LAMA3 overexpression enhances proliferation, migration and invasion in esophageal squamous cell carcinoma based on bioinformatics and experimental validation

Abstract

Esophageal squamous cell carcinoma (ESCC) is a highly lethal malignancy and remains a major public health burden worldwide, particularly in China, where both incidence and mortality rates are among the highest globally. Recent studies suggest that laminin subunit alpha 3 (LAMA3), a component of the basement membrane, may promote tumor progression; however, its specific role in ESCC remains unclear. In this study, we analyzed public RNA-sequencing data from TCGA and TIMER to evaluate LAMA3 expression and its clinical relevance in ESCC. We also validated LAMA3 protein and mRNA expression in clinical samples and cell lines using immunohistochemistry and RT-qPCR. Using siRNA, we established LAMA3-knockdown ESCC cell models and assessed cell proliferation, colony formation, migration, and invasion in vitro. LAMA3 expression was 2.4-fold higher in ESCC tumor tissues than in adjacent normal tissues (p < 0.001). High LAMA3 levels were associated with worse overall survival and disease-free survival (p < 0.05). Knockdown of LAMA3 suppressed cell proliferation by 57% (p < 0.001), migration by 49% (p < 0.001), and invasion by 47% (p < 0.001).Pathway enrichment analysis indicated involvement of LAMA3 and its co-expressed genes in cell adhesion, extracellular matrix organization, and the PI3K-AKT pathway. In summary, our results demonstrate that LAMA3 is a major promoter of ESCC progression and a potential biomarker and therapeutic target.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-08026-x.

Introduction

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer with poor prognosis and high mortality rates, especially in China^1,2. Despite advances in diagnosis and treatment, most ESCC patients are diagnosed at an advanced stage, resulting in low five-year survival rates³. These challenges highlight the need for reliable biomarkers and new therapeutic targets to improve patient outcomes⁴.

Laminin subunit alpha 3 (LAMA3) encodes a major component of the basement membrane and plays a crucial role in cell adhesion, migration, and tissue architecture^5–7. Abnormal LAMA3 expression has been reported in several cancers, where it often correlates with tumor progression and metastasis^8–12. However, the clinical significance and biological function of LAMA3 in ESCC remain poorly understood¹³.

In this study, we comprehensively investigated LAMA3 expression in ESCC using public transcriptomic datasets and validated these findings in patient samples and cell lines. We also explored the effects of LAMA3 knockdown on tumor cell behavior and analyzed the underlying molecular pathways. Our aim was to clarify the role of LAMA3 in ESCC progression and to assess its potential as a diagnostic, prognostic, and therapeutic biomarker.

Materials and methods

Bioinformatics analysis

RNA-seq data acquisition and processing

RNA-seq data and clinical information were retrieved from the TCGA-ESCA/ESCC project in The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov) and Genotype-Tissue Expression‌ (GTEx) databases (https://m.gtexportal.org/).A total of 174 esophageal squamous carcinoma tumor samples and 11 adjacent normal tissue samples were included. Raw sequencing data were processed through the STAR workflow and normalized to transcripts per million (TPM) format¹⁴. Clinically irrelevant or duplicate entries were systematically excluded. Univariate Cox regression was first performed to evaluate the association between LAMA3 expression (log2-transformed TPM values) and overall survival (OS). Variables with p < 0.1 in univariate analysis, including age (dichotomized at 60 years), clinical stage (I–IV), gender, and smoking history (pack-years ≥ 20vs.<20), were incorporated into multivariate Cox proportional hazards models. Variance inflation factors (VIF) were calculated to assess multicollinearity (VIF < 2.5 considered acceptable). Data analysis and visualization were conducted using R software (v4.2.1), with statistical calculations performed using the “stats” and “car” packages.

Identification of differentially expressed mRNAs (DEmRNAs)

Normalized and log-transformed TPM data were analyzed using the “limma” package¹⁵. Differentially expressed mRNAs were identified with thresholds of |log2 fold change (logFC)| > 1.5 and adjusted P-value < 0.05. mRNA expression patterns of LAMA3 were visualized using boxplots and paired scatterplots generated by the “ggplot2” package¹⁶.

