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Article Open Access

Proteomics reveals the regulation of ALG1 expression by lentivirus‑mediated THBS1 overexpression and its mechanism in meibomian gland carcinoma

  • Authors:
    • Wei Wang
    • Hetong Wang
    • Guijia Wu
    • Xun Liu
    • Limin Zhu
    • Fang Tian
    • Tingting Lin
  • View Affiliations / Copyright

    Affiliations: Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, P.R. China
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 517
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    Published online on: September 9, 2025
       https://doi.org/10.3892/ol.2025.15263
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Abstract

Meibomian gland carcinoma (MGC) is a highly aggressive eyelid malignancy with a poor prognosis. MicroRNA‑3907 (miR‑3907) is upregulated in MGC, where it promotes tumor progression by suppressing thrombospondin 1 (THBS1). The present study aimed to assess the downstream regulatory mechanisms of THBS1 in MGC. Utilizing 4D label‑free quantitative proteomics, chitobiosyldiphosphodolichol β‑mannosyltransferase 1 (ALG1) was identified as the most significantly differentially expressed downstream protein following lentivirus‑mediated overexpression of THBS1, with results corroborated by consistent sequencing data. Protein‑protein docking models predicted a stable interaction between THBS1 and ALG1, mediated by hydrogen bonds involving key amino acid residues such as ASN‑991 and MET‑58. Co‑immunoprecipitation and immunofluorescence assays confirmed the physical interaction and co‑localization of THBS1 and ALG1 in MGC cells. Notably, ALG1 mRNA levels were significantly elevated in MGC cells compared with in normal meibomian gland cells. Functional assays revealed that ALG1 knockdown markedly suppressed malignant cellular behaviors and modulated the expression of genes associated with epithelial‑mesenchymal transition, a key process in tumor metastasis. Conversely, ALG1 overexpression enhanced these malignant traits. Together, these findings highlight ALG1 as a critical downstream effector in the miR‑3907/THBS1/ALG1 regulatory axis and suggest its potential as a biomarker for targeted diagnosis and therapy in MGC. The present study provides new insights into the molecular mechanisms underlying MGC and offers a basis for developing personalized therapeutic strategies.
View Figures

Figure 1

Study flowchart. Screening of
differential target proteins in the downstream of THBS1 by
proteomics, followed by the functional validation of ALG1 in
meibomian gland carcinoma. THBS1, thrombospondin 1; ALG1,
chitobiosyldiphosphodolichol β-mannosyltransferase 1; nc, negative
control; oe, overexpression; miRNA/miR, microRNA.

Figure 2

Proteomic analysis following THBS1
overexpression. (A) Fluorescent imaging (×40; scale bar, 200 µm) of
NCoe and THBS1oe groups after
lentiviral-mediated THBS1 overexpression. (B) Evaluation of THBS1
overexpression using reverse transcription-quantitative PCR and
western blot analysis. (C) Hierarchical clustering heatmap of
differentially expressed proteins. (D) Volcano plot of DEPs: Green
dots indicate downregulated proteins, and red dots indicate
upregulated proteins. (E) GO and (F) KEGG pathway enrichment
analysis of DEPs. **P<0.01. THBS1, thrombospondin 1; NC,
negative control; oe, overexpression; DEP, differentially expressed
protein; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and
Genomes; FC, fold change.

Figure 3

Validation of the interaction between
THBS1 and ALG1. (A) Validation of ALG1 downregulation following
THBS1 overexpression via reverse transcription-quantitative PCR and
western blot analyses. (B) Protein docking model of THBS1 and ALG1
showing their interface residues (THBS1, pink; ALG1, yellow; and
hydrogen bonds, yellow dashed lines). (C) Immunofluorescence
co-localization analysis of THBS1 and ALG1 (magnification, ×400;
scale bar, 20 µm). (D) Immunoprecipitation analysis demonstrating
the specific interaction between THBS1 and ALG1. ***P<0.001.
ALG1, chitobiosyldiphosphodolichol β-mannosyltransferase 1; THBS1,
thrombospondin 1; NC, negative control; oe, overexpression; CoIP,
co-immunoprecipitation.

Figure 4

Regulatory role of ALG1 in the
Biological Behaviors of MGC Cells. (A) Differential expression
analysis of ALG1 in tumors and adjacent normal tissues across
several cancer types and subtypes using the Tumor Immune Estimation
Resource 2.0. (B) Comparison of ALG1 mRNA expression levels in
primary MG and MGC cells. (C) Fluorescence microscopy images (×40;
scale bar, 200 µm) of MGC cells following ALG1 knockdown
(ALG1si) and overexpression (ALG1oe) in
NCsi, NCoe and ALG1oe groups. (D)
Reverse transcription-quantitative PCR and (E) western blot
analyses of successful knockdown and overexpression of ALG1 in MGC
cells using siRNA and lentivirus, respectively. (F) Cell Counting
Kit-8 assay evaluating the effects of ALG1 regulation on MGC cell
proliferation. (G) Scratch wound-healing assay assessing the impact
of ALG1 modulation on cell migration (×40; scale bar, 200 µm). (H)
Transwell assay measuring the effects of ALG1 on MGC cell migration
and invasion (×100; scale bar, 100 µm). *P<0.05; **P<0.01;
***P<0.001; ****P<0.0001. ALG1, chitobiosyldiphosphodolichol
β-mannosyltransferase 1; MG, meibomian gland; MGC, MG carcinoma;
si, small interfering; oe, overexpression; NC, negative control;
TPM, transcripts per million; OD, optical density.

