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

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, is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of results in anti-tumoral effect. Therefore, inactivating has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the 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 structure to target - and - sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n--GuideA-puromycin and pSp-Cas9n--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 compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the edited sequence shows a 5 nt deletion in the region and a 1 nt deletion in the region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the miRNA gene and reduce expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.