Survival and ROC analysis

Prognostic relevance of LAMA3 in ESCC was evaluated using the “survival” package. Patients were stratified into high- and low-expression groups based on median LAMA3 expression. Kaplan-Meier survival curves were generated via the Kaplan-Meier Plotter platform (https://kmplot.com/). Receiver operating characteristic (ROC) analysis was performed using the “pROC” package, with validation datasets sourced from XIANTAO (https://www.xiantaozi.com/).

Single-gene correlation screening and PPI network construction

Pearson correlation analysis with Benjamini-Hochberg (BH) correction was applied to assess associations between LAMA3 and other molecules in ESCA. Significant interactions were filtered using thresholds of |Pearson correlation coefficient| > 0.3 and adjusted P < 0.05. Protein-protein interaction (PPI) networks for LAMA3 were constructed using the STRING database (http://string-db.org), and results were ranked by descending correlation strength.

Functional enrichment analysis via GO, KEGG, and GSEA

Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed to elucidate biological pathways associated with LAMA3^17-20. GO and KEGG analyses were automated using the “clusterProfiler” package, with results visualized as bubble plots²¹. For GSEA (v4.2.3), ESCA patients were divided into high- and low-expression cohorts based on median LAMA3 levels²². Gene sets with sizes between 5 and 5,000 were analyzed, and statistical significance was defined as P < 0.05.

Immune infiltration analysis

The CIBERSORT (https://cibersortx.stanford.edu/) algorithm was employed to quantify tumor-infiltrating immune cells (TIICs) in ESCC microenvironments²³. Correlations between LAMA3 expression and 22 TIIC subtypes were calculated using Pearson coefficients²⁴.

Immunohistochemical (IHC) analysis

A tissue microarray containing 1.5-mm cores of esophageal carcinoma specimens with matched clinicopathological information (Xinchao Biotechnology Co., Ltd.) was analyzed using tissue microarray technology and immunohistochemistry. The array comprised 50 esophageal squamous cell carcinoma specimens and 55 adjacent normal esophageal mucosa samples (Table S1). Stained sections were quantitatively evaluated using CaseViewer 2.4 software (3DHISTECH). The integrated optical density (IOD) was calculated by summing all pixel density values within the target area and dividing by the corresponding tissue area to obtain mean optical density (MOD) values. Protein expression levels of LAMA3 in tumor and adjacent tissues were systematically compared through this quantitative analysis.

Cell culture and transfection

Four human esophageal cancer cell lines (Eca-109, Kyse-30, Kyse-150, TE-1; Zhong Qiao Xin Zhou Co., Ltd.) were maintained in DMEM supplemented with 10% fetal bovine serum and 100 IU/mL penicillin/streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. Three specific siRNA sequences targeting LAMA3 (si-1, si-2, si-3) and scrambled siRNA control (NC: Negative Control) (Gene Pharma Co., Ltd.) were transfected into logarithmically growing cells using Lipofectamine 2000 (Thermo Fisher Scientific). Knockdown efficiency was validated at both mRNA and protein levels 24 and 48 h post-transfection²⁵. siRNA sequences:

LAMA3-si-1: 5’-CCCUCCAUACAAAGGUUGUAUTT-3’ (sense), 5’-AUACAACCUUUGTAUGGAGGGTT-3’ (antisense)

LAMA3-si-2: 5’-CGGGCACUAAGAACUCCUUUATT-3’ (sense), 5’-UAAAGGAGUUCUUAGUGCCCGTT-3’ (antisense)

LAMA3-si-3: 5’-CGGGAUCAUAAAGGCUUGUAUTT-3’ (sense), 5’-AUACAAGCCUUUAUGAUCCCGTT-3’ (antisense)

Quantitative real-time PCR (RT-qPCR)

