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

Scisciani, Cecilia; Vossio, Stefania; Guerrieri, Francesca; Schinzari, Valeria; De Iaco, Rossana; D’Onorio de Meo, Paolo; Cervello, Melchiorre; Montalto, Giuseppe; Pollicino, Teresa; Raimondo, Giovanni; Levrero, Massimo; Pediconi, Natalia

doi:10.1016/j.jhep.2011.11.017

« Previous Next »Journal of Hepatology
Volume 56, Issue 4 , Pages 855-861, April 2012

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

Cecilia Scisciani
- EAL Inserm U785, Sapienza University of Rome, Rome, Italy
- Laboratory of Gene Expression, Fond. A. Cesalpino, Rome, Italy
- Rome Oncogenomic Center, IRE, Rome, Italy
Stefania Vossio
- Laboratory of Gene Expression, Fond. A. Cesalpino, Rome, Italy
- Dept. of Biochemistry, University of Geneva, Geneva, Switzerland
Francesca Guerrieri
- EAL Inserm U785, Sapienza University of Rome, Rome, Italy
- Laboratory of Gene Expression, Fond. A. Cesalpino, Rome, Italy
- Rome Oncogenomic Center, IRE, Rome, Italy
Valeria Schinzari
- EAL Inserm U785, Sapienza University of Rome, Rome, Italy
- Laboratory of Gene Expression, Fond. A. Cesalpino, Rome, Italy
- Dept. of Internal Medicine, Sapienza University of Rome, Rome, Italy
Rossana De Iaco
- EAL Inserm U785, Sapienza University of Rome, Rome, Italy
- Laboratory of Gene Expression, Fond. A. Cesalpino, Rome, Italy
Paolo D’Onorio de Meo
- CASPUR, Consorzio interuniversitario per le Applicazioni di Supercalcolo per Università e Ricerca, Rome
Melchiorre Cervello
- IBIM, CNR, Palermo, Italy
Giuseppe Montalto
- Dept. of Clinical Medicine, University of Palermo, Palermo, Italy
Teresa Pollicino
- Dept. of Internal Medicine, University of Messina, Messina, Italy
Giovanni Raimondo
- Dept. of Internal Medicine, University of Messina, Messina, Italy
Massimo Levrero
- EAL Inserm U785, Sapienza University of Rome, Rome, Italy
- Laboratory of Gene Expression, Fond. A. Cesalpino, Rome, Italy
- Rome Oncogenomic Center, IRE, Rome, Italy
- Dept. of Internal Medicine, Sapienza University of Rome, Rome, Italy
- Corresponding authors. Address: Dept. of Internal Medicine (DMISM), Lab. of Gene Expression, Sapienza University, Rome, Italy. Tel.: +39 06 49970892; fax: +39 06 4452388.
Natalia Pediconi
- EAL Inserm U785, Sapienza University of Rome, Rome, Italy
- Laboratory of Gene Expression, Fond. A. Cesalpino, Rome, Italy
- Rome Oncogenomic Center, IRE, Rome, Italy
- Dept. of Internal Medicine, Sapienza University of Rome, Rome, Italy
- Corresponding authors. Address: Dept. of Internal Medicine (DMISM), Lab. of Gene Expression, Sapienza University, Rome, Italy. Tel.: +39 06 49970892; fax: +39 06 4452388.

Received 7 March 2011; received in revised form 18 October 2011; accepted 11 November 2011. published online 16 December 2011.

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 NFκB-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 NFκB sites. We showed that LTα and TNFα 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 IκBα (IκBSR) represses it. ChIP analysis showed that p65/NFκB is recruited on the miR-224 promoter and that its binding sharply increases after exposure to LPS, TNFα, and LTα. Altogether these findings link the inflammatory signals to NFκB-mediated activation of miR-224 expression. An antago-miR specific for miR-224 blocked LPS and LTα 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/NFκB as a direct transcriptional regulator of miR-224 expression and link miR-224 up-regulation with the activation of the LPS, LTα, and TNFα inflammatory pathways and cell migration/invasion in HCC.

Abbreviations: miR, microRNA, HCC, hepatocellular carcinoma, NFκB, nuclear factor kappa-B, TNFα, tumor necrosis factor-alpha, LTα, Lymphotoxin-alpha (TNFβ), LPS, lipopolysaccharide, IκBα, nuclear factor kappa-B inhibitor alpha, IκK, nuclear factor-kappa-B inhibitor kinase