Abstract 4086: Ultraconserved non-coding RNAs are involved in human hepatocellular cancer growth
Authors: Braconi Chiara , Kogure Takayuki , Valeri Nicola , Huang Nianyuan , Gasparini Pierluigi , Nuovo Gerard , Terracciano Luigi , Croce Carlo M. , Patel Tushar 
Published: February 2011

Abstract
Background: Non-coding RNAs (ncRNA) have recently been implicated in carcinogenesis. Highly conserved ncRNA expression across species suggests a biological function. A group of ncRNAs that are greater than 200 bp and highly conserved across species (ultraconserved regions) has been recently shown to be differentially expressed in human cancers but their role in hepatocarcinogenesis remains unclear. Our aim was to evaluate the expression and potential role of these ultraconserved RNA (ucRNA) in hepatocellular cancer (HCC).
Methods: The global expression of 481 ucRNAs was analyzed in human non-malignant (HH) or malignant (HepG2) hepatocytes using a custom microarray. Expression of selected ucRNA was verified by real time PCR in HCC cell lines and by in situ hybridization in 30 human HCC tissues. Cells were transfected with siRNAs or plasmids over-expressing ucRNA to reduce or enhance their cellular expression. Anchorage dependent growth was evaluated using viable cell assay and anchorage independent growth was assessed by growth in soft agar using a fluorometric assay. Cell cycle progression was determined by flow cytometry.
Results: 56 ucRNAs were aberrantly expressed with 33 increased and 23 decreased in HepG2 cells compared to HH (p&lt;0.05). Of these, uc.338 was the most up-regulated (6.9-fold). uc.338 was significantly increased in several HCC cell lines (HepG2, Huh-7, PLC/PRF-5, SK-Hep-1, SNU-182, and SNU-449) compared to HH by real-time PCR. uc.338 expression was also detected in 73% of human HCC. Although uc.338 is located within the PCBP2 gene, the expression of PCBP2 mRNA did not correlate with that of uc.338 and inhibition of uc.338 did not reduce PCBP2 mRNA, suggesting independent regulation of these genes. Over-expression of uc.338 increased growth rate of HH compared to cells transfected with an empty vector. Huh-7 and HepG2 cells transfected with anti-uc.338 had slower growth rate compared to cells transfected with control-siRNA. Growth in soft agar was decreased by 40% in HepG2 cells transfected with anti-uc.338 compared to controls. Transfection with anti-uc.338 also increased the number of cells in sub-G1 phase and decreased the number of cells in S phase by 30 ± 1.3 % (p&lt;0.001) compared to controls. To confirm the conservation of uc.338 in different species, we assessed its expression in murine BNL-CL2 cells (embryonic liver mouse cells) and BNL-SVA.8 cells (SV40-transformed BNL-CL2 cells with an increased growth potential). uc.338 was over-expressed by 2-fold in BNL-SVA.8 cells compared to BNL-CL2 cells (p:0.001). Inhibition of uc.338 by siRNA reduced anchorage-dependent growth by 65% at 72 hours in BNL-SVA.8 cells (p: 0.0001).
Conclusion: These studies identify a novel non-coding RNA uc.338 that is increased in expression and modulates cell growth in HCC in a species-independent fashion. These studies indicate a functional role for ultraconserved RNA genes and expand the role of non-coding RNA in HCC.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4086.