Total RNA was isolated using an RNA extraction kit (Golden Bridge Co., Ltd.) and quantified by NanoDrop spectrophotometry (Thermo Fisher Scientific). Reverse transcription was performed using the HiScript II Reverse Transcriptase kit (Novizan Co., Ltd.). LAMA3 expression was analyzed using β-actin as endogenous control with the following cycling parameters: 95 °C for 10 s, 60 °C for 20 s (40 cycles). Relative quantification was calculated using the 2 − ΔΔCt method. Primer sequences (Biosune Co., Ltd.):

LAMA3: Forward 5’-GCGAATTCAATGAAACTAGACATGACTGGG-3’, Reverse 5’-TAGGATCCTCAGACACACAGGTCCCCACT-3’

β-actin: Forward 5’-CATGTACGTTGCTATCCAGGC-3’, Reverse 5’-CTCCTTAATGTCACGCACGAT-3’

Western blot analysis

Total proteins were extracted using RIPA lysis buffer and quantified via BCA assay. Equal protein amounts were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk. Membranes were incubated overnight at 4 °C with primary antibodies against LAMA3 (1:5,000; Proteintech) and α-Tubulin (1:5,000; Proteintech), followed by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000) incubation for 2 h at room temperature. Protein bands were visualized using ECL substrate and quantified by ImageJ software.

Cell proliferation assay (CCK-8)

Cells were seeded in 96-well plates (1 × 10⁴ cells/well) and cultured under standard conditions. CCK-8 reagent (Beyotime Co., Ltd.) was added at 0, 24, 48, and 72 h timepoints. Absorbance at 450 nm was measured after 4 h incubation using a microplate reader.

Colony formation assay

Single-cell suspensions (1,000 cells/well) were plated in 6-well plates and cultured for 14 days. Colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted. Colony formation rate was calculated as: (Number of colonies/Inoculated cell number) × 100%.

Transwell migration assay

Cell suspensions (4 × 10⁵ cells/mL in serum-free medium) were added to upper chambers (LABSELECT). Lower chambers contained complete medium. After 48 h incubation, migrated cells were fixed, stained, and counted under light microscopy.

Wound healing assay

Confluent monolayers in 6-well plates (2 × 10⁵ cells/well) were scratched with sterile pipette tips. Wound closure was monitored at 0, 24, and 48 h using phase-contrast microscopy. Healing rate was calculated as: [(Initial area − Area at time t)/Initial area] × 100%.

Cell invasion assay

Cell invasion was assessed using the transwell assay. Boyden chamber plates with an 8 μm pore size were used to evaluate cell migration. In the upper compartment, 1 × 10⁵ cells were seeded and cultured in 200 µL serum-free medium. Meanwhile, 20% FBS medium was added to the lower chamber. After 24 h of incubation, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.The number of invading cells was quantified using an Olympus micro­scope.

Statistical analysis

All experiments were performed in triplicate. Data were analyzed using GraphPad Prism 10 and SPSS 29.0.1.0. TCGA data processing employed R packages. Statistical significance (P < 0.05) was determined by chi-square test (IHC), independent samples t-test (two-group comparisons), and one-way analysis of variance (one-way ANOVA) for comparisons involving three or more groups. For multiple-group comparisons, one-way ANOVA was first conducted to assess overall significance, with false discovery rate (FDR) correction applied (α = 0.05). If the ANOVA indicated statistically significant differences, Tukey’s honestly significant difference (HSD) test was subsequently performed for pairwise group comparisons to control for Type I error inflation due to multiple testing.

