Transcriptional regulation of miR-224 upregulated in human HCCs by NFκB inflammatory pathways

Background & Aims: miR-224 is up-regulated in human HCCs as compared to both paired peri-tumoral cirrhotic tissues and cirrhotic livers without HCC. Here, we have cloned the miR-224 regulatory region and characterized its transcriptional regulation by the NF kappa B-dependent inflammatory pathways. Methods: Mature miRNA expression was evaluated by a 2 step stem-loop real-time RT-PCR. The recruitment of polymerase II and transcription factors on the pre-miR-224 promoter has been assessed by ChIPSeq and ChIP. Results: We found miR-224 levels strongly up-regulated in both peri-tumoral cirrhotic livers and HCC samples as compared to normal livers. In silico analysis of the putative miR-224 promoter revealed multiple NF kappa B sites. We showed that LT alpha and TNF alpha activate transcription from the miR-224 promoter and of endogenous miR-224 expression in HCC cell lines, whereas the expression of miR-224 target API5 was reduced. Exogenously expressed p65/RelA activates the miR-224 promoter and a dominant negative form of I kappa B alpha (I kappa BSR) represses it. ChIP analysis showed that p65/NF kappa B is recruited on the miR-224 promoter and that its binding sharply increases after exposure to LPS, TNF alpha, and LT alpha. Altogether these findings link the inflammatory signals to NF kappa B-mediated activation of miR-224 expression. An antagomiR specific for miR-224 blocked LPS and LT alpha stimulated HCC cells migration and invasion. Conversely, the IKK inhibitor BMS-345541 blocks pre-miR-224-induced cellular migration and invasion. Conclusions: Our results identify p65/NF kappa B as a direct transcriptional regulator of miR-224 expression and link miR-224 up-regulation with the activation of the LPS, LT alpha, and TNF alpha inflammatory pathways and cell migration/invasion in HCC. (c) 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.