Abstract 1178: Involvement of MEG3, a long non-coding RNA, in hepatocellular cancer (HCC)
Authors: Braconi Chiara , Kogure Takayuki , Valeri Nicola , Nuovo Gerard , Costinean Stefan , Croce Carlo Maria , Patel Tushar 
Published: June 2012

Introduction: The mammalian genome is replete with long non coding RNAs (ncRNAs). Although emerging evidence supports the involvement of ncRNAs in cancer, the expression and functional role of ncRNAs in HCC has not been systematically evaluated. Experimental procedures: Genome-wide expression of &gt;23,000 ncRNAs was analyzed in normal hepatocytes and 3 HCC cell lines by microarray analysis. Expression of Maternally Expressed Gene 3 (MEG3), was verified by real time PCR in human HCC cell lines and by in situ hybridization (ISH) in human HCC tissues. Methylation dependent regulation of MEG3 was evaluated by treatment with 5-Aza-2-Azacytidine (5-Aza), siRNA-mediated inhibition of DNA Methyltransferases (DNMT) and methylation specific PCR (msPCR) of the promoter. Cells were transfected with 1ug full-length MEG3 or empty vector controls, or with 100nM miR-29 or miRNA control. Anchorage dependent growth was evaluated using a viable cell assay and anchorage independent growth by assessing growth in soft agar. Cell cycle progression and apoptosis were determined by flow cytometry. A hepatocyte-specific-miR-29a/b1-knock out (KO) mouse was developed by breeding miR-29a/b1 loxP/loxP and Albumin-Cre mice. Results: 712 (∼3%) ncRNAs were down-regulated and 218 (∼1%) ncRNAs were up-regulated in HCC cells compared to non-malignant hepatocytes. We confirmed that the expression of the ncRNA MEG3 was markedly reduced in HepG2, PLC/PRF-5, Huh-7, and Hep-3B cells compared to normal hepatocytes by real-time PCR. An intense cytoplasmic expression of MEG3 was noted by ISH in non-neoplastic liver whereas expression was absent or very weak in HCC tissues. Enforced expression of MEG3 in HCC cells significantly decreased both anchorage dependent and anchorage independent cell growth. Over-expression of MEG3 did not affect progression through the cell cycle, but increased apoptosis. MEG3 expression was significantly increased in HCC cells after 5 days-treatment with 5-Aza, or with siRNA to DNMT1 and 3b. Hypermethylation of the promoter of MEG3 in HCC cells was confirmed by msPCR. The role of miRNA dependent regulation of MEG3 expression was studied by evaluating the involvement of miR-29, which can modulate DNMT 1 and 3 expression. MiR-29a is the most abundantly expressed isoform of miR-29 in liver cell lines and tissues. The expression of MEG3 and miR-29a were directly correlated with an r of 0.88 (p: 0.04) in liver cells. Over-expression of mir-29a increased expression of MEG3 in human HCC cells. GTL2, the murine homolog of MEG3, was reduced in liver tissues from hepatocyte-specific miR-29a/b1 KO mice compared to wild type controls. Conclusions: These data show that the expression of ncRNA such as MEG3 can be altered in HCC. The expression of MEG3 can be modulated by a miRNA dependent manner, likely through methylation-dependent suppression at the MEG3 promoter. We speculate that ncRNA such as MEG3 may be useful potential targets for 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 1178. doi:10.1158/1538-7445.AM2011-1178