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Letters to the Editor
rated fatty acids. It acts through a direct interaction with the AU-rich elements (ARE) located in the 3’UTR of hep- cidin mRNA.12 Importanly, we observed that HuR mRNA expression is increased in the liver in response to Tm treatment (Online Supplementary Figure S1F). HuR was thus a good candidate for the control of hepcidin mRNA stability not only in response to fatty acids but also to ER stress. In HepG2 cells transfected with a siRNA pool
directed against human HuR, HuR silencing reduced HuR mRNA expression (Figure 2F) and, prevented a significant increase of hepcidin mRNA in response to Tm (Figure 2G). In order to determine if, in response to Tm, HuR regulates hepcidin mRNA stability through direct binding to hepcidin 3’UTR, we performed RNA-CLIP experi- ments with HuR or IgG control antibodies followed by quantitative RT-PCR with primers specific for the hep-
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Figure 2. Endoplasmic reticulum stress stabilizes hepcidin mRNA through HuR in vitro. HepG2 cells were treated with mock (blue boxes) or tunicamycin (Tm) (red boxes) and analyzed for (A) ATF3 mRNA expression; (B) HAMP mRNA expression; (C) ID1 mRNA expression and (D) P-Smad 5 relative to total Smad 5 protein expression. Values shown are the result of three independent experiments performed in duplicate. (E) HepG2 cells were treated with mock (black box) or acti- nomycin D alone (blue box) or together with Tm (red box) for 6 hours (h) and analyzed for HAMP mRNA expression. (F-G) HepG2 cells were transfected either with a control pool of small interfering RNA (siRNA) or a pool of siRNA designed to silence human HuR and subjected to either treatment with mock (blue box) or Tm (red box) for 6 h. The results of four experiments were analyzed by repeated measures one-way ANOVA. Fold-change compared to cells transfected with siRNA control (ctl) and treated with mock are shown on the graphs. Total lysates from HepG2 cells treated with mock or Tm were subjected to cross-linking immunoprecipitation CLIP-seq RNA-CLIP with either HuR or control normal IgG antibodies. RNA was collected from the immunoprecipitates and analyzed for (H) HAMP 3’untranslated region (UTR) sequence and (I) a non-relevant sequence (BMPR1A) enrichment by quantitative RT-PCR. One representative experiment over three independent experiments is presented. Estimates of the fold changes in gene expression ( 2−DDCt) are shown on the graphs. *P<0.05; **P<0.01; ****P<0.0001. NS: not sigificant.
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haematologica | 2021; 106(4)