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Tytuł:
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Targeting the Highly Expressed microRNA miR-146b with CRISPR/Cas9n Gene Editing System in Thyroid Cancer.
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Autorzy:
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Santa-Inez DC; Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil.
Fuziwara CS; Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil.
Saito KC; Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil.
Kimura ET; Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil.
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Źródło:
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International journal of molecular sciences [Int J Mol Sci] 2021 Jul 27; Vol. 22 (15). Date of Electronic Publication: 2021 Jul 27.
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Typ publikacji:
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Journal Article
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Język:
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English
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Imprint Name(s):
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Original Publication: Basel, Switzerland : MDPI, [2000-
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MeSH Terms:
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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
Animals ; Cell Line ; Cell Movement/genetics ; Cell Survival/genetics ; Heterografts ; Humans ; Mice ; Neoplasm Transplantation
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References:
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Grant Information:
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430756/2018-6 Conselho Nacional de Desenvolvimento Científico e Tecnológico; 430756/2018-6 Conselho Nacional de Desenvolvimento Científico e Tecnológico; 2019/25116-8 Fundação de Amparo à Pesquisa do Estado de São Paulo; 2019/17282-5 Fundação de Amparo à Pesquisa do Estado de São Paulo; 2019/19865-8 Fundação de Amparo à Pesquisa do Estado de São Paulo; 2020/10403-9 Fundação de Amparo à Pesquisa do Estado de São Paulo; Code 001 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; PNPD 88887.374682/2019-00 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; NapmiR Universidade de São Paulo
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Contributed Indexing:
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Keywords: CRISPR/Cas9n; anaplastic thyroid cancer; gene editing; miR-146b; microRNA
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Substance Nomenclature:
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0 (MIRN146 microRNA, human)
0 (MicroRNAs)
0 (RNA, Neoplasm)
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Entry Date(s):
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Date Created: 20210807 Date Completed: 20210909 Latest Revision: 20210909
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Update Code:
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20240105
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PubMed Central ID:
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PMC8348963
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DOI:
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10.3390/ijms22157992
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PMID:
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34360757
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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.
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