Abstract
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, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B 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 pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-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 miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.
Highlights
The CRISPR/Cas9 (CRISPRAssociated Protein 9) is a revolutionary methodology to modify genes using a single-guideRNA and the endonuclease Cas9 [1]
We show that miR-146b is highly expressed in anaplastic thyroid cancer (ATC) cell lines, and we design two sgRNAs cloned in a plasmidial CRISPR/Cas9 nickase (Cas9n) system to target the
We demonstrate that gene editing of MIR146B with CRISPR/Cas9n is effective in disrupting the MIR146B gene sequence and reducing miR-146b expression, leading to an overall anti-tumoral effect in the ATC cell line
Summary
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR. Associated Protein 9) is a revolutionary methodology to modify genes using a single-guide. MiRNAs are considered excellent markers for tumor classification and prognosis [9,10] In this context, we used the CRISPR/Cas9n system in a previous study to inactivate the oncogenic miRNA cluster miR-17-92, composed of six distinct miRNAs, resulting in anti-tumoral effect in an ATC cell line [11]. Different studies have shown that miR-146b regulates thyroid tumorigenesis by targeting genes involved in tumor migration, invasion, and thyroid cell differentiation [18,19,20], and high levels of miR-146b-5p are associated with aggressive thyroid cancer [21,22]. We demonstrate that gene editing of MIR146B with CRISPR/Cas9n is effective in disrupting the MIR146B gene sequence and reducing miR-146b expression, leading to an overall anti-tumoral effect in the ATC cell line
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