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J Biol Chem. 2017 Mar 3;292(9):3614-3623. doi: 10.1074/jbc.M116.749689. Epub 2017 Jan 19.

MicroRNA-145 Modulates N6-Methyladenosine Levels by Targeting the 3'-Untranslated mRNA Region of the N6-Methyladenosine Binding YTH Domain Family 2 Protein.

Author information

1
From the State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071, China.
2
State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071, China, and.
3
Department of Clinical Laboratory, Key Laboratory of Cancer Prevention and Therapy, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China yli08@tmu.edu.cn.
4
From the State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071, China, zhangxd@nankai.edu.cn.

Abstract

N6-Methyladenosine (m6A) is a prevalent modification present in the mRNAs of higher eukaryotes. YTH domain family 2 (YTHDF2), an m6A "reader" protein, can recognize mRNA m6A sites to mediate mRNA degradation. However, the regulatory mechanism of YTHDF2 is poorly understood. To this end, we investigated the post-transcriptional regulation of YTHDF2. Bioinformatics analysis suggested that the microRNA miR-145 might target the 3'-untranslated region (3'-UTR) of YTHDF2 mRNA. The levels of miR-145 were negatively correlated with those of YTHDF2 mRNA in clinical hepatocellular carcinoma (HCC) tissues, and immunohistochemical staining revealed that YTHDF2 was closely associated with malignancy of HCC. Interestingly, miR-145 decreased the luciferase activities of 3'-UTR of YTHDF2 mRNA. Mutation of predicted miR-145 binding sites in the 3'-UTR of YTHDF2 mRNA abolished the miR-145-induced decrease in luciferase activity. Overexpression of miR-145 dose-dependently down-regulated YTHDF2 expression in HCC cells at the levels of both mRNA and protein. Conversely, inhibition of miR-145 resulted in the up-regulation of YTHDF2 in the cells. Dot blot analysis and immunofluorescence staining revealed that the overexpression of miR-145 strongly increased m6A levels relative to those in control HCC cells, and this increase could be blocked by YTHDF2 overexpression. Moreover, miR-145 inhibition strongly decreased m6A levels, which were rescued by treatment with a small interfering RNA-based YTHDF2 knockdown. Thus, we conclude that miR-145 modulates m6A levels by targeting the 3'-UTR of YTHDF2 mRNA in HCC cells.

KEYWORDS:

N6-methyladenosine (m6A); RNA methylation; RNA modification; YTH domain family 2 (YTHDF2); mRNA decay; mRNA degradation; miR-145; microRNA (miRNA); microRNA mechanism

PMID:
28104805
PMCID:
PMC5339747
DOI:
10.1074/jbc.M116.749689
[Indexed for MEDLINE]
Free PMC Article

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