti-sense, 5'-ACGUGACACGUUCGGAGAAdTdT-3'. Both the LINC01314 siRNA and its NC were designed and synthesized by GenePharma (Shanghai, China).

The synthetic LINC01314 complementary DNA (cDNA) was cloned into the pcDNA3.1-EGFP plasmid (Invitrogen, USA) to generate the pcDNA3.1-EGFP-LINC01314 construct, and its structure and fidelity were confirmed by restriction mapping and sequencing. A corresponding NC pcDNA3.1-EGFP construct was similarly generated. Plasmid purification was performed using the PureLink HiPure Plasmid Midiprep Kit (Invitrogen, USA).

Cells in 6-well plates were transfected with either 0.1 nmol of synthetic LINC01314 siRNA or its NC, or with 2 µg of pcDNA3.1-EGFP-LINC01314 or its corresponding NC, and subsequently subjected to further functional assays. Total RNA was harvested 24 hours post-transfection for qRT-PCR analysis, and total protein was collected 48 hours post-transfection for western blot analysis.

Wound-healing assay

A wound-healing assay was conducted to assess the migration potential of transfected cells. Following transfection, cells were grown to near confluence in 6-well plates containing RPMI-1640 supplemented with 10% FBS. In each well, a linear wound was generated by scraping the monolayer with a 200-µL pipette tip. Suspended cells were removed by washing twice with phosphate-buffered saline (PBS; Hyclone, USA), and the wounded monolayer was subsequently incubated in serum-free RPMI-1640 for 24 hours. Photographs of the wound area were captured under a microscope at 0 and 24 hours post-wounding. The migration index was defined as the distance traversed by the cell monolayer over 24 hours to close the wound. All experiments were performed in triplicate.

Cell viability assay

Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; Bimake, Texas, USA) according to the manufacturer’s protocol. Briefly, 3,000 cells per well were seeded into 96-well plates and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. At 12, 24, 48, and 72 hours, 10 µL of CCK-8 reagent was added to each well, followed by a 2-hour incubation. Absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad, California, USA). All experiments were performed in triplicate.

Cell migration and invasion assays

To investigate the potential role of LINC01314 in modulating metastasis in pulmonary adenocarcinoma cell lines A549 and H1299, migration and invasion assays were performed. For the migration assays, 2×10⁴ cells suspended in 200 µL of serum-free medium were seeded into the upper chamber of a transwell insert with an 8-µm pore size (BD Biosciences, San Diego, CA, USA). For the invasion assays, 2×10⁴ cells in 200 µL of serum-free medium were seeded into an upper chamber pre-coated with Matrigel (BD, USA) according to the manufacturer’s protocol, while the lower chamber was filled with medium containing 20% FBS. After 24 hours of incubation at 37 °C, cells remaining on the upper side of the membrane were removed, and those that had migrated to the lower surface were fixed in 95% ethanol and stained with crystal violet. Cells from five randomly selected fields were then counted at 200× magnification. All experiments were performed in triplicate.

RNA isolation and qRT-PCR

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol. The RNA was then reverse-transcribed into cDNA using the PrimeScript RT Master Mix (Takara, Tokyo, Japan). The purity and concentration of both RNA and cDNA were assessed by measuring the optical density at 260/280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Relative expression levels of LINC01314 were calculated using the 2^−ΔΔCt method. Quantitative PCR was performed on a StepOne thermocycler (Life Technologies, Carlsbad, CA, USA) with SYBR Green Mix (ABI, Foster City, CA, USA) under the following cycling conditions: 50 °C for 2 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control.

Protein isolation and western blotting

Total protein was extracted from transfected cells. The cells were washed twice with ice-cold PBS and lysed using radioimmunoprecipitation assay (RIPA) buffer (KeyGene, Wageningen, Holland) supplemented with protease inhibitors at 4 °C for 30 minutes. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat milk for 2 hours at room temperature and then incubated with primary antibodies at 4 °C overnight. Subsequently, the membrane was washed three times with Tris-buffered saline containing 0.1% Tween-20 and incubated with a horseradish peroxidase-conjugated secondary antibody for 2 hours at room temperature. The expression of GAPDH (Bioworld Technology, Louis Park, MN, USA), E-cadherin, ZO-1, vimentin, MMP9, and MMP2 (Cell Signaling Technology, Beverly, MA, USA) was determined according to the manufacturer’s protocol. Enhanced chemiluminescence detection reagent (Thermo Scientific, Waltham, MA, USA) was used to visualize the signals, and protein levels were quantified by normalization to GAPDH. All experiments were performed in triplicate with three technical replicates.