Results

Bioinformatics analysis of LAMA3 in ESCA

Pan-Cancer and ESCA-Specific expression patterns

Integrated analysis of TCGA and GTEx datasets revealed elevated LAMA3 expression in multiple malignancies, including ACC, BRCA, CESC, CHOL, COAD, DLBC, ESCA, FPPP, HNSC, KICH, KIPAN, KIRC, KIRP, LAML, LIHC, OV, PAAD, PCPG, PRAD, STAD, and THYM. Conversely, reduced expression was observed in GBM, LGG, LUAD, READ, SKCM, TGCT, THCA, UCEC, and UCS (Fig. 1A; Table 1). Paired-sample analysis confirmed these trends (Fig. 1B). ESCA-specific analysis further validated elevated LAMA3 expression in tumor tissues relative to both normal (Fig. 1C) and adjacent non-tumor tissues (Fig. 1D). Radar plot quantification demonstrated significantly greater LAMA3 expression areas in tumor tissues compared to normal counterparts across 33 cancer types (Fig. 1E).

Fig. 1.

LAMA3 Expression in TCGA Database (A) Pan-cancer expression profile of LAMA3 across TCGA and GTEx datasets. (B) LAMA3 expression in paired tumor and adjacent normal tissue samples. (C-D) LAMA3 expression in esophageal carcinoma (ESCA): (C) unpaired samples, (D) paired samples. (E) Radar plot depicting LAMA3 expression differences between tumor and non-tumor tissues. ∗p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant.

Table 1.

The abbreviations of cancers in the TCGA.

ACC	Adrenocortical carcinoma	LUSC	Lung squamous cell carcinoma
BLCA	Bladder Urothelial Carcinoma	MESO	Mesothelioma
BRCA	Breast invasive carcinoma	OV	Ovarian serous cystadenocarcinoma
CESC	Cervical squamous cell carcinoma and endocervical adenocarcinoma	PAAD	Pancreatic adenocarcinoma
CHOL	Cholangiocarcinoma	PCPG	Pheochromocytoma and Paraganglioma
COAD	Colon adenocarcinoma	PRAD	Prostate adenocarcinoma
ESCA	Esophageal carcinoma	READ	Rectum adenocarcinoma
GBM	Glioblastoma multiforme	SARC	Sarcomav
HNSC	Head and Neck squamous cell carcinoma	SKCM	Skin Cutaneous Melanoma
KICH	Kidney Chromophobe	STAD	Stomach adenocarcinoma
KIRC	Kidney renal clear cell carcinoma	TGCT	Testicular Germ Cell Tumors
KIRP	Kidney renal papillary cell carcinoma	THCA	Thyroid carcinoma
LAML	Acute Myeloid Leukemia	THYM	Thymoma
LGG	Brain Lower Grade Glioma	UCEC	Uterine Corpus Endometrial Carcinoma
LIHC	Liver hepatocellular carcinoma	UCS	Uterine Carcinosarcoma
LUAD	Lung adenocarcinoma	UVM	Uveal Melanoma

Diagnostic and prognostic significance of LAMA3

ROC curve analysis (TCGA/GTEx cohort) identified strong diagnostic potential for LAMA3 in ESCA (AUC = 0.889; Fig. 2A). Patients were stratified into high- and low-expression groups based on median LAMA3 levels. Kaplan-Meier survival curves revealed significantly reduced overall survival (OS) and disease-free survival (DFS) in the high-expression group (P < 0.05; Fig. 2C-D). A prognostic nomogram incorporating LAMA3 expression demonstrated robust predictive accuracy for ESCA outcomes (Fig. 2B).

Co-expression network and functional enrichment analysis of LAMA3 in ESCC

To further elucidate the potential biological functions of LAMA3 in ESCC, we performed a co-expression analysis based on TCGA-ESCC data. As shown in Fig. 3A, the top 30 genes most significantly correlated with LAMA3 expression were identified and visualized in a heatmap. These genes, including LAMC2, LAMB3, ITGB4, EHBP1L1, and ITGA3, are primarily involved in cell adhesion, extracellular matrix organization, and cytoskeleton remodeling. Notably, many of these co-expressed genes have been previously implicated in tumor cell migration and invasion, suggesting a potential role for LAMA3 in promoting malignant phenotypes in ESCC. Downstream GO and KEGG pathway enrichment analyses of these co-expressed genes revealed significant enrichment in pathways such as focal adhesion, PI3K-AKT signaling, MAPK signaling, and regulation of the actin cytoskeleton (Fig. 3B-E). These results support the hypothesis that LAMA3 and its co-expressed genes participate in key oncogenic signaling pathways and structural remodeling processes that facilitate tumor cell proliferation, migration, and invasion.

