Targeting the Highly Expressed microRNA miR-146b with CRISPR/Cas9n Gene Editing System in Thyroid Cancer

• Main Authors: Daniel Casartelli de Santa-Inez, Cesar Seigi Fuziwara, Kelly Cristina Saito, Edna Teruko Kimura
• Format: Article
• Language: English
• Published: MDPI AG 2021-07-01
• Series: International Journal of Molecular Sciences
• Subjects: CRISPR/Cas9n; <i>miR-146b</i>; anaplastic thyroid cancer; microRNA; gene editing
• Online Access: https://www.mdpi.com/1422-0067/22/15/7992

Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, <i>miR-146b-5p</i> is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of <i>miR-146b-5p</i> results in anti-tumoral effect. Therefore, inactivating <i>miR-146b</i> has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the <i>MIR146B</i> gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the <i>pre-mir-146b</i> structure to target <i>miR-146b</i>-<i>5p</i> and <i>miR-146b</i>-<i>3p</i> sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-<i>miR-146b</i>-GuideA-puromycin and pSp-Cas9n-<i>miR-146b</i>-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of <i>miR-146b-5p</i> compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the <i>MIR146B</i> edited sequence shows a 5 nt deletion in the <i>miR-146b-5p</i> region and a 1 nt deletion in the <i>miR-146b-3p</i> region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the <i>MIR146B</i> miRNA gene and reduce <i>miR-146b-5p</i> expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.