RNA sequencing (RNA-seq) data and gene chip data collecting and analysis

RNA-Seq data for lung adenocarcinoma were downloaded from TCGA-Lung Adenocarcinoma Project (TCGA-LUAD) via the GDC Data Portal (https://portal.gdc.cancer.gov/). Gene chip data were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/) using the search term “lung cancer”, restricted to Homo sapiens and series entries. Datasets were selected based on the following criteria: (I) inclusion of normal tissue samples to enable comparison of LINC01314 expression between tumor and non-tumor tissues; (II) presence of lung adenocarcinoma samples; and (III) a minimum total sample size of 30. Based on these criteria, three datasets were selected: GSE31210, GSE19188, and GSE30219. Detailed dataset characteristics, including sample size and type, are provided in Table S1.

The cancer patients in available datasets were stratified into two groups based on median LINC01314 expression (LINC01314-high and LINC01314-low, cut-off value available in Table S1). Differentially expressed genes (DEGs) were computed using limma package (11) for gene chip data or DESeq2 package (12) for RNA-Seq data with a cutoff of absolute value of log₂fold change (logFC) over 1 and adjusted P value less than 0.05 in R (version 4.4.2). DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using the clusterProfiler package (13) in R with the GO analysis restricted to BP. Genes were ranked by logFC and analyzed via Gene Set Enrichment Analysis (GSEA) using hallmark gene sets from the Molecular Signatures Database (MSigDB, v2024.1; http://www.gsea-msigdb.org) with the clusterProfiler package. Significant pathways were defined by P<0.05, adjusted P<0.25, and absolute value of normalized enrichment score (NES) >1.

To assess the prognostic significance of LINC01314 expression in lung adenocarcinoma, overall survival (OS) and progression-free survival (PFS) were analyzed using online KM Plotter (http://kmplot.com/analysis/) with automatic optimal cutoff selection for GEO datasets, and the R survival package with the median expression value as the cutoff for the TCGA-LUAD dataset. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Statistical analyses

Continuous variables were expressed as means ± standard deviations (SDs) or medians [with interquartile ranges (Q1, Q3)] and were compared using a t-test or Wilcoxon rank sum test, as appropriate. Categorical data were presented as counts and proportions, and differences were assessed using the Chi-squared test or Fisher’s exact test. All statistical analyses were performed and visualized using R (version 4.4.2), and statistical significance was set at P<0.05.

Results

LINC01314 downregulation and its association with clinicopathological characteristics

In a previous study, we employed microarray analysis and qRT-PCR to validate the expression of LINC01314 in pulmonary adenocarcinoma patients (10). In the current study, we further analyzed the TCGA-LUAD cohort and GEO datasets to confirm the expression profile of LINC01314 in pulmonary adenocarcinoma. Consistent with our previous findings, LINC01314 expression was significantly downregulated in pulmonary adenocarcinoma patients, both in the overall TCGA-LUAD dataset and in paired samples (Figure 1A,1B). A similar expression profile was observed in all available GEO datasets, including GSE31210, GSE19188, and GSE30219 (Figure 1C-1E).

Figure 1 LINC01314 expression in lung adenocarcinoma datasets. LINC01314 expression was systematically evaluated across multiple lung adenocarcinoma cohorts. In the TCGA-LUAD cohort, (A) depicts the expression levels in the entire sample set, whereas (B) shows expression in paired tumor-normal samples. (C-E) present data from GEO datasets GSE19188, GSE30219, and GSE31210, respectively. Consistently, LINC01314 expression is uniformly low across all datasets. Intergroup comparisons were performed using t-tests. LINC01314, long intergenic non-protein coding RNA 1314; LUAD, lung adenocarcinoma; TCGA-LUAD, The Cancer Genome Atlas-Lung Adenocarcinoma.

