Molecular biology reports · 2026
CRISPR/Cas9-mediated miR-21 editing in high-grade urothelial carcinoma cells and its biological effects.
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Yi Lin Lee Maria Carolina, Camargo Juliana Alves, Caetano Vilas Boas Giovana, Silva Karina Serafim, Silva Luana Pereira, Mioshi Carolina Mie, Pimenta Ruan, Silva Iran Amorim, Ramos Moreira Leite Katia, Nahas William Carlos, Dos Reis Sabrina T
Аннотация
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.
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.