Supplementary Fig. S3 from Hypoxia Causes Downregulation of Dicer in Hepatocellular Carcinoma, Which Is Required for Upregulation of Hypoxia-Inducible Factor 1α and Epithelial–Mesenchymal Transition

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posted on 2023-03-31, 20:06 authored by Ahmed Atef Ibrahim, Christian Schmithals, Erik Kowarz, Verena Köberle, Bianca Kakoschky, Thomas Pleli, Otto Kollmar, Scarlett Nitsch, Oliver Waidmann, Fabian Finkelmeier, Stefan Zeuzem, Horst-Werner Korf, Tobias Schmid, Andreas Weigert, Bernd Kronenberger, Rolf Marschalek, Albrecht Piiper

Supplementary Fig. S3. Doxycyline-induced overexpression of Dicer in HCC cells. HepG2 (A) and Huh-7 (B) cells stably transfected with the inducible Dicer construct were incubated with or without doxycycline (Dox) for 24 h. C: HepG2 cells with the inducible Dicer construct were incubated for the indicated duration with or without doxycycline. A-C: The lysates were analyzed by immunoblotting using anti-Dicer and anti-β-actin antibodies. D: Western blot analysis of Dicer protein expression in Dicer-transgenic HepG2 xenografts from mice treated for 7 days with doxycycline (n = 3) or vehicle (n = 4), that were used for the experiment displayed in Fig. 5F. A-D: β-actin was used as endogenous control.

Funding

DAAD

Deutsche Krebshilfe

History

ARTICLE ABSTRACT

Purpose: A role of Dicer, which converts precursor miRNAs to mature miRNAs, in the tumor-promoting effect of hypoxia is currently emerging in some tumor entities. Its role in hepatocellular carcinoma (HCC) is unknown.Experimental Design: HepG2 and Huh-7 cells were stably transfected with an inducible Dicer expression vector and were exposed to hypoxia/normoxia. HepG2-Dicer xenografts were established in nude mice; hypoxic areas and Dicer were detected in HCC xenografts and HCCs from mice with endogenous hepatocarcinogenesis; and epithelial–mesenchymal transition (EMT) markers were analyzed by immunohistochemistry or by immunoblotting. The correlation between Dicer and carbonic anhydrase 9 (CA9), a marker of hypoxia, was investigated in resected human HCCs.Results: Hypoxia increased EMT markers in vitro and in vivo and led to a downregulation of Dicer in HCC cells. The levels of Dicer were downregulated in hypoxic tumor regions in mice with endogenous hepatocarcinogenesis and in HepG2 xenografts. In human HCCs, the levels of Dicer correlated inversely with those of CA9, indicating that the negative regulation of Dicer by hypoxia also applies to HCC patients. Forced expression of Dicer prevented the hypoxia-induced increase in hypoxia-inducible factor 1α (HIF1α), HIF2α, hypoxia-inducible genes (CA9, glucose transporter 1), EMT markers, and cell migration.Conclusions: We here identify downmodulation of Dicer as novel essential process in hypoxia-induced EMT in HCC and demonstrate that induced expression of Dicer counteracted hypoxia-induced EMT. Thus, targeting hypoxia-induced downmodulation of Dicer is a promising novel strategy to reduce HCC progression. Clin Cancer Res; 23(14); 3896–905. ©2017 AACR.