We also investigated the association between LINC01314 expression and clinicopathological characteristics. In the TCGA-LUAD dataset, patients with low LINC01314 expression were significantly younger (P=0.007) and exhibited a higher T stage (P<0.001) than those with high expression. No significant differences were observed in gender, smoking history, ethnicity, synchronous malignancy, mass location, N stage, or M stage. Although patients with low LINC01314 expression tended to have a more advanced tumor node metastasis (TNM) stage, this difference did not reach statistical significance (P=0.10). In the GSE30219 dataset, no significant differences were found with respect to age, gender, T stage, N stage, or M stage. In contrast, the GSE31210 dataset revealed that patients with low LINC01314 expression had a significantly higher TNM stage (P=0.02), while no significant differences were found in age, gender, smoking history, or gene alteration status (Table 1).

Association of LINC01314 expression with survival outcomes in pulmonary adenocarcinoma

We further explored the relationship between LINC01314 expression and survival outcomes using publicly available datasets. The results, summarized in Table 2 and illustrated in Figure 2, show that patients with high LINC01314 expression had significantly improved PFS [hazard ratio (HR) =0.74, 95% confidence interval (CI): 0.56–0.97, P=0.03] and OS (HR =0.72, 95% CI: 0.54–0.96, P=0.02) in the TCGA-LUAD dataset (Figure 2A,2B). In the GEO datasets, patients with low LINC01314 expression exhibited shorter OS (Figure 2C,2D, Figure S1), with statistical significance observed in the GSE31210 cohort (Figure 2D). Additionally, consistent with the TCGA-LUAD findings, patients with low LINC01314 expression in the GSE31210 and GSE30219 datasets demonstrated poorer PFS, although these differences did not reach statistical significance (Figure S1).

Figure 2 Prognostic significance of LINC01314 expression in lung adenocarcinoma. Kaplan-Meier survival analyses were performed based on LINC01314 expression levels. In the TCGA-LUAD cohort, (A,B) illustrate OS and progression-free survival (PFS), respectively. In the GEO datasets, (C) depicts OS for GSE19188; (D) represents OS for GSE31210. Collectively, lower LINC01314 expression is significantly associated with poorer survival outcomes. Intergroup comparisons were performed using log-rank tests. GEO, gene expression omnibus; HR, hazard ratio; LINC01314, long intergenic non-protein coding RNA 1314; OS, overall survival; PFS, progression-free survival; TCGA-LUAD, The Cancer Genome Atlas-Lung Adenocarcinoma.

LINC01314 inhibits cell migration and invasion

To elucidate the role of LINC01314 in lung adenocarcinoma, we performed both knockdown and overexpression assays. A549 and H1299 cells with reduced or enhanced LINC01314 expression were generated, and the relative expression levels were confirmed by qRT-PCR (Figure 3).

Figure 3 Effects of LINC01314 modulation on proliferation, migration, and invasion in lung adenocarcinoma cells. (A) qRT-PCR validation confirms effective knockdown of LINC01314 in A549 and H1299 lung adenocarcinoma cells. (B,C) CCK-8 assays demonstrate that LINC01314 knockdown significantly enhances cellular proliferation in A549 (B) and H1299 (C) cells compared with the NC. (D) qRT-PCR analysis verifies successful overexpression of LINC01314 in both cell lines. (E,F) CCK-8 assays indicate that LINC01314 overexpression markedly suppresses proliferation in A549 (E) and H1299 (F) cells relative to controls. (G-J) Wound healing assays show that reduced LINC01314 expression accelerates cell migration (G,H), whereas overexpression impairs migratory capacity (I,J) compared with NC cells. (K-N) Transwell assays reveal that knockdown of LINC01314 enhances both migratory (K,L) and invasive (M,N) abilities. (O-R) Conversely, overexpression of LINC01314 significantly attenuates migration (O,P) and invasion (Q,R). Data are presented as the mean ± standard deviation from at least three independent experiments. Intergroup comparisons were performed using t-tests. Crystal violet staining; scale bars =200 µm. *, P<0.05. CCK-8, cell counting kit-8; LINC01314, long intergenic non-protein coding RNA 1314; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

As shown in Figure 3, the CCK-8 assay demonstrated that cells with reduced LINC01314 expression exhibited significantly higher absorbance at 450 nm, reflecting enhanced proliferation (Figure 3B,3C). In contrast, cells overexpressing LINC01314 displayed lower optical density at 450 nm, indicative of a diminished proliferative capacity (Figure 3E,3F).

