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. Over-expression 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 was rescued by the 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.
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