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Functional and Biological Characterization of the LGR5Δ5 Splice Variant in HEK293T Cells
Matthias Kappler1, Laura Thielemann1, Markus Glaß2
1Department of Oral and Maxillofacial Plastic Surgery, Faculty of Medicine, Martin Luther University Halle-Wittenberg, 06120 Halle, Germany.
International Journal of Molecular Sciences
|January 8, 2025
Summary
The LGR5Δ5 isoform, a variant of the Wnt pathway regulator LGR5, does not restore Wnt activity but enhances radiosensitization. LGR5Δ5 influences cytoskeleton and angiogenesis genes, unlike full-length LGR5.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a key regulator of the Wnt pathway, found in stem cells and overexpressed in carcinomas.
- The LGR5Δ5 isoform, previously identified, is linked to prognosis and metastasis in oral squamous cell carcinoma (OSCC) and soft tissue sarcoma (STS).
Purpose of the Study:
- To investigate the distinct functions of the LGR5Δ5 isoform compared to full-length LGR5 (LGR5FL) in Wnt pathway activity, cell migration, and radiosensitization.
- To elucidate the molecular mechanisms underlying LGR5Δ5 and LGR5FL functions using a HEK293T cell model.
Main Methods:
- CRISPR/Cas9 gene editing was employed to knockout LGR5 and LGR4.
- HEK293T cells were utilized for functional assays, including Wnt pathway activity assessment, cell migration studies, and radiosensitization experiments.
- RNA sequencing was performed to analyze gene expression changes induced by LGR5 isoforms.
Main Results:
- CRISPR/Cas9 knockout of LGR5/LGR4 abolished Wnt activity, which LGR5Δ5 could not restore, unlike LGR5FL.
- LGR5FL overexpression reduced cell migration, while LGR5Δ5 did not affect it.
- CRISPR/Cas9 knockout induced radiosensitization, further enhanced by LGR5FL or LGR5Δ5 overexpression.
- LGR5Δ5 significantly increased R-spondin 1 (RSPO1) levels and regulated genes involved in cytoskeleton, extracellular matrix, and angiogenesis.
- LGR5FL was associated with the regulation of collagens and histone proteins.
Conclusions:
- LGR5Δ5 functions differently from LGR5FL, notably not restoring Wnt pathway activity but contributing to radiosensitization.
- LGR5Δ5 plays a role in regulating genes associated with cellular structure, matrix interaction, and blood vessel formation.
- LGR5FL is implicated in regulating collagen and histone proteins, suggesting distinct roles in cellular processes and potentially cancer progression.
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