Wound‑healing assays further revealed that the migratory capacity of the cell monolayer—quantified as the distance covered—was significantly increased in cells with LINC01314 knockdown compared to NC and untreated control cells (Figure 3G,3H). Conversely, overexpression of LINC01314 resulted in a marked decrease in wound‑healing capacity relative to the NC and control (Figure 3I,3J).

Moreover, migration and invasion assays demonstrated that cells with LINC01314 knockdown exhibited significantly enhanced migratory (Figure 3K,3L) and invasive (Figure 3M,3N) abilities (P<0.05) compared with the NC and control cells. In contrast, cells overexpressing LINC01314 showed reduced migratory (Figure 3O,3P) and invasive (Figure 3Q,3R) capacities relative to the NC and control (P<0.05). Collectively, these data suggest that decreased expression of LINC01314 promotes proliferation, migration, and invasion in pulmonary adenocarcinoma cells.

Impact of LINC01314 on cell division, extracellular matrix (ECM), and EMT inhibition

To explore the potential mechanisms underlying LINC01314 function, we stratified each dataset based on the median expression of LINC01314 and performed differential gene expression analysis to identify DEGs. These DEGs were then subjected to KEGG pathway and Gene Ontology biological process (GO-BP) enrichment analyses, with the top 10 significant terms presented in Figure 4. In the GSE19188 dataset, no significantly enriched terms were identified. In contrast, in GSE30219, KEGG analysis revealed that the DEGs were associated with the cell cycle, calcium signaling pathway, and neuroactive ligand-receptor interaction, while GO-BP analysis indicated enrichment in ECM organization, non-membrane-bounded organelle assembly, and mitosis-related processes (e.g., mitotic nuclear division and cell cycle phase transition). In GSE31210, KEGG analysis showed that the DEGs were related to ECM-receptor interaction, several system-related pathways (including renin secretion, protein digestion and absorption, and adrenergic signaling in cardiomyocytes), and signal transduction, whereas GO-BP analysis highlighted terms related to ECM organization, connective tissue development, and cell growth. Similarly, analysis of the TCGA-LUAD dataset demonstrated that DEGs were enriched in pathways such as ECM-receptor interaction, lipid metabolism, cell cycle, and protein digestion and absorption (KEGG), as well as in ECM organization, regulation of membrane potential, and mitosis-related processes (GO-BP). Collectively, these enrichment analyses suggest that the common DEGs are primarily involved in regulation of the ECM and cell proliferation. Furthermore, GSEA using the hallmark gene sets from the MSigDB was performed on all four datasets. Ranking by normalized NES and selection of the top 10 terms (Figure 4C) revealed that E2F targets, G2/M checkpoint, epithelial-to-mesenchymal transition (EMT), glycolysis, and Myc targets V1 were altered in at least three datasets. Notably, the EMT pathway was the only pathway consistently activated across all four datasets when LINC01314 expression was downregulated, suggesting that reduced LINC01314 expression may promote EMT.

Figure 4 Functional enrichment analysis stratified by LINC01314 expression. Datasets were stratified into high and low LINC01314 expression groups. DEGs were identified between these groups, and their log₂fold changes were calculated. (A) GO enrichment analysis revealed that common DEGs are predominantly involved in ECM organization and cell proliferation. (B) KEGG pathway analysis corroborated these findings. (C) GSEA demonstrated that pathways including E2F targets, G2/M checkpoint, EMT, glycolysis, and Myc targets V1 were significantly altered in at least three independent datasets. Notably, the EMT pathway was consistently activated across all four datasets in the context of reduced LINC01314 expression. DEGs, differentially expressed genes; ECM, extracellular matrix; EMT, epithelial-to-mesenchymal transition; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; LINC01314, long intergenic non-protein coding RNA 1314; NES, normalized enrichment score; TCGA-LUAD, The Cancer Genome Atlas-Lung Adenocarcinoma.

