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Updated: Jun 20, 2026
CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis
Published on: April 25, 2022
CRISPR/Cas9-mediated miR-21 editing in high-grade urothelial carcinoma cells and its biological effects
Maria Carolina Yi Lin Lee1, Juliana Alves Camargo1, Giovana Caetano Vilas Boas1
1Laboratory of Medical Investigation (LIM55), Urology Department, University of São Paulo Medical School, Av. Dr. Arnaldo 455, 2° floor, room 2145 - Cerqueira Cesar, São Paulo, Brazil.
Background:
Urothelial carcinoma, the predominant form of bladder cancer, represents a global public health challenge due to its high rates of recurrence and progression. At the molecular level, microRNA-21 (miR-21) has been characterized as an "oncomir" because of its ability to negatively regulate tumor suppressor genes, thereby promoting tumor survival and progression. In this context, the CRISPR/Cas9 system has emerged as a precise genome-editing tool.
Objective:
To investigate the biological effects of miR-21 modulation using CRISPR/Cas9-mediated genome editing in the T24 high-grade invasive urothelial carcinoma cell line.
Methods:
The CRISPR/Cas9 system was delivered as a ribonucleoprotein (RNP) complex. Editing efficiency was assessed using quantitative reverse transcription PCR (RT-qPCR). Functional effects were evaluated through gene expression assays, cell migration assays, as well as Matrigel invasion assays. The presence of the Cas9 protein was confirmed by immunofluorescence.
Results:
CRISPR/Cas9 treatment targeting miR-21 showed a trend toward reduced miR-21 expression (p = 0.0563), although this did not reach statistical significance. A statistically significant increase in MASPIN (p < 0.0001) and PDCD4 (p = 0.0239), as well as a trend toward increased PTEN expression (p = 0.055), was observed following treatment. Functionally, a significant reduction in the migratory capacity of edited cells was observed after 48 h (p = 0.0334). The presence of Cas9 was successfully confirmed in transfected cells.
Conclusion:
These findings suggest that CRISPR/Cas9-mediated modulation of miR-21 may influence tumor suppressor pathways and reduce the migratory potential of urothelial carcinoma cells.
Insights
CRISPR/Cas9 gene editing targeting microRNA-21 (miR-21) in urothelial carcinoma cells showed a trend toward reduced miR-21 expression. This approach may influence tumor suppressor pathways and decrease cancer cell migration.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Urothelial carcinoma, a common bladder cancer, recurs and progresses frequently.
- MicroRNA-21 (miR-21) acts as an oncomir by suppressing tumor suppressor genes.
- CRISPR/Cas9 is a precise genome-editing technology.
Purpose of the Study:
- To assess the effects of modulating miR-21 using CRISPR/Cas9 in urothelial carcinoma cells.
- To investigate impacts on tumor suppressor genes and cell behavior.
Main Methods:
- CRISPR/Cas9 delivered as ribonucleoprotein (RNP) complexes.
- Assessed editing efficiency via RT-qPCR.
- Evaluated functional effects through gene expression, cell migration, and invasion assays.
- Confirmed Cas9 presence using immunofluorescence.
Main Results:
- CRISPR/Cas9 targeting miR-21 showed a trend toward reduced miR-21 expression (p=0.0563).
- Significant increases in MASPIN (p<0.0001) and PDCD4 (p=0.0239), and a trend for PTEN (p=0.055) were observed.
- Cell migration was significantly reduced by 48h (p=0.0334).
- Cas9 presence was confirmed.
Conclusions:
- CRISPR/Cas9-mediated miR-21 modulation may impact tumor suppressor pathways.
- This approach shows potential in reducing urothelial carcinoma cell migration.
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