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Enhancing RECK Expression Through miR-21 Inhibition: A Promising Strategy for Bladder Carcinoma Control

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Abstract

Bladder carcinoma (BC) is the tenth most frequent malignancy worldwide, with high morbidity and mortality rates. Despite recent treatment advances, high-grade BC and muscle-invasive BC present with significant progression and recurrence rates, urging the need for alternative treatments. The microRNA-21 (miR-21) has superexpression in many malignancies and is associated with cellular invasion and progression. One of its mechanisms of action is the regulation of RECK, a tumor suppressor gene responsible for inhibiting metalloproteinases, including MMP9. In a high-grade urothelial cancer cell line, we aimed to assess if miR-21 downregulation would promote RECK expression and decrease MMP9 expression. We also evaluated cellular migration and proliferation potential by inhibition of this pathway. In a T24 cell line, we inhibited miR-21 expression by transfection of a specific microRNA inhibitor (anti-miR-21). There were also control and scramble groups, the last with a negative microRNA transfected. After the procedure, we performed a genetic expression analysis of miR-21, RECK, and MMP9 through qPCR. Migration, proliferation, and protein expression were evaluated via wound healing assay, colony formation assay, flow cytometry, and immunofluorescence.After anti-miR-21 transfection, miR-21 expression decreased with RECK upregulation and MMP9 downregulation. The immunofluorescence assay showed a significant increase in RECK protein expression (p < 0.0001) and a decrease in MMP9 protein expression (p = 0.0101). The anti-miR-21 transfection significantly reduced cellular migration in the wound healing assay (p < 0.0001). Furthermore, in the colony formation assay, the anti-miR-21 group demonstrated reduced cellular proliferation (p = 0.0008), also revealed in the cell cycle analysis by flow cytometry (p = 0.0038). Our results corroborate the hypothesis that miR-21 is associated with BC cellular migration and proliferation, revealing its potential as a new effective treatment for this pathology.

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Data Availability

The datasets and materials used in this study are available upon request from the corresponding author. Researchers interested in accessing the data or materials for the purpose of academic or research inquiries are encouraged to contact Nayara Izabel Viana at niviana@usp.br. We are committed to promoting transparency and facilitating scientific collaboration, and we will make every effort to provide the necessary data and materials in a timely manner.

Abbreviations

BC:

Bladder cancer

EMT:

Epithelial to mesenchymal transition

MIBC:

Muscle invasive bladder cancer

miRNA:

MicroRNA

MMP:

Metalloproteinases

oncomiRs:

Oncogenic miRs

TAM–Tumor:

Associated macrophages

TS miRs:

Tumor suppressor miRs

UC:

Urothelial carcinoma

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We declare that we have not received any funding for the preparation of this article.

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Authors and Affiliations

  1. Laboratorio de Investigação Médica 55 (LIM55), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil

    Paulo Rodolfo Moraes dos Santos, Paulo Ricardo da Silva Gomes, Poliana Romão, Feres Camargo Maluf, Vanessa Ribeiro Guimarães, Patrícia Candido, Juliana Alves de Camargo, Gabriel Arantes dos Santos, Iran Silva, Katia Ramos Moreira Leite, Sabrina T. Reis, Ruan Pimenta & Nayara Izabel Viana

  2. Faculdade de Medicina, Universidade Anhembi Morumbi, São Paulo, SP, Brazil

    Paulo Rodolfo Moraes dos Santos

  3. Faculdade de Medicina, Universidade Federal do Pará, Belém, PA, Brazil

    Paulo Ricardo da Silva Gomes

  4. Moriah Institute of Science and Education (MISE), Hospital Moriah, São Paulo, SP, Brazil

    Patrícia Candido & Sabrina T. Reis

  5. Laboratory of Renal Physiology, Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil

    Guilherme Lopes Gonçalves

  6. Uro-Oncology Group, Urology Department, University of Sao Paulo Medical School and Institute of Cancer Estate of Sao Paulo (ICESP), Sao Paulo, Brazil

    William Nahas

  7. D’Or Institute for Research and Education (IDOR), Sao Paulo, Brazil

    Ruan Pimenta

  8. Universidade do Estado de Minas Gerais - UEMG, Passos, MG, Brazil

    Nayara Izabel Viana

  9. Precision Immunology Institute, Department of Immunology and Immunotherapy, and Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, United States

    Ruan Pimenta

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  1. Paulo Rodolfo Moraes dos Santos
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Contributions

PRM, PRSG, PR and FCM. wrote the main manuscript text; PR and RP. prepared figures. PRM, PR, VRG, PC, GLG, JAC, GAS, IS, RP and NIV contributed to study design and performed the in vitro experiments. IS, KRML, WN, STR contributed to intellectual input. KRML, WN, STR, NIV supervised the experiments, contributed to the study design, drafting and critical reading. RP contributed to statistical analysis, drafting and critical reading. All authors reviewed the manuscript.

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Correspondence to Nayara Izabel Viana.

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dos Santos, P.R.M., da Silva Gomes, P.R., Romão, P. et al. Enhancing RECK Expression Through miR-21 Inhibition: A Promising Strategy for Bladder Carcinoma Control. Biochem Genet (2024). https://doi.org/10.1007/s10528-024-10714-8

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