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Int J Oncol. 2016 Jan;48(1):421-9. doi: 10.3892/ijo.2015.3248. Epub 2015 Nov 18.

The mechanism of adenosine-mediated activation of lncRNA MEG3 and its antitumor effects in human hepatoma cells.

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Department of Gastroenterology, Second Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.
Key Laboratory of Infectious Diseases and Immunopathology of Guangdong Province, Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.
Department of Microbiology and Immunology, Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.


Long non-coding RNA MEG3 is suggested to function as a tumor suppressor. However, the activation mechanism of MEG3 is still not well understood and data are not available on its role under adenosine-induced apoptosis. In this study, HepG2 cells were treated with adenosine or 5-Aza‑cdR. Methylation status of MEG3 promoter was detected by methylation specific PCR (MSP) and MEG3 expression was determined by qRT-PCR. PcDNA3.1-MEG3 recombinant plasmid was constructed and transfected to hepatoma HepG2 and Huh7 cells. Cell growth, morphological changes, cell-cycle distribution and apoptosis were analyzed by MTT assay, fluorescence microscopy and flow cytometry. The mRNA and protein expression levels were detected by qRT-PCR and western blot analysis. MEG3 binding proteins were screened by the improved MS2 biotin tagged RNA affinity purification method. The co-expression network of MEG3 was generated by GO analysis and ILF3 was identified as MEG3 binding protein by RNA pulldown and western blot analysis. Both adenosine and 5-Aza-CdR increased MEG3 mRNA expression and the CpG island of MEG3 gene in HepG2 cells was typical hypermethylation. Ectopic expression of MEG3 inhibited hepatoma cell growth in a time-dependent manner, resulted in cell cycle arrest and induced apoptosis. Ectopic expression of MEG3 increased p53, caspase-3 mRNA and protein levels, decreased MDM2 and cyclin D1 mRNA and protein levels, as well as ILF3 protein expression in HepG2 cells. These findings are the first to identify that adenosine increases MEG3 expression by inhibition of DNA methylation and its antitumor effects is involved in MEG3 activation. ILF3 may participate in the anticancer regulation of MEG3 by interacting with MEG3.

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