Oncogene (2011) 30, 4750–4756 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc SHORT COMMUNICATION microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer C Braconi1, T Kogure1,2, N Valeri1, N Huang1, G Nuovo3, S Costinean1, M Negrini4, E Miotto4, CM Croce1 and T Patel1,2 1College of Medicine, and the Ohio State University Comprehensive Cancer Center, Ohio State University, Columbus, OH, USA; 2Mayo Clinic, Jacksonville, FL, USA; 3Department of Pathology, Ohio State University, Columbus, OH, USA and 4Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy The human genome is replete with long non-coding RNAs Introduction (lncRNA), many of which are transcribed and likely to have a functional role. Microarray analysis of 423 000 Recent large scale complementary DNA cloning lncRNAs revealed downregulation of 712 (B3%) lncRNA projects have identified that a large portion of the in malignant hepatocytes, among which maternally mammalian genome is transcribed, although only a expressed gene 3 (MEG3) was downregulated by 210-fold small fraction of these transcripts represent protein- relative to expression in non-malignant hepatocytes. coding genes (Kawaji et al., 2011). The functional role MEG3 expression was markedly reduced in four human for most of these transcripts remains obscure and their hepatocellular cancer (HCC) cell lines compared with relevance to human physiology and disease is undefined. normal hepatocytes by real-time PCR. RNA in situ Although the role of protein-coding genes as oncogenes hybridization showed intense cytoplasmic expression of or onco-suppressors has been extensively studied, the MEG3 in non-neoplastic liver with absent or very weak involvement of non-protein coding genes in tumor expression in HCC tissues. Enforced expression of MEG3 pathogenesis and growth is less well characterized. in HCC cells significantly decreased both anchorage- Their role in human cancers warrants evaluation. dependent and -independent cell growth, and induced Amongst the non-coding RNA, microRNAs have apoptosis. MEG3 promoter hypermethylation was identi- been extensively studied and their role in carcinogenesis fied by methylation-specific PCR and MEG3 expression has been established (Croce, 2009). Several non-coding was increased with inhibition of methylation with either RNAs (ncRNAs) with functional involvement in the 5-Aza-2-Deoxycytidine, or siRNA to DNA Methyltrans- liver have been identified, although the repertoire of ferase (DNMT) 1 and 3b in HCC cells. MiRNA- long ncRNA (lncRNA) is not clear. A large and dependent regulation of MEG3 expression was studied growing body of literature shows the involvement of by evaluating the involvement of miR-29, which can miRNAs in hepatocarcinogenesis (Meng et al., 2007; modulate DNMT 1 and 3. Overexpression of mir-29a Coulouarn et al., 2009; Gramantieri et al., 2009; Ji et al., increased expression of MEG3. GTL2, the murine 2009; Su et al., 2009; Wang et al., 2009; Braconi et al., homolog of MEG3, was reduced in liver tissues from 2010; Pineau et al., 2010). In contrast few long hepatocyte-specific miR-29a/b1 knock-out mice compared non-coding RNA have been identified (reviewed in with wild-type controls. These data show that methyla- Huarte and Rinn, 2010). Long ncRNA that have been tion-dependent tissue-specific regulation of the lncRNA implicated as having a functional role in hepato- MEG3 by miR-29a may contribute to HCC growth and carcinogenesis or liver cancer growth include HULC, highlight the inter-relationship between two classes of H19 and TUC338 (Matouk et al., 2007; Panzitt et al., non-coding RNA, miRNAs and lncRNAs, and epigenetic 2007; Braconi et al., 2011). regulation of gene expression. The mechanisms of expression of lncRNAs are Oncogene (2011) 30, 4750–4756; doi:10.1038/onc.2011.193; unknown, although there is evidence for regulation published online 30 May 2011 by similar mechanisms as for protein coding genes. Long ncRNAs frequently originate from intronic regions Keywords: MEG3; methylation; HCC; non-coding and are independently transcribed (Louro et al., 2009). RNA Likewise, their functional role remains obscure. It has been postulated that ncRNAs can act transcriptionally or post-transcriptionally; however, mechanisms that underlie such regulatory behaviors still remain to be fully understood. Long ncRNAs can mediate epigenetic changes by recruiting chromatin-remodeling complexes Correspondence: Professor T Patel, Department of Transplantation, to specific genomic loci. Other long ncRNAs have been Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA. E-mail: [email protected] shown to regulate transcription, whereas a few long Received 8 December 2010; revised 3 April 2011; accepted 