Characterization of HULC, a Novel Gene With Striking Up-Regulation in Hepatocellular Carcinoma, as Noncoding RNA

Abstract

Background & Aims: Recent studies have highlighted the role of noncoding RNAs (ncRNAs) in carcinogenesis, and suggested that this class of genes might be used as biomarkers in cancer. We searched the human genome for novel genes including ncRNAs related to hepatocellular carcinoma (HCC). Methods: An HCC-specific gene library was generated and screened for deregulated genes with 46 HCCs, 4 focal nodular hyperplasias, and 7 cirrhoses utilizing cDNA arrays. Sequencing of library clones identified a novel ncRNA as the most up-regulated gene in HCC. This gene was also cloned from different monkeys and characterized by quantitative RT-PCR, Northern blot analysis and in situ hybridization. Structural and functional studies included comparative sequence and protein expression analyses, quantitative RT-PCR of polysomal preparations, and siRNA-mediated knockdown experiments. Results: The most up-regulated gene in HCC named highly up-regulated in liver cancer (HULC) was characterized as a novel mRNA-like ncRNA. HULC RNA is spliced and polyadenlyated, and resembles the mammalian LTR transposon 1A. It does not contain substantial open reading frames, and no native translation product was detected. HULC is present in the cytoplasm, where it copurifies with ribosomes. siRNA-mediated knockdown of HULC RNA in 2 HCC cell lines altered the expression of several genes, 5 of which were known to be affected in HCC, suggesting a role for HULC in post-transcriptional modulation of gene expression. Conclusions: HULC is the first ncRNA with highly specific up-regulation in HCC. Because HULC was detected in blood of HCC patients, a potential use as novel biomarker can be envisaged. Background & Aims: Recent studies have highlighted the role of noncoding RNAs (ncRNAs) in carcinogenesis, and suggested that this class of genes might be used as biomarkers in cancer. We searched the human genome for novel genes including ncRNAs related to hepatocellular carcinoma (HCC). Methods: An HCC-specific gene library was generated and screened for deregulated genes with 46 HCCs, 4 focal nodular hyperplasias, and 7 cirrhoses utilizing cDNA arrays. Sequencing of library clones identified a novel ncRNA as the most up-regulated gene in HCC. This gene was also cloned from different monkeys and characterized by quantitative RT-PCR, Northern blot analysis and in situ hybridization. Structural and functional studies included comparative sequence and protein expression analyses, quantitative RT-PCR of polysomal preparations, and siRNA-mediated knockdown experiments. Results: The most up-regulated gene in HCC named highly up-regulated in liver cancer (HULC) was characterized as a novel mRNA-like ncRNA. HULC RNA is spliced and polyadenlyated, and resembles the mammalian LTR transposon 1A. It does not contain substantial open reading frames, and no native translation product was detected. HULC is present in the cytoplasm, where it copurifies with ribosomes. siRNA-mediated knockdown of HULC RNA in 2 HCC cell lines altered the expression of several genes, 5 of which were known to be affected in HCC, suggesting a role for HULC in post-transcriptional modulation of gene expression. Conclusions: HULC is the first ncRNA with highly specific up-regulation in HCC. Because HULC was detected in blood of HCC patients, a potential use as novel biomarker can be envisaged. Noncoding RNAs (ncRNAs) have emerged as a new class of functional transcripts in eukaryotic cells, and were grouped into 3 subclasses according to their number of nucleotides.1Mattick J.S. Non-coding RNAs: the architects of eukaryotic complexity.EMBO Rep. 2001; 2: 986-991Crossref PubMed Scopus (626) Google Scholar, 2Costa F.F. 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Elevated expression of PCGEM1, a prostate-specific gene with cell growth-promoting function, is associated with high-risk prostate cancer patients.Oncogene. 2004; 23: 605-611Crossref PubMed Scopus (220) Google Scholar BC200 RNA overexpression has recently been correlated with the progression of breast tumors and proposed as a new molecular marker for breast carcinomas.14Iacoangeli A. Lin Y. Morley E.J. Muslimov I.A. Bianchi R. Reilly J. Weedon J. Diallo R. Bocker W. Tiedge H. BC200 RNA in invasive and preinvasive breast cancer.Carcinogenesis. 2004; 25: 2125-2133Crossref PubMed Scopus (136) Google Scholar Increased expression of the MALAT-1 gene indicates a worse clinical outcome in lung cancer patients, and further the potential role of ncRNAs in P. Diederichs S. Wang W. Boing S. Metzger R. Schneider P.M. Tidow N. Brandt B. Buerger H. Bulk E. Thomas M. Berdel W.E. Serve H. Muller-Tidow C. MALAT-1, a novel non-coding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer.Oncogene. 