Expression and functional role of a transcribed noncoding RNA with an ultraconserved element in hepatocellular carcinoma Chiara Braconia, Nicola Valerib, Takayuki Kogurea, Pierluigi Gasparinib, Nianyuan Huanga, Gerard J. Nuovoc, Luigi Terraccianod, Carlo M. Croceb, and Tushar Patela,1 Departments of aInternal Medicine, bMolecular Virology, Immunology, and Medical Genetics, and cPathology, College of Medicine, Ohio State University, Columbus, OH 43210; and dMolecular Pathology Division, Institute of Pathology, University Hospital Basel, 4003 Basel, Switzerland Edited by Raymond L. White, University of California at San Francisco, Emeryville, CA, and approved December 9, 2010 (received for review July 28, 2010) Although expression of non–protein-coding RNA (ncRNA) can be shown to be involved in the modulation of cell proliferation and altered in human cancers, their functional relevance is unknown. apoptosis (12–15). Recent studies revealing the presence of Ultraconserved regions are noncoding genomic segments that are several hundred long transcribed ncRNAs raise the possibility 100% conserved across humans, mice, and rats. Conservation of that many ncRNAs contributing to cancer remain to be discov- gene sequences across species may indicate an essential functional ered (16). Other than microRNAs, however, only a handful of role, and therefore we evaluated the expression of ultraconserved ncRNAs have been implicated in hepatocarcinogenesis (17–20). RNAs (ucRNA) in hepatocellular cancer (HCC). The global expres- For the most part, the function of these ncRNAs is unknown. sion of ucRNAs was analyzed with a custom microarray. Expression Sequence conservation across species has been postulated to in- was verified in cell lines by real-time PCR or in tissues by in situ dicate that a given ncRNA may have a cellular function (21, 22). A genome-wide survey identified several hundred ncRNAs with hybridization using tissue microarrays. Cellular ucRNA expression > was modulated with siRNAs, and the effects on global gene ex- a size 200 bp that showed a remarkable conservation with 100% pression and growth of human and murine HCC cells were evalu- identity across the human, mouse, and rat genomes (23). These highly conserved ncRNAs have been named ultraconserved ele- ated. Fifty-six ucRNAs were aberrantly expressed in HepG2 cells ments and are conserved across many other species as well, with compared with nonmalignant hepatocytes. Among these ucRNAs, MEDICAL SCIENCES 99% of these RNAs showing high levels of conservation within the greatest change was noted for ultraconserved element 338 the dog, 97% within the chicken, and 67% within the fugu (uc.338), which was dramatically increased in human HCC com- genomes. Their wide distribution in the genome and lack of pared with noncancerous adjacent tissues. Although uc.338 is natural variation in the human population suggested that these PCBP2 partially located within the poly(rC) binding protein 2 ( ) ultraconserved regions have a biological function that is essential gene, the transcribed ncRNA encoding uc.338 is expressed inde- for normal cells (24, 25). pendently of PCBP2 and was cloned as a 590-bp RNA gene, termed Recent genome-wide expression profiling studies showed that TUC338. Functional gene annotation analysis indicated predomi- transcribed ultraconserved RNAs (ucRNAs) exhibit distinct nant effects on genes involved in cell growth. These effects were profiles in various human cancers (26, 27). Aberrant expression experimentally demonstrated in both human and murine cells. of specific ucRNA has been linked with leukemia and several siRNA to TUC338 decreased both anchorage-dependent and an- solid tumors. The ultraconserved element 73 (uc.73), for exam- chorage-independent growth of HCC cells. These studies identify ple, modulates apoptosis and cell proliferation in colorectal a critical role for TUC338 in regulation of transformed cell growth cancer cells (26). A correlation of some ucRNA with clinical and of transcribed ultraconserved ncRNA as a unique class of prognostic factors, such as Myc amplification, has been reported genes involved in the pathobiology of HCC. in neuroblastoma (28). Although rare, single-nucleotide poly- morphisms in ultraconserved regions have been reported in liver cancer | hepatocarcinoma | exaptation | transposon familial breast cancer, chronic lymphocytic leukemia, and co- lorectal cancer (29, 30). The functional role of ucRNAs in hu- epatocellular carcinogenesis involves a complex interaction man cancer behavior and development is unknown, and our aims Hof genes resulting in variable modulation of key pathways were to evaluate their expression and potential involvement in involved in tumor cell growth. By using molecular techniques growth regulation in human HCC. fi for global genomic pro ling, the transcriptome in hepatocellular Results and Discussion cancers (HCCs) has been described, and several genes that are differentially activated have been identified. The major focus of Aberrant Expression of Selected ucRNAs in Malignant Hepatocytes. fi fi attention in these efforts has been on the characterization of ex- Genome-wide expression pro ling identi ed 56 ucRNAs, rep- pression of protein-coding genes and their use for determining resenting 11% of all ucRNAs analyzed, that were aberrantly and fi < clinical outcomes (1–9). However, the majority of the human ge- signi cantly (P 0.05) expressed in malignant HepG2 cells nome consists of non–protein-coding RNA (ncRNA). Increasing compared with nonmalignant human hepatocytes (Fig. 1A). Of evidence points to an important functional or regulatory role of these, 33 were increased by 1.3- to 6.9-fold, whereas 23 were ncRNA in cellular processes as well as a contribution of aberrant decreased by 0.8- to 0.3-fold in malignant hepatocytes. Exonic ncRNA expression to disease phenotypes. ucRNAs were not selectively enriched in malignant cells, and the Along with the highly abundant transfer and ribosomal RNAs, ncRNAs include microRNAs that modulate mRNA expression, small nucleolar RNAs that guide chemical modification of RNA Author contributions: C.B. and T.P. designed research; C.B., N.V., T.K., P.G., N.H., G.J.N., molecules, siRNAs that account for the interference pathway, and T.P. performed research; L.T. and C.M.C. contributed new reagents/analytic tools; C.B. Piwi-RNAs that are linked to transcriptional gene silencing of analyzed data; and C.B. and T.P. wrote the paper. retrotransposons, and long ncRNAs, whose role is still unknown The authors declare no conflict of interest. (10, 11). The role played by the ncRNA genome in malignant This article is a PNAS Direct Submission. transformation and tumor growth in HCC is being increasingly 1To whom correspondence should be addressed. E-mail: [email protected]. recognized. We and others have recently provided data from This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. profiling studies in which several microRNAs were identified and 1073/pnas.1011098108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1011098108 PNAS Early Edition | 1of6 Downloaded by guest on October 4, 2021 exonic A B non exonic A B uc.338 possibly exonic uc.20 uc.189 100 35 * 0.00 90 * 844.cu 431.cu 30 80 0.01 100 70 HCC 25 * 29 23 60 90 Cirrhotic liver 20 0.02 80 50 Cases, % Non cirrhotic 70 40 15 liver 30 p value 60 0.03 42 54 10 50 20 0.04 40 10 5 uc.338 positivity, % of % of positivity, cells uc.338 uc.24 % Percentage, 30 0 0 20 29 negative weak moderate strong 0.05 23 non cirrhotic cirrhotic HCC 10 5-19 20-49 50 liver liver 0 0.06 s uc.338 positivity, % of cells d A e -3 -2 -1 0 1 2 3 4 fi N i R d c Fold change, o 2 u m G 1 scrambled probe log2 s p 8 C D A e 4 probe anti-uc.338 HepG2 / HH N H ll 5 R n a c i u 4 3 HCC Fig. 1. ucRNAs are aberrantly expressed in malignant hepatocytes. (A) 2 increased Genome-wide expression profiling was performed in HepG2 malignant cells 1 0 and normal human hepatocytes (HH). Fifty-six ucRNAs were aberrantly ex- score uc.338 adjacent -1 pressed in malignant hepatocytes with P < 0.05, with 12 ucRNAs increased non cancerous -2 and 7 decreased by greater than twofold. The ratio of expression of these liver -3 ucRNAs in malignant cells relative to nonmalignant cells is plotted against the -4 decreased -5 P value. Selected ucRNAs with a greater than threefold change in expression are annotated. (B) The genomic locations of the ucRNA as exonic, nonexonic, Fig. 3. uc.338 is overexpressed in human HCC tissues. uc.338 expression was or possibly exonic relative to protein-coding genes is depicted for all ucRNAs evaluated in a total of 221 HCCs, 72 cases of noncirrhotic liver tissues, and 97 and for the group of ucRNAs that are aberrantly expressed in malignant cases of cirrhotic adjacent liver tissues. Paraffin-embedded, formalin-fixed hepatocytes. Selective enrichment of a specific group of ucRNA based on liver tissues were incubated with LNA–anti-uc.338. (A) uc.338 expression was their genomic relationship to known protein-coding genes was not observed. classified as negative, weak, moderate, or strong based on the percentage of cells with detectable staining for uc.338. The proportion of cases of HCC, cirrhotic liver, or noncirrhotic liver within each class is depicted in the col- proportion of exonic regions in aberrantly expressed ucRNA umns. (B) The mean and 95% confidence intervals of uc.338 expression in (29% exonic, 42% nonexonic) was similar to those of all ultra- noncirrhotic liver, cirrhotic liver, and HCC tissues is shown. *P < 0.05. (C) conserved regions (Fig. 1B). The greatest change was noted for uc.338 expression was compared between 156 HCCs and their corresponding ultraconserved element 338 (uc.338), which was increased in adjacent liver tissues. Picture of representative cases are shown.