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TGFBR2 Regulates Hedgehog Pathway and Cervical Cancer Cell Proliferation and Migration by Mediating SMAD4
Jialing Yuan1,2, Ke Yi1,2, Lingyun Yang1,2
1Key Laboratory of Birth Defects and Related Diseases of Women and Children, Sichuan University, Ministry of Education, Chengdu 610041, China.
Abstract:
TGFBR2 serves as an initial regulator of the TGF-β signaling pathway, and loss or reduction of its expression can lead to uncontrolled cell growth. This study was conducted to further explore the mechanism of TGFBR2/SMAD4 on the migration and proliferation of CC cells. Here, TGFBR2 and SMAD4 expressions in CC cells and control cells were measured. The expression patterns of TGFBR2 and SMAD4 in CC cells were verified in the TCGA database. After CC cells were transfected with pcDNA3.1-TGFBR2 or pcDNA3.1-SMAD4, or cotransfected with pcDNA3.1-TGFBR2 and si-SMAD4, Co-IP was utilized for identification of the interaction between TGFBR2 and SMAD4, CCK-8 assay for the assessment of CC cell proliferation, and flow cytometry for the performance of cell cycles. After that, the migration ability of CC cells was examined by cell scratch assay. The expression levels of Hedgehog signaling pathway-related proteins (GLI1 and PTCH) were assayed by Western blot. Lowly expressed TGFBR2 and SMAD4 in CC cells were displayed by the TCGA database. Overexpression of TGFBR2 restrained CC cell migration and proliferation abilities, while the coeffect of TGFBR2 overexpression and SMAD4 knockdown reversed these trends. Besides, highly expressed PTCH and lowly expressed GLI1 were found in CC cells with overexpression of TGFBR2 or SMAD4. The Hedgehog signaling inhibitor (GANT58) can substantially hinder the development of CC cells. Cells in pcDNA3.1-TGFBR2 + si-SMAD4 + GANT58 group had suppressed abilities of cell proliferation and migration than those in pcDNA3.1-TGFBR2 + si-SMAD4 group. Hedgehog pathway agonist (SAG) reversed the inhibitory effect of pcDNA3.1-TGFBR2 or pcDNA3.1-SMAD4 on CC cell biological function. Collectively, TGFBR2 restrains the migration and proliferation abilities of CC cells via mediating SMAD4 to partially block the Hedgehog signaling pathway.
Insights
Transforming growth factor beta receptor 2 (TGFBR2) restrains colorectal cancer (CC) cell migration and proliferation by regulating SMAD4 and partially inhibiting the Hedgehog signaling pathway.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Transforming growth factor beta receptor 2 (TGFBR2) is a key regulator of the TGF-β signaling pathway.
- Loss of TGFBR2 expression is linked to uncontrolled cell growth, suggesting its role in cancer development.
- The precise mechanism of TGFBR2 and SMAD4 in colorectal cancer (CC) cell migration and proliferation requires further elucidation.
Purpose of the Study:
- To investigate the mechanism by which TGFBR2 and SMAD4 influence the migration and proliferation of colorectal cancer (CC) cells.
- To explore the interaction between TGFBR2, SMAD4, and the Hedgehog signaling pathway in CC.
- To validate the expression patterns of TGFBR2 and SMAD4 in CC using the TCGA database.
Main Methods:
- Expression analysis of TGFBR2 and SMAD4 in CC cells and TCGA database.
- Cell transfection and co-transfection experiments to manipulate TGFBR2 and SMAD4 levels.
- Co-immunoprecipitation (Co-IP) to assess protein interactions.
- CCK-8 assay, flow cytometry, and cell scratch assay to evaluate cell proliferation, cell cycle, and migration.
- Western blot analysis to measure Hedgehog signaling pathway proteins (GLI1, PTCH).
- Treatment with Hedgehog signaling inhibitor (GANT58) and agonist (SAG).
Main Results:
- TCGA data revealed low expression of TGFBR2 and SMAD4 in CC cells.
- Overexpression of TGFBR2 inhibited CC cell migration and proliferation; SMAD4 knockdown reversed this effect.
- TGFBR2 and SMAD4 modulated the expression of Hedgehog pathway proteins (PTCH and GLI1).
- Hedgehog pathway inhibition (GANT58) suppressed CC cell proliferation and migration.
- Hedgehog pathway activation (SAG) counteracted the inhibitory effects of TGFBR2 or SMAD4 manipulation.
Conclusions:
- TGFBR2 acts as a tumor suppressor in CC by inhibiting cell migration and proliferation.
- TGFBR2 exerts its effects by mediating SMAD4 to partially block the Hedgehog signaling pathway.
- Targeting the TGFBR2/SMAD4 axis and the Hedgehog pathway presents a potential therapeutic strategy for CC.
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