2003; 22: 8031-8041Crossref PubMed Scopus (1799) Google Scholar carcinoma is of the of and a J. P. the cancer J 2001; PubMed Scopus Google Scholar a and with a complex most the of liver by hepatitis C virus liver a variety of J.W. of human hepatocellular Genet. PubMed Scopus Google Scholar The of these liver diseases to the development of liver which HCC the highly HCC, several also in the The most in the liver is focal nodular which is characterized by of and that are to be related to liver T. R. V. in focal nodular of the PubMed Scopus Google Scholar the evidence for a ncRNAs and and a for novel which are associated with the molecular of HCC, not in gene and HCC-specific gene and cDNA identified a novel ncRNA as the most up-regulated gene in the and named it HULC up-regulated in liver to its expression a of this first ncRNA associated with HCC. were from the the of of from tumors and from were in with in and in The was by the of the of RNA was from 3 HCC and from 3 liver and was cDNA and were the cDNA were cloned into and into For and the was were from the HCC cDNA cDNA clones genes with in cell and were from the for and were included as for clones were and according to P. R. K. C. S. R. E. N. J. to cDNA 2000; PubMed Scopus Google Scholar and a were the to of were used for RNA with included a liver as In a of RNA of were with different of The RNAs were with 3 5 by were to the for to P. R. K. C. S. R. E. N. J. to cDNA 2000; PubMed Scopus Google Scholar and were a were by and and for were in the 3 and 5 by were by from the According to their the in the 2 was were of was by the The was used to for with In and and 7 the the of of were and a RNA The first the RNA not to RNA were by quantitative RT-PCR and of HULC and RNA were from RNA genes expression levels in the with where the first were of siRNA-mediated HULC knockdown in the and cell were with 2 different and 2 different for HULC RNA expression levels from 2 were by quantitative RT-PCR in the knockdown to the present the of The of cDNA cDNA was used for the of and from RNA, from RNA from a of HCC and from RNA with The sequence for the HULC has been into the with the number of RNA were a and to a by The HULC to was with J. Xu T. Zhang J. W. G. G. M. Xu W. J.R. Z. into hepatocellular by gene expression profiles of hepatocellular carcinoma with of Natl Acad Sci U S A. 2001; PubMed Scopus Google the was used for and and were detected by For the of blood from blood for which was 5 of blood was in 5 were in and to in a in a for and RNA from cell from the by of and for and was the For the of RNA from were the to of were used for RNA with according to the RNA from was as A. B. K. H. gene in 2005; PubMed Scopus Google Scholar cDNA was from of RNA from 3 including the liver from the cDNA were used with of cDNA and were in were generated in with and and the were The were HULC HULC HULC were from to the gene, RNA were as in situ was as P. M. A. C. K. H. as for J Pathol. PubMed Scopus Google Scholar The was generated by in of were in and of the were with a and with were in with and the and were a in for 2 were from the and RNA were cDNA was from RNA and to quantitative RT-PCR were in 2 and was and a with a were with the and for The were to the putative were detected with the were in with and in and were with 4 2 were into the in were from and analysis 2 different of the HULC RNA were used for the for HULC and for HULC involved a and a as and sequence as was according to the were to with and in For of a for in were by of and was to were of a were and to RNA of RNA was and the of was by quantitative were to hybridization. The of RNA from was the with a RNA were to of RNA the The cDNA product was to genes according to the were the For cell and for knockdown 2 were were the and analysis were used to genes affected by siRNA-mediated Expression were by and the up-regulated genes were to functional to and affected by the For a gene expression generated HCC-specific cDNA by Z. gene expression Natl Acad Sci U S A. PubMed Scopus Google Scholar from the HCC cDNA library in with and genes were used for the of HCC-specific cDNA a of cDNA of 46 HCCs, 4 7 and 2 were with a of liver be as The most of up-regulated genes a with and 2 genes that are known to be highly up-regulated the of human HCC J.W. of human hepatocellular Genet. PubMed Scopus Google Scholar, L. J.P. R. C. C. of in human liver PubMed Google Scholar, E. of the and of are in liver and Res. Google Scholar the liver in of HCCs, and by 3 HCC-specific library this was named HULC expression levels were to a up-regulated in In to the in liver HULC was up-regulated in liver HULC is commonly overexpressed in neoplastic of a of normal and their were for HULC RNA expression by quantitative RT-PCR HULC was in most of the normal and not in the of the