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| Author(s): |
Daniel Casartelli de Santa Inez
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2021-12-08 |
| Examining board members: |
Edna Teruko Kimura;
Vanessa Morais Freitas;
Ileana Gabriela Sanchez de Rubio;
Carolina Ferraz da Silva
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| Advisor: | Edna Teruko Kimura; Cesar Seigi Fuziwara |
| Abstract | |
MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of target mRNAs and are commonly deregulated in cancer. In thyroid cancer, miR-146b is the most overexpressed miRNA, associated with oncogenesis and tumor aggressiveness, leading to clinical and pathological characteristics of worse prognosis. Therefore, we aim to understand the role of miR-146b in an aggressive model of thyroid cancer using the CRISPR/Cas9n gene editing methodology. The KTC2 strain (human anaplastic carcinoma) expressing a high level of miR-146b was cotransfected with plasmids pSp-Cas9n-miR-146b-guideA-puromycin and pSp-Cas9n-miR-146b-guideB-GFP, containing guide RNAs designed to flank the precursor region of miR-146b, preventing pre-miR formation. Following the transfection, two clones were selected, KTC2-Cl1 and KTC2-Cl3, and also a control cell line, KTC2-CTR, cotransfected with both plasmids with no guide RNA. After selection of clones and MIR146B editing confirmed by sequencing, miR-146b expression was analyzed by real-time PCR from the total RNA extracted, and cell counting, viability, migration, colony and xenotransplantation assays were performed. The KTC2 Cl1 and Cl3 strains showed a decrease in the expression of miR-146b in relation to the control, also showing reduced proliferation, cell viability, migration, and colony formation. Furthermore, in the xenograph experiment, no tumor growth was observed of the KTC2-Cl1 cells, injected into the flank of nude mice. Thus, a modulation of miR-146b expression by gene editing using CRISPR / Cas9n proved to be efficient as a molecular tool for understanding the role of miRNAs in thyroid cancer. (AU) | |
| FAPESP's process: | 19/19865-8 - Effect of miR-146b silencing in thyroid cancer |
| Grantee: | Daniel Casartelli de Santa Inez |
| Support Opportunities: | Scholarships in Brazil - Master |