Figure 5

Regulatory role of ALG1 in apoptosis
and epithelial-mesenchymal transition of MGC Cells. (A) Flow
cytometry analysis of cell apoptosis and quantification of the
apoptosis rate following ALG1 knockdown. (B) RT-qPCR analysis of
pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2
expression levels after ALG1 knockdown. (C) Flow cytometry analysis
of cell apoptosis and quantification of apoptosis rate after ALG1
overexpression. (D) RT-qPCR analysis of pro-apoptotic protein Bax
and anti-apoptotic protein Bcl-2 expression levels after ALG1
overexpression. (E) Evaluation of the downregulation of Bax and the
upregulation of Bcl-2 after ALG1 overexpression by western blot
analysis. (F) Statistical analysis of the grayscale values of the
downregulation of Bax and the upregulation of Bcl-2 after ALG1
overexpression. (G) RT-qPCR analysis of epithelial-mesenchymal
transition-related genes (E-Cadherin, N-Cadherin, and Vimentin)
after ALG1 knockdown. (H) RT-qPCR analysis of
epithelial-mesenchymal transition-related genes (E-Cadherin,
N-Cadherin, and Vimentin) after ALG1 overexpression. *P<0.05;
**P<0.01; ***P<0.001; ****P<0.0001. RT-qPCR, reverse
transcription-quantitative PCR; ALG1, chitobiosyldiphosphodolichol
β-mannosyltransferase 1; NC, negative control; si, small
interfering; oe, overexpression.
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Copy and paste a formatted citation
Spandidos Publications style
Wang W, Wang H, Wu G, Liu X, Zhu L, Tian F and Lin T: Proteomics reveals the regulation of ALG1 expression by lentivirus‑mediated THBS1 overexpression and its mechanism in meibomian gland carcinoma. Oncol Lett 30: 517, 2025.
APA
Wang, W., Wang, H., Wu, G., Liu, X., Zhu, L., Tian, F., & Lin, T. (2025). Proteomics reveals the regulation of ALG1 expression by lentivirus‑mediated THBS1 overexpression and its mechanism in meibomian gland carcinoma. Oncology Letters, 30, 517. https://doi.org/10.3892/ol.2025.15263
MLA
Wang, W., Wang, H., Wu, G., Liu, X., Zhu, L., Tian, F., Lin, T."Proteomics reveals the regulation of ALG1 expression by lentivirus‑mediated THBS1 overexpression and its mechanism in meibomian gland carcinoma". Oncology Letters 30.5 (2025): 517.
Chicago
Wang, W., Wang, H., Wu, G., Liu, X., Zhu, L., Tian, F., Lin, T."Proteomics reveals the regulation of ALG1 expression by lentivirus‑mediated THBS1 overexpression and its mechanism in meibomian gland carcinoma". Oncology Letters 30, no. 5 (2025): 517. https://doi.org/10.3892/ol.2025.15263
Copy and paste a formatted citation
x
Spandidos Publications style
Wang W, Wang H, Wu G, Liu X, Zhu L, Tian F and Lin T: Proteomics reveals the regulation of ALG1 expression by lentivirus‑mediated THBS1 overexpression and its mechanism in meibomian gland carcinoma. Oncol Lett 30: 517, 2025.
APA
Wang, W., Wang, H., Wu, G., Liu, X., Zhu, L., Tian, F., & Lin, T. (2025). Proteomics reveals the regulation of ALG1 expression by lentivirus‑mediated THBS1 overexpression and its mechanism in meibomian gland carcinoma. Oncology Letters, 30, 517. https://doi.org/10.3892/ol.2025.15263
MLA
Wang, W., Wang, H., Wu, G., Liu, X., Zhu, L., Tian, F., Lin, T."Proteomics reveals the regulation of ALG1 expression by lentivirus‑mediated THBS1 overexpression and its mechanism in meibomian gland carcinoma". Oncology Letters 30.5 (2025): 517.
Chicago
Wang, W., Wang, H., Wu, G., Liu, X., Zhu, L., Tian, F., Lin, T."Proteomics reveals the regulation of ALG1 expression by lentivirus‑mediated THBS1 overexpression and its mechanism in meibomian gland carcinoma". Oncology Letters 30, no. 5 (2025): 517. https://doi.org/10.3892/ol.2025.15263
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