neoplastic The of HULC RNA levels in prostate and was as as in HCC, where the HULC RNA expression normal and neoplastic by a of The quantitative RT-PCR the cDNA although the levels of up-regulation were in the cDNA these a highly specific up-regulation of HULC RNA expression levels in HCC. In situ the specific and expression of HULC RNA in the of HCC, it was not detected in and liver Northern blot analysis identified HULC with a length of HULC RNA levels were in HCC than in liver which is with cDNA and quantitative RT-PCR a gene and to the HULC transcriptional and and of cDNA cDNA were Sequencing of the HULC sequence to a human analysis of the sequence to the of the HULC transcriptional The HULC gene a and and 2 and from the and The genomic the HULC is of LTR mammalian LTR transposon indicating the of a this A. Identification of a of mammalian Acids Res. 2: Scopus Google Scholar that to no cDNA in the as from the abundance of the HULC identified 5 to HULC in the genomic and and the genome of these were from a of liver and cDNA a of from including in of not HULC in for a in for HULC in cloned and HULC from 3 monkeys and HULC were with quantitative RT-PCR from liver RNA from these human Sequencing of the that HULC were highly conserved in these the of and the and in the In analysis of was J. D. T.R. J. Brosius J. The BC200 RNA gene and its neural expression are conserved in PubMed Scopus Google Scholar The human HULC a of with of 5 in In the to was and to 2 was from the HULC This indicates of HULC and that it is transcriptional to have a HULC The sequence and of the HULC transcriptional in are highly for several to to for the and for the no is for the HULC transcriptional of the HULC RNA sequence is the of in the potential reading frames the HULC in translation of the HULC RNA not the of a substantial in the HULC The with a functional translation is nt and a gene product of the sequence does not contain known protein does it to in the lack of than suggested that the HULC RNA might not be J.S. the the of RNAs in complex 2003; 25: PubMed Scopus Google Scholar in translation to protein product was generated from the HULC RNA In translation as as overexpression of HULC in not not protein product to than 4 generated to 2 from this potential sequence to a putative protein by this these studies were of HCC and and cell with the putative HULC expression In was a HULC protein not be detected by analysis of from levels of HULC RNA from with a HULC expression the putative HULC sequence was to the of to a the detected the sequence in the protein indicating that were to to the used for The of HULC was further by analysis of with quantitative In with the by the of HULC were detected in the not Because 2 human ncRNAs, and were to be associated with and to J. D. T.R. J. Brosius J. The BC200 RNA gene and its neural expression are conserved in PubMed Scopus Google Scholar, H. P. S. Y. of in cancer to of and Res. Google Scholar, M. B. A. A. S. B. C. The protein with RNA and the translation of specific 2003; PubMed Scopus Google Scholar further HULC RNA also to the ribosomes. of RNA from the by and quantitative RT-PCR analysis of RNA from a of HULC RNA in the a first into a role of HULC in hepatocellular carcinogenesis, the of siRNA-mediated knockdown of HULC expression was in 2 cell lines by transcriptional For that and were for with 2 different and HULC different of the HULC different HULC and a were included in this to the and of HULC to C was used as and for of the not RNA from 2 of these was to quantitative RT-PCR for of HULC knockdown of and were for HULC and 2 in cells, HULC knockdown in was and HULC expression by and with the of the not these the expression of the marker genes and protein B. G. Zhang Y. Wang J.R. Lin Z. G. of by PubMed Scopus Google Scholar was not affected not For transcriptional RNAs from 2 knockdown in and with the 2 different HULC and the 2 different were to the of genes that were in as as in to the 2 which several have been in the of liver different features, searched for these and HULC and these the which not in Cell with to in HULC cell cell associated protein cell protein gene protein protein to protein RNA 4 that have been in the of liver cancer. in cell lines with to protein protein protein cell and with cell and in HULC associated protein are grouped with their and function according to that have been in the of liver cancer. in cell lines with to in a new are grouped with their and function according to In a blood from with liver and 4 HCC were by quantitative RT-PCR for the of HULC RNA in of The for 3 of 4 HCC of HULC RNA HULC expression levels in blood were in blood 3 and in blood 4 than in the of known liver For 2 of the HCC of blood and blood and from tumors and liver were for a of HULC expression in blood with HULC expression in liver HCC were from the and liver the and HULC expression levels in were by quantitative HULC expression in HCC was 5 and than in the liver not HCC-specific cDNA identified a novel mRNA-like as of the most up-regulated genes in HCC. 