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MiR-221 Is Specifically Elevated in PC3 Cells and its Deletion Reduces Adhesion, Motility and Growth
D Alwyn Dart1,2, Sarah Koushyar3,4, Ben E Lanning3
1Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, U.K. a.dart@imperial.ac.uk.
Background/Aim:
MiR-221, often described both as an oncogenic microRNA and as a tumour suppressor, targets mRNAs involved in carcinogenesis. While other oncogenic microRNAs showed correlations with prostate cancer cell lines' aggressiveness, miR-221 showed an unusual overexpression in PC3.
Materials And Methods:
CRISPR was used to delete miR-221 from PC3 cells. Analysing the characteristics of PC3miR-221del cells, a reduced growth rate and expression of cell-cycle genes was observed. In global gene expression/ontology analysis of PC3miR-221del cells, cell-cell and cell-substrate adhesion pathways were found to be greatly affected. In addition, reduced levels of adhesion, invasion and motility for PC3miR-221del cells, a change in F-actin localisation and a reduction of EMT markers were observed.
Results:
The tumour suppressor gene, DIRAS3, was a predicted target of miR-221. In PC3miR-221del cells DIRAS3 was up-regulated at the gene and protein level. Ectopic expression of DIRAS3 in PC3wt cells recapitulated the cellular morphology changes seen in PC3miR-221del cells. DIRAS3 3'UTR was more stable in PC3miR-221del cells, as measured by semi-quantitative PCR and luciferase fusion reporter assays.
Conclusion:
MiR-221 promotes aggressiveness of PC3 cells by down-regulating DIRAS3, and promoting epithelial-to-mesenchymal transition.
Insights
MicroRNA-221 (miR-221) promotes prostate cancer aggressiveness by suppressing DIRAS3 and enhancing epithelial-to-mesenchymal transition in PC3 cells. Deleting miR-221 reduced cell growth and invasion.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- MicroRNA-221 (miR-221) has a dual role as an oncogene and tumor suppressor, targeting mRNAs in carcinogenesis.
- While other oncogenic microRNAs correlate with prostate cancer aggressiveness, miR-221 is unusually overexpressed in PC3 cells.
Purpose of the Study:
- To investigate the role of miR-221 in prostate cancer aggressiveness.
- To elucidate the molecular mechanisms by which miR-221 affects PC3 cell behavior.
Main Methods:
- CRISPR-Cas9 was employed to delete miR-221 from PC3 cells, generating PC3miR-221del cells.
- Global gene expression analysis, cell-cycle assays, adhesion, invasion, and motility assays were performed.
- Target validation involved assessing DIRAS3 expression, 3'UTR stability, and ectopic DIRAS3 expression.
Main Results:
- PC3miR-221del cells exhibited reduced growth rates, cell-cycle gene expression, adhesion, invasion, and motility.
- Global gene expression analysis revealed significant alterations in cell-cell and cell-substrate adhesion pathways.
- The tumor suppressor gene DIRAS3 was identified as a direct target of miR-221, with its upregulation in PC3miR-221del cells.
Conclusions:
- MiR-221 promotes PC3 cell aggressiveness by downregulating the tumor suppressor DIRAS3.
- MiR-221 facilitates epithelial-to-mesenchymal transition (EMT), contributing to cancer progression.
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