Effect of gene editing and expression modulation of MIR146B in thyroid cancer.

Author(s)

Daniel Casartelli de Santa Inez

• 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

• 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