Functional pathway enrichment and immune infiltration analyses of LAMA3 in ESCC

To explore the molecular pathways associated with LAMA3 in ESCC, Gene Set Enrichment Analysis (GSEA) was performed. The results indicated that high LAMA3 expression was significantly enriched in pathways related to focal adhesion, PI3K-AKT-mTOR signaling, alpha 6 beta 4 signaling, JAK-STAT signaling, and calcium signaling (Fig. 4A-B). These pathways are closely associated with tumor cell adhesion, migration, and signal transduction.

Fig. 2.

Diagnostic and Prognostic Role of LAMA3 in ESCC(A) Receiver operating characteristic (ROC) curve illustrating the diagnostic performance of LAMA3 in ESCC.(B) Prognostic nomogram incorporating LAMA3 expression for ESCC outcome prediction.(C-D) Kaplan-Meier (KM) survival analysis of LAMA3 expression in ESCC: (C) Overall survival (OS), (D) disease-free survival (DFS).

Furthermore, analysis of the tumor immune microenvironment revealed that LAMA3 expression was significantly correlated with the infiltration levels of specific immune cell subsets (Fig. 4C-D). In particular, LAMA3 was positively correlated with CD56^dim^ NK cells (R = 0.224, P < 0.01) and Th1 cells (R = 0.177, P < 0.05), but negatively correlated with plasmacytoid dendritic cells (pDC) (R = -0.164, P < 0.05). These results suggest that LAMA3 may play a role in modulating tumor immunity and influencing immune cell composition within the ESCC microenvironment.

Differential LAMA3 expression in esophageal carcinoma tissues

Immunohistochemical (IHC) analysis of pathologically confirmed esophageal carcinoma specimens (n = 55) revealed stronger LAMA3 immunoreactivity in tumor tissues compared to adjacent non-tumor tissues (Fig. 5A, C-D; Table S1). Quantitative evaluation demonstrated significantly higher LAMA3 expression in tumor tissues (23.90 ± 35.13) versus adjacent tissues (15.65 ± 13.01, P < 0.05; Fig. 5B).

Fig. 3.

GO and KEGG Pathway Analysis of LAMA3 Co-Expressed Genes in ESCC(A) Heatmap of genes co-expressed with LAMA3 in ESCC.(B) KEGG pathway enrichment analysis.(C-E) Gene Ontology (GO) enrichment analysis: (C) Biological Process (BP), (D) Cellular Component (CC), (E) Molecular Function (MF).

LAMA3 expression in normal and malignant esophageal cell lines

RT-qPCR analysis identified differential LAMA3 mRNA levels across cell lines (Fig. 6A). Kyse-30 and Kyse-150 cells exhibited significantly elevated LAMA3 expression compared to normal esophageal epithelial cells (HEEC; P < 0.001). These two cell lines were subsequently selected for knockdown experiments.

Fig. 4.

GSEA and Immune Infiltration Analysis of LAMA3 in ESCC (A) Gene Set Enrichment Analysis (GSEA) of LAMA3-related signaling pathways. (B) Dendrogram of enriched signaling pathways identified by GSEA. (C) Bubble plot illustrating immune infiltration patterns associated with LAMA3 expression. (D) Stacked bar chart showing the proportion of immune cell populations stratified by LAMA3 expression levels. ∗p < 0.05, ∗∗ p < 0.01; ns, not significant.

Establishment of LAMA3-knockdown models

Kyse-30 and Kyse-150 cells were transfected with three siRNA sequences (si-1, si-2, si-3). qPCR analysis at 24 h post-transfection confirmed significant LAMA3 mRNA reduction versus siRNA-negative control (si-NC; P < 0.05; Fig. 6B-C). Western blot at 48 h validated protein-level knockdown, with si-1 and si-2 showing the most robust suppression (Fig. 6D-E). Due to the poor effect of the siLAMA3-3 group, we did not use it as an experimental subject later, and the other two sequences were used for subsequent phenotypic assays.

