Page 49 - ebook HCC

46 PROGRAMME AND ABSTRACTS GENEVA, SWITZERLAND EASL HCC SUMMIT 47
FEBRUARY 13 - 16, 2014

OPPOSING PPARG AND ROSIGLITAZONE
PATHWAYS CONVERGE ON RUVBL1 IN HCC
CELL LINES

Tommaso Mello 1, 2 , Mirko Tarocchi , Fabio Perini , Francesca Buccoliero , Despite its ability to activate PPARg transcriptional activity in Hepa1-6 and HepG2
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Giada Marroncini , Ceni Elisabetta , Simone Polvani , Sara Tempesti , cells, RGZ had negligible effects on Ruvbl-1 promoter activity. Moreover, RGZ does not
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Stefano Milani , Andrea Galli 1, 2 destabilize Ruvbl1 mRNA, as tested by the 3’-UTR reporter assay. Analysis of Ruvbl1
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pre-mRNA suggests that RGZ may impair Ruvbl1 mRNA maturation thereby reducing its
1 Biomedical, Clinical and Experimental Sciences, University of Florence, expression.
2 Gastroenterology Unit, Careggi University Hospital, Florence, Italy
Both PPARg or Ruvbl1 silencing impair HCC cell growth in vitro and reduce the number
Corresponding author’s e-mail: tommaso.mello@unifi.it and size of tumors in the orthotopic mice model. PPARg downregulation reduced HCC cell
viabilty, which was recovered through the overexpression of Ruvbl1.
Introduction: Ruvbl1 is an AAA+helicase involved in many cellular processes including
BASIC SPEAKERS ABSTRACTS cancers including HCC, where its expression correlate with a poor prognosis. We expression. Knockdown of Ruvbl-1 or PPARgamma effectively reduces HCC cell growth BASIC SPEAKERS ABSTRACTS
Conclusions: RGZ and PPARg antagonize each other in the regulation of Ruvbl1
cell growth and oncogenic transformation. Ruvbl1 is overexpressed in several human
in vitro and in vivo. Since Ruvbl1 regulates several cellular processes crucial for cancer,
previously identified Ruvbl1 as a gene downregulated by Rosiglitazone (RGZ) in a
the net outcome of PPARg and RGZ antagonism may potentially elicit either pro- or anti-
transgenic PPARg-conditional knock-out mice prone to HCC. However, the role of PPARg
and RGZ in the regulation of Ruvbl1 are currently unknown.
cancer effects.
Aims: To investigate the RGZ-PPARg axis in the regulation of Ruvbl1 and its biological
relevance to HCC cell growth.
Methodology: Cell lines: Hepa1-6 and HepG2. Ruvbl1 expression was evaluated by
qPCR, western blot and IHC. Ruvbl1 promoter analysis was performed by CHIP and
reporter assays. mRNA stability was evaluated by 3’-UTR reporter assay. Modulation of
Ruvbl1 and PPARg was performed by RNAi and overexpression. HCC cell growth was
evaluated in vitro by thymidine incorporation and ATP measurement, and in vivo through
a syngenic-orthotopic mice model.

Results: RGZ reduces Ruvbl-1 expression in HCC cell lines. Surprisingly, also
PPARg silencing significantly reduces both Ruvbl1 expression and promoter activity.
Overexpression of PPARg activates Ruvbl1 promoter and increases Ruvbl1 mRNA and
protein levels. In silico analysis identified several putative PPAR Response Elements
on the Ruvbl1 promoter. By progressive deletions we show that funcional PPREs are
localized in two distinct promoter regions, consistent with the in silico prediction. PPARg
binding on Ruvbl1 promoter was confirmed by CHIP in HepG2 cells.