Effects of LINC01314 on the EMT and MMPs expression

To further confirm the impact of LINC01314 on the EMT pathway, we performed western blot analysis. The results demonstrated that LINC01314 knockdown led to decreased expression of the epithelial markers E-cadherin and ZO-1, accompanied by an increase in vimentin expression (Figure 5A-5C). In contrast, LINC01314 overexpression resulted in increased levels of E-cadherin and ZO-1 together with reduced vimentin expression, suggesting that LINC01314 suppresses EMT. Furthermore, knockdown of LINC01314 was associated with elevated expression of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) (Figure 5D-5F), whereas overexpression correlated with decreased levels of these matrix metalloproteinases (MMPs).

Figure 5 Modulation of EMT markers and MMP expression by LINC01314 in lung adenocarcinoma cells. (A-C) Western blot analysis in A549 and H1299 cells transfected with LINC01314 siRNA demonstrated that knockdown of LINC01314 leads to a significant decrease in the epithelial markers E-cadherin and ZO-1, accompanied by an increase in the mesenchymal marker vimentin and upregulation of matrix metalloproteinases MMP2 and MMP9. (D-F) In contrast, overexpression of LINC01314 via the pcDNA3.1-EGFP-LINC01314 plasmid resulted in increased levels of E-cadherin and ZO-1 and a concomitant reduction in vimentin, MMP2, and MMP9 expression. Densitometric quantification was performed by normalizing the intensity of target protein bands to that of GAPDH. Data are expressed as the mean ± standard deviation from at least three independent experiments. Intergroup comparisons were performed using t-tests. *, P<0.05 compared with the negative control. EMT, epithelial-to-mesenchymal transition; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LINC01314, long intergenic non-protein coding RNA 1314; MMP, matrix metalloproteinase; NC, negative control; siRNA, small interfering RNA.

Discussion

The occurrence and progression of tumors are widely regarded as resulting from the cooperative action of multiple genes (14). Among these, certain genes exhibit similar expression patterns across different tumor types, thereby influencing the biological behavior of tumor cells through specific signaling pathways. In particular, some of these genes play pivotal roles in tumor progression, and their inhibition may suppress malignant phenotypes, thereby arresting tumor development. Therapeutic strategies that target these critical genes—either by inhibiting or enhancing their activity—are collectively known as targeted therapies and have been implemented in clinical practice. For example, targeted therapy against epidermal growth factor receptor (EGFR) has demonstrated significant clinical success and is recommended as a first-line treatment for patients harboring EGFR driver mutations (15).

LINC01314 is one of many long intergenic non-coding RNAs that emerged during the post-genomic era as high-throughput sequencing and transcriptome analyses expanded the catalog of nonproteincoding transcripts, with comprehensive datasets from the early 2010s postulating its initial annotation alongside numerous other lncRNAs (16,17). In a study by Chen et al. (18), LINC01314 was recognized as one of the top 10 lncRNAs with the highest diagnostic value in lung squamous cell carcinoma, demonstrating its potential to distinguish early from advanced stages of the disease. Similarly, Morovat et al. (19) reported that LINC01314 may aid in the diagnosis of thyroid cancer. In the present study, we found that LINC01314 was significantly downregulated in lung adenocarcinoma and was associated with a poor prognosis, suggesting its potential as a prognostic factor.

In the present study, we evaluated the functional impact of modulating LINC01314 expression in pulmonary adenocarcinoma cell lines A549 and H1299 by transfecting cells with LINC01314-targeting siRNA or an overexpression plasmid. Our results demonstrated that overexpression of LINC01314 significantly inhibited cell proliferation, migration, and invasion, whereas its downregulation enhanced these oncogenic characteristics.