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Y. as a in 2004; PubMed Scopus Google Scholar of a in the was by in H. S. T. O. R. Y. T. Y. Y. analysis of gene expression in human hepatocellular cDNA of genes involved in and Res. 2001; Google Scholar indicating that HULC is not to HCC in as in such a expression has not been for a of The of HULC as ncRNA is several and The HULC sequence does not a substantial open reading in translation not protein a HULC protein not a in HCC a of HULC RNA, eukaryotic protein expression were to a and a protein was with the the HULC sequence was to the and not to the of HULC is as a protein was by structural of HULC RNA the which usually not found in not In this it was also to the length of the HULC The of clones by different was to the RNA length detected by Northern of HULC genes in 3 of the HULC sequence the not in the further and which indicates the and of functional the HULC RNA for the of the HULC gene is the that the first of the HULC sequence of LTR mammalian LTR transposon A. Identification of a of mammalian Acids Res. 2: Scopus Google Scholar We that a mammalian LTR transposon from J. D. T.R. J. Brosius J. The BC200 RNA gene and its neural expression are conserved in PubMed Scopus Google Scholar a of HULC was not the the HULC gene to and This that HULC has been by the monkeys from J. D. T.R. J. Brosius J. The BC200 RNA gene and its neural expression are conserved in PubMed Scopus Google Scholar by J. and from the RNA to the 2003; 3: PubMed Scopus Google Scholar are into that expression of J. a for and Natl Acad Sci U S A. PubMed Scopus Google Scholar might be for The of this is further by the conserved of are most to of the of the sequence is of a functional of this more than 2 of the HULC were for their the and these not the of which are for in the the functional of conserved in the HULC sequence the function of HULC were from siRNA-mediated knockdown of HULC in and cells, which in a and of several genes, of which have been in the of liver cancer. This indicates that not be HULC gene, a more role for The of HULC with the a to its of as it is of the of which have recently been found to be associated with the the complex that is to and can as translational Verhaegh G.W. D. Schalken J.A. a and specific marker to prostate Res. Google Scholar In to such ncRNAs, for which a to the has been F.F. Non-coding RNAs: new players in eukaryotic biology.Gene. 2005; 357: 83-94Crossref PubMed Scopus (282) Google Scholar and which have as involved in carcinogenesis, HULC as for the of mRNA-like ncRNA with ribosomes. studies no HULC and its putative indicating that the of HULC its not be the by which HULC of a to be HULC expression was also in which is a liver from of to T. R. V. in focal nodular of the PubMed Scopus Google Scholar does not to HCC, are several affected in which also a role in in cell cell of a cell and The up-regulation of HULC expression in also HULC expression with suggesting that HULC to modulation of gene expression in than involved in the of the highly HCC is no of are no in to this in more the of HULC with the and knockdown a role of HULC in the post-transcriptional of gene expression. The recent of new of ncRNAs in of cancer and progression the role of these transcripts in the of and several potential for and biomarkers for for example, is the most prostate ncRNA M.J. van Bokhoven A. Verhaegh G.W. Smit F.P. Karthaus H.F. Schalken J.A. Debruyne F.M. Ru N. Isaacs W.B. DD3: a new prostate-specific gene, highly overexpressed in prostate cancer.Cancer Res. 1999; 59: 5975-5979PubMed Google Scholar and a and specific marker for the of J.W. E.J. complex in 2004; PubMed Scopus Google Scholar is deregulated in breast W. Bocker W. Brosius J. Tiedge H. Expression of neural BC200 RNA in human tumours.J Pathol. 1997; 183: 345-351Crossref PubMed Scopus (174) Google Scholar and BC200 RNA overexpression was recently as a new molecular marker for a in breast carcinomas.14Iacoangeli A. Lin Y. Morley E.J. Muslimov I.A. Bianchi R. Reilly J. Weedon J. Diallo R. Bocker W. Tiedge H. BC200 RNA in invasive and preinvasive breast cancer.Carcinogenesis. 2004; 25: 2125-2133Crossref PubMed Scopus (136) Google Scholar In this the potential role of HULC as a novel biomarker is its expression and by the that HULC RNA can be detected in the blood of HCC and in by H. P. S. Y. of in cancer to of and Res. Google Scholar, R. R. L. R. J.P. M. development the of 2003; PubMed Scopus Google Scholar The A. J. and E. for J. K. K. H. M. D. M. A. R. and M. for and W. and M. G. for the We also and of the of of for human and associated