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Targeting the Highly Expressed microRNA miR-146b with CRISPR/Cas9n Gene Editing System in Thyroid Cancer.
- Source :
-
International journal of molecular sciences [Int J Mol Sci] 2021 Jul 27; Vol. 22 (15). Date of Electronic Publication: 2021 Jul 27. - Publication Year :
- 2021
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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.
- Subjects :
- Animals
Cell Line
Cell Movement genetics
Cell Survival genetics
Heterografts
Humans
Mice
Neoplasm Transplantation
CRISPR-Cas Systems
Gene Editing
Gene Targeting
MicroRNAs biosynthesis
MicroRNAs genetics
RNA, Neoplasm biosynthesis
RNA, Neoplasm genetics
Thyroid Carcinoma, Anaplastic genetics
Thyroid Carcinoma, Anaplastic metabolism
Thyroid Carcinoma, Anaplastic pathology
Thyroid Neoplasms genetics
Thyroid Neoplasms metabolism
Thyroid Neoplasms pathology
Subjects
Details
- Language :
- English
- ISSN :
- 1422-0067
- Volume :
- 22
- Issue :
- 15
- Database :
- MEDLINE
- Journal :
- International journal of molecular sciences
- Publication Type :
- Academic Journal
- Accession number :
- 34360757
- Full Text :
- https://doi.org/10.3390/ijms22157992