Ultraconserved RNA Research Articles

Functional Modulation of Gene Expression by Ultraconserved Long Non-coding RNA TUC338 during Growth of Human Hepatocellular Carcinoma

SummaryTUC338 is an ultraconserved long non-coding RNA that contributes to transformed cell growth in hepatocellular carcinoma (HCC). Genomic regions of TUC338 occupancy were enriched in unique or known binding motifs homologous to the tumor suppressors Pax6 and p53. Genes involved in cell proliferation were enriched within a 9-kb range of TUC338-binding sites. TUC338 RNA-based purification was used to isolate chromatin for mass spectrometry, and the plasminogen activator inhibitor-1 RNA-binding protein (PAI-RBP1) was identified as a TUC338 RNA-binding partner. The PAI-RBP1 target gene plasminogen activator inhibitor-1 (PAI-1) itself could also be post-transcriptionally regulated by TUC338. Thus modulation of transformed cell growth by TUC338 may involve binding to PAI-RBP1 as well as to sequence-defined cis-binding sites to modulate gene expression. These findings suggest that ultraconserved RNAs such as TUC338 can function in a manner analogous to transcription factors to modulate cell proliferation and transformed cell growth in HCC.

Abstract 4086: Ultraconserved non-coding RNAs are involved in human hepatocellular cancer growth

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<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<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.