Fig. 6.

LAMA3 Expression Levels and Knockdown Validation in ESCC Cell Lines (A) LAMA3 expression across different ESCC cell lines. (B-C) RT-qPCR validation of LAMA3 knockdown: (B) Kyse-30, (C) Kyse-150. (D-E) Western blot confirmation of LAMA3 knockdown: (D) Kyse-30, (E) Kyse-150. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

LAMA3 regulates ESCC cell proliferation

CCK-8 assay

Quantitative analysis demonstrated time-dependent proliferation inhibition in LAMA3-deficient cells. Kyse-30 knockdown groups exhibited 57.3% ± 8.9% (NC: 1.79 ± 0.07 vs KD: 0.76 ± 0.11) (NC: Negative Control), and (KD: Knockdown), 16.2% ± 5.7% (NC: 1.85 ± 0.09 vs KD: 1.55 ± 0.18), and 25.4% ± 4.3% (NC: 3.41 ± 0.26 vs KD: 2.54 ± 0.28) reductions in viability at 24 h, 48 h, and 72 h, respectively (all P < 0.001; Fig. 7A). Kyse-150 cells showed comparable suppression (44.1% ± 6.2% [NC: 1.73 ± 0.05 vs KD: 0.97 ± 0.21], 35.7% ± 5.9% [NC: 1.75 ± 0.08 vs KD: 1.13 ± 0.22], 42.8% ± 7.1% [NC: 3.25 ± 0.25 vs KD: 1.86 ± 0.31]; P < 0.001; Fig. 7B), confirming sustained anti-proliferative effects.

Fig. 7.

Impact of LAMA3 Knockdown on ESCC Cell Proliferation(A-B) CCK-8 assay assessing cell viability: (A) Kyse-30, (B) Kyse-150.(C-F) Colony formation assay evaluating proliferative capacity: (C, E) Kyse-30, (D, F) Kyse-150.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Colony formation assay

Clonogenic capacity was profoundly impaired in knockdown groups. Kyse-30 siLAMA3-1/siLAMA3-2 groups formed 567 ± 1.51 and 582 ± 1.73 colonies versus 796 ± 2.46 in NC (P < 0.001; Fig. 7C, E). Kyse-150 displayed similar suppression patterns (627 ± 1.66 and 645 ± 1.71 vs. 851 ± 2.67; P < 0.001; Fig. 7D, F). Residual colonies in knockdown groups exhibited 31.6% ± 3.2% (Kyse-30) and 26.3% ± 2.9% (Kyse-150) reductions in pixel area versus NC (Fig. 7C-D, insets), indicating compromised proliferative potential.

LAMA3 modulates ESCC cell migration

Wound healing assay

Scratch closure was systematically impaired in LAMA3-deficient cells. Kyse-30 siLAMA3-1/siLAMA3-2 groups achieved 18.8% ± 3.6% and 15.2% ± 1.4% wound closure at 24 h versus 40.8% ± 2.9% in NC (Δ = -53.9% and − 62.7%, P < 0.001; Fig. 8A, C). Kyse-150 knockdown cells showed 29.3% ± 0.7% and 28.3% ± 3.6% closure versus 44.4% ± 4.6% in NC (Δ = -34.0% and − 36.3%, P < 0.001; Fig. 8B, D). Persistent inhibition was observed at 48 h with 25.2% ± 4.0% (Kyse-30) and 48.4% ± 4.1% (Kyse-150) closure in knockdown groups versus 46.3% ± 3.2% and 56.8% ± 2.4% in respective NC controls.

Fig. 8.

