| Full text | |
| Author(s): |
De Santa-Inez, Daniel Casartelli
[1]
;
Fuziwara, Cesar Seigi
[1]
;
Saito, Kelly Cristina
[1]
;
Kimura, Edna Teruko
[1]
Total Authors: 4
|
| Affiliation: | [1] Univ Sao Paulo, Inst Biomed Sci, Dept Cell & Dev Biol, BR-05508000 Sao Paulo - Brazil
Total Affiliations: 1
|
| Document type: | Journal article |
| Source: | INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES; v. 22, n. 15 AUG 2021. |
| Web of Science Citations: | 0 |
| 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. (AU) | |
| FAPESP's process: | 19/25116-8 - Modulation of microRNAs and its regulatory target network with therapeutic potential in thyroid cancer: application of gene editing with CRISPR/Cas9 |
| Grantee: | Edna Teruko Kimura |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 20/10403-9 - Transcriptional and post-transcriptional control in aggressive cancer and metastasis |
| Grantee: | Cesar Seigi Fuziwara |
| Support Opportunities: | Scholarships in Brazil - Young Researchers |
| FAPESP's process: | 19/17282-5 - Transcriptional and post-transcriptional control in aggressive cancer and metastasis |
| Grantee: | Cesar Seigi Fuziwara |
| Support Opportunities: | Research Grants - Young Investigators Grants |