Transcriptional regulation of miR-224 upregulated in human HCCs by NF¦ÊB inflammatory pathways

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• 作者：Cecilia Scisciani¹ ; ² ; ³ ; Stefania Vossio² ; ⁵ ; Francesca Guerrieri¹ ; ² ; ³ ; Valeria Schinzari¹ ; ² ; ⁴ ; Rossana De Iaco¹ ; ² ; Paolo D&rsquo ; Onorio de Meo⁶ ; Melchiorre Cervello⁷ ; Giuseppe Montalto⁸ ; Teresa Pollicino⁹ ; Giovanni Raimondo⁹ ; Massimo Levrero¹ ; ² ; ³ ; ⁴ ; ^{massimo.levrero@uniroma1.it} ; Natalia Pediconi¹ ; ² ; ³ ; ⁴ ; ^{natalia.pediconi@uniroma1.it}
• 关键词：miR ; microRNA ; HCC ; hepatocellular carcinoma ; NF¦ÊB ; nuclear factor kappa-B ; TNF¦Á ; tumor necrosis factor-alpha ; LT¦Á ; Lymphotoxin-alpha (TNF¦Â) ; LPS ; lipopolysaccharide ; I¦ÊB¦Á ; nuclear factor kappa-B inhibitor alpha ; I¦ÊK ; nuclear factor-kappa-B inhibit
• 刊名：Journal of Hepatology
• 出版年：2012
• 出版时间：April, 2012
• 年：2012
• 卷：56
• 期：4
• 页码：855-861
• 全文大小：733 K

文摘

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