Impact of LAMA3 Knockdown on ESCC Cell Proliferation(A-B) CCK-8 assay assessing cell viability: (A) Kyse-30, (B) Kyse-150.(C-F) Colony formation assay evaluating proliferative capacity: (C, E) Kyse-30, (D, F) Kyse-150.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Transwell migration assay

Transwell analysis showed that migrating cells were reduced to 66.7% (Kyse-30) and 38.8% (Kyse-150) in the siLAMA3 group compared to the NC group, respectively (P < 0.001; Fig. 8E-F). The migration rate was inversely proportional to the level of LAMA3 expression.

LAMA3 suppresses ESCC cell invasion

Transwell invasion assays demonstrated significant suppression of invasive potential. Kyse-30 siLAMA3-1/siLAMA3-2 groups showed 115 ± 4.97 and 121 ± 9.07 invading cells versus 226.33 ± 7.02 in NC (Δ = -49.2% and − 46.5%, P < 0.001; Fig. 9A-B). Kyse-150 knockdown cells exhibited 131 ± 5.52 and 145.33 ± 7.37 invading cells versus 257.67 ± 15.52 (Δ = -49.1% and − 43.6%, P < 0.001).

Fig. 5.

Immunohistochemical Staining of LAMA3 in Esophageal Carcinoma Tissues (A) Representative immunohistochemical (IHC) staining images of LAMA3 in 55 ESCC and 50 paraneoplastic. (B) Quantification of LAMA3 expression in ESCC versus adjacent tissues. (C-D) Detailed immunohistochemical images: (C)paraneoplastic, (D) ESCC tissue.

Fig. 9.

Effect of LAMA3 Knockdown on ESCC Cell Invasion (A) Transwell invasion assay evaluating invasive capacity in Kyse-30 cells. (B) Quantification of invasion assay results across different ESCC cell lines.

Discussion

Esophageal cancer remains a malignancy with high incidence and mortality rates globally, particularly in China, where ESCC constitutes over 90% of cases²⁶. Despite advancements in medical technology, early diagnosis and effective treatment of esophageal cancer remain significant challenges²⁷. The progression of this disease is influenced by various factors, including genetic predisposition, environmental exposure, and lifestyle choices. Studies have indicated that tumor microenvironment alterations and enhanced cell proliferation and migration play crucial roles in esophageal cancer pathogenesis, emphasizing the need for novel biomarkers to improve prognosis and treatment outcomes^28,29.

This study investigated the expression of LAMA3 in ESCC and its impact on tumor cell proliferation and migration. Bioinformatics analyses of TCGA and TIMER datasets revealed that LAMA3 was significantly upregulated in ESCC tissues and negatively correlated with patient survival. Immunohistochemical and cellular assays further confirmed the oncogenic role of LAMA3 in ESCC progression. These findings provide theoretical evidence supporting LAMA3 as a potential biomarker for early diagnosis, prognosis assessment, and targeted therapy in ESCC^30–32. However, it should be noted that the current conclusions are primarily derived from clinical datasets and in vitro models, which cannot fully recapitulate the dynamic interactions between tumor cells and the immune microenvironment in vivo.

This study characterized the functional impact of LAMA3 overexpression on ESCC progression, demonstrating its significant association with enhanced tumor aggressiveness through regulation of cell proliferation, migration, and apoptotic resistance (p < 0.01). While our findings establish LAMA3 as a critical phenotypic regulator, the molecular mechanisms underlying these effects (e.g., downstream signaling cascades or epigenetic modifiers) remain to be fully elucidated through future mechanistic studies³³. Supporting earlier findings, LAMA3 has been identified as an oncogenic factor in various cancers, promoting tumor cell growth and migration through pathways like the PI3K-Akt signaling cascade³⁴. + Intriguingly, LAMA3 exhibits tissue-specific expression patterns in ESCC. Our bioinformatics enrichment analyses suggest potential associations between LAMA3 overexpression and the activation of pathways such as PI3K-AKT and JAK-STAT. However, these results are based solely on computational prediction and correlative analysis, and direct downstream validation (e.g., western blot for p-AKT, E-cadherin, or vimentin) was not performed in this study. Therefore, the functional relevance of these pathways in ESCC remains speculative and requires further experimental verification. We acknowledge this limitation and recommend that future studies perform mechanistic experiments to confirm these findings in ESCC models, as previously reported in other cancer types^35–38. Specifically, LAMA3 appears to enhance the adhesion of tumor cells to the extracellular matrix, which aids in their migration and invasion, thereby laying the groundwork for LAMA3-targeted therapies^39–41.