18 April 2011; ncRNAs are antisense transcripts, which may regulate published online 30 May 2011 mRNA dynamics at a post-transcriptional level (Mercer MEG3 is an oncosuppressor in HCC C Braconi et al 4751 et al., 2009; Orom et al., 2010). The mechanisms by which of MEG3 in human liver tissues by in situ hybridization. ncRNA expression is altered in cancers are similar to those MEG3 staining was intensively positive in non-neoplas- for protein coding genes, and include epigenetic mecha- tic liver, whereas expression was remarkably reduced in nisms (Lujambio et al., 2007, 2008; Saito et al., 2010). human HCC tissues (Supplementary Figure 1). These In order to begin to understand the involvement data indicate that MEG3 expression is downregulated in of lncRNAs in human hepatocellular cancer (HCC), malignant liver cancer cell lines and tissues. we sought to identify lncRNAs that are altered in expression in HCC, and characterize their expression and functional involvement in tumor growth. We Functional consequences of deregulated MEG3 expression identified the maternally expressed gene-3 (MEG3), as To assess the potential functional role of MEG3 in one of the most significantly downregulated lncRNAs hepatic epithelia, we overexpressed the full length in malignant hepatocytes, and report a functional tissue- of MEG3 complementary DNA in HCC cells. PLC/ specific mechanism of regulation of MEG3 expression PRF/5 cells were transfected with MEG3-CMV6-XL5 via microRNA-29a-dependent regulation of promoter or empty-CMV6-XL5 vectors. MEG3 expression was methylation. increased by Beightfold after 24 h of transfection in cells transfected with vector overexpressing MEG3 in comparison with those transfected with an empty vector (Figure 2a). Cells with enforced expression of MEG3 Results and discussion had a decreased anchorage-dependent growth in compar- ison with cells transfected with empty vector (Figure 2b). Deregulated expression of the long non-coding RNA The capacity of the cells to grow in absence of anchorage MEG3 in human HCC is a typical feature of cancer cells and was further verified To identify long non-coding RNAs that are altered by assessing the capacity of forming colonies in soft agar. in expression in malignant hepatocytes, we performed Anchorage-independent growth was reduced by 40% in a hybridization-based microarray analysis of ncRNA cells with enforced expression of MEG3 (Figure 2c). expression (Arraystar, Rockville, MD, USA). The Next, we assessed the effect of MEG3 on progression expression of several lncRNAs was noted to be altered through the cell cycle. Cells transfected with MEG3 between malignant and normal human hepatocytes showed an increased accumulation in the sub G0/1 phase (Supplementary Table 1). MEG3 was among the top without any significant modulation of the checkpoint most significantly downregulated lncRNAs. MEG3 is an G0/1 or G2/M (Figure 3a). These data suggested that imprinted gene maternally expressed in humans that MEG3 may modulate apoptosis. Thus we assessed the encodes a long non-coding RNA of B1700 nucleotides rate of late apoptosis by terminal transferase dUTP nick- (Zhou et al., 2007). Although expressed in several end labeling assay after MEG3 overexpression in HCC diverse isoforms in fetal liver, MEG3 is expressed in cells. Interestingly apoptotic cells (BrdU þ /PI þ ) were adult liver as a few isoforms (Hagan et al., 2009; Zhang significantly increased after transfection with MEG3 in et al., 2010b). The potential role and involvement of comparison with transfection with an empty vector MEG3 in liver cancers is unknown. Using quantitative (Figure 3b). reverse transcriptase PCR, we verified that the expres- Deregulated expression of MEG3 in malignant sion of MEG3 was reduced in four separate HCC cell hepatocytes, and in association with liver preneoplastic lines relative to the expression in non-malignant foci and tumors, suggests a role in tumorigenesis. There hepatocytes (Figure 1a). Next, we assessed the expres- is data suggesting that MEG3 can function as a tumor- sion of MEG3 in a series of human primary HCC and suppressor. Expression of MEG3 is lost in pituitary adjacent liver tissues. MEG3 was remarkably reduced in tumors, mengiomas and myelomas (Zhang et al., 2003, expression in 81% of HCC in comparison with adjacent 2010a; Benetatos et al., 2008, 2010). Moreover, expres- cirrhotic tissue (Figure 1b). We also assessed expression sion of MEG3 has been shown to suppress growth and Figure 1 MEG3 expression is reduced in HCC. (a) MEG3 expression was assessed by real time PCR using SYBRGreen (Clontech, Mountain View, CA, USA) in human normal hepatocytes (HH) and HCC cells and expression was normalized to that of GAPDH.