ABSTRACT
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.
Subject(s)
Carcinoma, Hepatocellular/physiopathology , Liver Neoplasms/physiopathology , MicroRNAs/physiology , NF-kappa B/physiology , Signal Transduction/physiology , Transcription, Genetic/physiology , Up-Regulation/physiology , Aged , Carcinoma, Hepatocellular/pathology , Case-Control Studies , Cell Line, Tumor , Cell Movement/drug effects , Female , Humans , Lipopolysaccharides/pharmacology , Liver/pathology , Liver/physiology , Liver Cirrhosis/pathology , Liver Cirrhosis/physiopathology , Liver Neoplasms/pathology , Lymphotoxin-alpha/pharmacology , Male , Middle Aged , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Cell specification in the nervous system requires patterning genes dictating spatio-temporal coordinates as well as fate determinants. In the case of neurons, which are controlled by the family of proneural transcription factors, binding specificity and patterned expression trigger both differentiation and specification. In contrast, a single gene, glide cell deficient/glial cell missing (glide/gcm), is sufficient for all fly lateral glial differentiation. How can different types of cells develop in the presence of a single fate determinant, that is, how do differentiation and specification pathways integrate and produce distinct glial populations is not known. By following an identified lineage, we here show that glia specification is triggered by high glide/gcm expression levels, mediated by cell-specific protein-protein interactions. Huckebein (Hkb), a lineage-specific factor, provides a molecular link between glide/gcm and positional cues. Importantly, Hkb does not activate transcription; rather, it physically interacts with Glide/Gcm thereby triggering its autoregulation. These data emphasize the importance of fate determinant cell-specific quantitative regulation in the establishment of cell diversity.