Additionally, this study emphasizes LAMA3’s potential role within the tumor immune microenvironment, as its overexpression correlates with increased infiltration of several immune cells, particularly tumor-associated macrophages and natural killer (NK) cells⁴². This suggests that LAMA3 may play a role in immune evasion and resistance to chemotherapy. Previous research has shown that tumor cells can manipulate immune cell infiltration to influence tumor progression, highlighting LAMA3’s potential as an immunoregulatory factor and a target for therapy in ESCC. Given LAMA3’s oncogenic role in ESCC, future research should further assess its clinical value as a prognostic marker. The observed link between LAMA3 overexpression and poor survival outcomes points to its potential as a prognostic biomarker. Targeting LAMA3 could represent a new therapeutic strategy to enhance outcomes for ESCC patients. These findings are consistent with earlier studies that have associated LAMA3 expression levels with prognosis in various cancers. Overall, this research not only enhances our understanding of LAMA3’s involvement in tumor progression but also offers new perspectives for clinical treatment approaches⁴³. The integration of clinical observations with mechanistic cellular data lends credibility to LAMA3’s role in ESCC progression and enhances the translational significance of these findings.

Despite the significant findings, this study has certain limitations. It primarily relied on in vitro experiments using cell lines and tissue samples, which means it lacks large-scale preclinical and clinical validation. Specifically, the absence of in vivo experimental models limits the exploration of LAMA3’s systemic effects on tumor progression and immune microenvironment regulation. To address this gap, future studies should incorporate xenograft models using ESCC cell lines with LAMA3 knockdown or overexpression, combined with longitudinal monitoring of tumor growth, metastasis, and immune cell infiltration (e.g., via flow cytometry or multiplex immunohistochemistry). Furthermore, genetically engineered mouse models (GEMMs) with conditional LAMA3 knockout in esophageal epithelium, particularly in combination with carcinogen-induced or TP53-mutant backgrounds, could elucidate its tissue-specific roles in tumor initiation and immune evasion.

Conclusion

Our study demonstrates that LAMA3 is upregulated 2.4-fold in ESCC tumor tissues compared to adjacent normal tissues (p < 0.001). High LAMA3 expression is associated with significantly poorer overall and disease-free survival (p < 0.05). Silencing LAMA3 by siRNA reduced ESCC cell proliferation by 57%, migration by 49%, and invasion by 47% (all p < 0.001). These quantitative results establish LAMA3 as an oncogenic driver in ESCC and support its utility as both a diagnostic and prognostic biomarker. Taken together, our findings identify LAMA3 as a promising target for early detection, risk stratification, and the development of new therapeutic strategies in ESCC. Further studies should clarify its mechanistic role within the tumor microenvironment.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

XW , YFC and JBW designed the study. QL , LL conducted the statistical analysis. XW , QL and LL contributed to data analysis and interpretation. XW drafed the manuscript, which was reviewed and revised by XW , QL, LL , YFC and JBW. All authors have approved the submitted version of the article.

Funding

This work was supported by Shandong Province Medical and Health Science and Technology Program (No. 202309030443).

Data availability

All data supporting the findings of this study have been deposited in public repositories or are included in the article and its Supplementary Information. All Western blot membranes were digitally scanned prior to any cropping or reprobing, and the original images are provided in an attachment to the article.

Declarations

Ethics approval and consent to participate

This study was discussed and approved by the Ethics Committee of Shandong Provincial Third Hospital, Shandong University (No. DWKYLL-2023019).

Consent for publication

All authors consent to publication.

Competing interests

The authors declare no competing interests.