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In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines
Published on: March 5, 2019
Mast cell-derived exosomal miR-181a-5p modulated trophoblast cell viability, migration, and invasion via YY1/MMP-9
1Maternity Department, Ningbo Women & Children's Hospital, Ningbo City, Zhejiang Province, China.
Background:
Mast cells regulate the process of preeclampsia (PE). Since we previously identified mast cells specifically expressing miR-181a-5p in the placenta of PE patients, it is plausible to examine the effect and mechanism of mast cell-derived exosomal miR-181a-5p on trophoblast cells.
Methods:
The miR-181a-5p and YY1 levels were determined by quantitative real-time reverse transcription-polymerase chain reaction. Exosomes were identified by transmission electron microscopy, Western blot, and PKH-26 labeling. Mast cells or trophoblast cell malignant phenotype were detected using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, wound healing, and Transwell assays. Quantification of YY1 and metastasis-related proteins was performed using Western blot. TargetScan, JASPAR, dual-luciferase reporter genes, and chromatin immunoprecipitation were exploited to verify the relationship between miR-181a-5p, YY1, and MMP-9.
Results:
MiR-181a-5p was overexpressed in mast cells of PE patients. Overexpressed miR-181a-5p restrained mast cell viability. Mast cell exosomes were successfully isolated, containing high expressions of CD63 and HSP70 and low expression of Calnexin and could be transported to the cytoplasm of trophoblast cells. Mast cell exosomes attenuated the viability, migration, and invasion of HTR-8/SVneo cells, inhibited YY1, N-cadherin, Vimentin, and MMP-9 protein expressions, and promoted E-cadherin protein expression. The effect of exosomes was enhanced by miR-181a-5p mimic but was reversed by miR-181a-5p inhibitor. MiR-181a-5p targeted YY1 which bound to the MMP-9 promoter. Overexpressed YY1 in HTR-8/SVneo cells accelerated the malignant phenotype of the cells and reversed the regulatory effects of exosomal miR-181a-5p.
Conclusion:
Mast cell-derived exosomal miR-181a-5p modulates HTR-8/SVneo cell viability, migration, and invasion via YY1/MMP-9.
Insights
Mast cell exosomes carrying miR-181a-5p regulate trophoblast cell behavior by targeting YY1/MMP-9. This finding offers insights into preeclampsia mechanisms and potential therapeutic strategies.
Area of Science:
- Cell Biology
- Molecular Biology
- Reproductive Medicine
Background:
- Mast cells play a role in preeclampsia (PE).
- miR-181a-5p is found in mast cells of PE patients.
- Investigating mast cell-derived exosomal miR-181a-5p in trophoblast cells is crucial.
Purpose of the Study:
- To determine the effect of mast cell-derived exosomal miR-181a-5p on trophoblast cells.
- To elucidate the underlying molecular mechanism involving YY1 and MMP-9.
Main Methods:
- Quantitative real-time PCR for miR-181a-5p and YY1.
- Exosome characterization (TEM, Western blot, PKH-26).
- Cellular assays (MTT, wound healing, Transwell) to assess trophoblast phenotype.
- Western blot for protein expression.
- Bioinformatic and molecular techniques (TargetScan, dual-luciferase, ChIP) to validate molecular interactions.
Main Results:
- Mast cell-derived exosomes carrying miR-181a-5p were successfully isolated and internalized by trophoblast cells.
- Exosomal miR-181a-5p attenuated trophoblast cell viability, migration, and invasion.
- miR-181a-5p targeted YY1, which in turn regulated MMP-9 expression, influencing trophoblast cell behavior.
Conclusions:
- Mast cell-derived exosomal miR-181a-5p modulates trophoblast cell viability, migration, and invasion.
- The mechanism involves the YY1/MMP-9 pathway.
- This highlights a novel regulatory axis in preeclampsia pathogenesis.
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