We further investigated the underlying mechanisms using bioinformatics analyses. GO and KEGG pathway enrichment analyses consistently identified ECM organization as a prominent process across multiple datasets. Moreover, GSEA revealed that the EMT pathway was activated in all datasets upon reduced LINC01314 expression.

The ECM actively governs cell behavior by providing both mechanical and biochemical cues. On one hand, changes in ECM organization—such as increased stiffness or altered composition (e.g., variations in collagen, fibronectin, or vitronectin levels)—can initiate the EMT (20). Mechanical stress sensed by cell surface receptors (e.g., β1 and β3 integrins) subsequently activates intracellular signaling pathways (such as TGF-β, PI3K-AKT, and focal adhesion kinase pathways) that downregulate epithelial markers like E-cadherin and upregulate mesenchymal markers such as vimentin (21). Thus, a remodeled or rigidified ECM serves as both a trigger and facilitator of EMT, promoting a shift toward a more migratory and invasive cellular phenotype (22). On the other hand, once epithelial cells undergo EMT, they no longer passively receive ECM-derived signals; instead, they actively remodel their microenvironment. EMT-transformed cells secrete MMPs and other enzymes that degrade and reorganize ECM components, thereby loosening cell-cell adhesions and altering tissue architecture to create pathways that support invasion and metastasis (23). Moreover, because the ECM can sequester and later release growth factors such as TGF-β, its degradation further sustains and amplifies EMT signaling, creating a feedback loop that reinforces cancer cell aggressiveness (24).

The bioinformatics analysis indicated that LINC01314 may function via the EMT pathway. These findings were validated by western blot analysis, which demonstrated that knockdown of LINC01314 induced changes in EMT marker expression—resulting in reduced levels of E‑cadherin and ZO-1 and increased expression of vimentin. In contrast, overexpression of LINC01314 led to elevated E‑cadherin and ZO-1 levels and diminished vimentin expression. Furthermore, LINC01314 knockdown was associated with increased expression of MMP2 and MMP9, whereas its overexpression correlated with decreased levels of these MMPs. These correlations between LINC01314 and the EMT pathway have also been observed in previous studies. For example, Lv et al. (25) reported that overexpression of LINC01314 reduced the migration of hepatoblastoma cells, and Tang et al. (26) demonstrated that overexpression inhibited migration and invasion of gastric cancer cells—corresponding to decreased N-cadherin and increased E-cadherin levels, which are well-known EMT markers (27). While LINC01314 has also been linked to the Wnt/β-catenin pathway in gastric cancer (26), our comprehensive GSEA across all 50 pathways included in the hallmark gene set did not reveal significant activation of this pathway in lung adenocarcinoma datasets under predefined thresholds. Accordingly, we focused on the EMT pathway, which was consistently enriched across all datasets. This prioritization was further influenced by limited research resources. Future studies are warranted to elucidate the potential involvement of Wnt/β-catenin signaling in LINC01314-mediated effects in lung adenocarcinoma.

Overall, our study indicates that LINC01314 is downregulated in lung adenocarcinoma and that its reduced expression is associated with poor prognosis. Moreover, LINC01314 modulates the biological behavior of lung adenocarcinoma cells—potentially exerting its effects via the EMT pathway—thereby suggesting its promise as a therapeutic target.

Conclusions

Our findings demonstrate that modulating the expression of LINC01314 in vitro significantly alters the proliferation, migration and invasion potential of pulmonary adenocarcinoma cell lines via regulation of EMT-related genes. These results indicate that LINC01314 may serve as a valuable diagnostic and prognostic marker for pulmonary adenocarcinoma and a promising target for lncRNA-mediated therapies.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-867/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-867/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-867/prf

Funding: The present study was supported by the Bethune Charitable Foundation (No. BCF-XD-ZL-20220118-003), Promoting New Life Public Welfare Projects (No. GYLZH22), Lung Cancer Targeted Therapy Standardized Diagnosis and Treatment Project (No. PM202410130102) and the Science Pre-research Foundation of the Second Affiliated Hospital of Soochow University (No. SDFEYHT2224).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-867/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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