Promoter hypermethylation influences the suppressive role of maternally expressed 3, a long non-coding RNA, in the development of epithelial ovarian cancer

Sheng,Xiujie; Li,Jianqi; Yang,Lei; Chen,Zhiyi; Zhao,Qin; Tan,Linyu; Zhou,Yanqing; Li,Juan

doi:10.3892/or.2014.3208

Promoter hypermethylation influences the suppressive role of maternally expressed 3, a long non-coding RNA, in the development of epithelial ovarian cancer

Authors:
- Xiujie Sheng
- Jianqi Li
- Lei Yang
- Zhiyi Chen
- Qin Zhao
- Linyu Tan
- Yanqing Zhou
- Juan Li
View Affiliations

Affiliations: Department of Obstetrics and Gynecology, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, P.R. China, The Institute for Chemical Carcinogenesis, Guangzhou Medical University, Guangzhou 510182, P.R. China, Guangzhou Institute of Obstetrics and Gynecology, Guangzhou 510182, P.R. China
Published online on: May 22, 2014 https://doi.org/10.3892/or.2014.3208
Pages: 277-285

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Maternally expressed 3 (MEG3) is a long non-coding RNA that can activate p53 and inhibit tumorigenesis and progression of various types of cancers. However, the role of MEG3 in epithelial ovarian cancer (EOC) is still unknown. The aim of the present study was to confirm whether MEG3 is downregulated in human EOC, determine its possible mechanism of action and elucidate the role of MEG3 in EOC. Differences in the expression of MEG3 and in the methylation status of the MEG3 promoter between EOC and normal ovary were analyzed using RT-PCR and methylation-specific PCR (MSP), respectively. MTT and EdU assays and flow cytometric analysis were used to assess the growth of ovarian cancer cells after overexpression of MEG3. The target genes regulated by MEG3 were detected with the Dual Luciferase Reporter system. The expression levels of target genes were confirmed using RT-PCR and western blotting. In contrast to normal ovarian tissues, the expression of MEG3 was absent or decreased in most EOC tissues as well as in human EOC cell lines, and the promoter of the MEG3 gene was highly methylated in both cancer tissues and cell lines. Treatment with 5-aza-2-deoxycytidine reversed the promoter hypermethylation and increased MEG3 expression. In addition, ectopic expression of MEG3 suppressed the proliferation and growth of OVCAR3 cells and promoted apoptosis. Finally, MEG3 activated p53 in OVCAR3 cells. In conclusion, our data suggest that MEG3 is epigenetically silenced in EOC due to promoter hypermethylation, which may contribute to the development of EOC.

View Figures

View References

1	Itamochi H: Targeted therapies in epithelial ovarian cancer: molecular mechanisms of action. World J Biol Chem. 1:209–220. 2010. View Article : Google Scholar : PubMed/NCBI
2	Gibb EA, Brown CJ and Lam WL: The functional role of long non-coding RNA in human carcinomas. Mol Cancer. 10:382011. View Article : Google Scholar : PubMed/NCBI
3	Miyoshi N, Wagatsuma H, Wakana S, et al: Identification of an imprinted gene, Meg3/Gtl2 and its human homologue MEG3, first mapped on mouse distal chromosome 12 and human chromosome 14q. Genes Cell. 5:211–220. 2000.
4	Zhang X, Zhou Y, Mehta KR, et al: A pituitary-derived MEG3 isoform functions as a growth suppressor in tumor cells. J Clin Endocrinol Metab. 88:5119–5126. 2003. View Article : Google Scholar : PubMed/NCBI
5	Zhou Y, Zhang X and Klibanski A: MEG3 noncoding RNA: a tumor suppressor. J Mol Endocrinol. 48:R45–R53. 2012. View Article : Google Scholar
6	Zhao J, Dahle D, Zhou Y, Zhang X and Klibanski A: Hypermethylation of the promoter region is associated with the loss of MEG3 gene expression in human pituitary tumors. J Clin Endocrinol Metab. 90:2179–2186. 2005. View Article : Google Scholar : PubMed/NCBI
7	Zhang X, Gejman R, Mahta A, et al: Maternally expressed gene 3, an imprinted noncoding RNA gene, is associated with meningioma pathogenesis and progression. Cancer Res. 70:2350–2358. 2010. View Article : Google Scholar
8	Astuti D, Latif F, Wagner K, et al: Epigenetic alteration at the DLK1-GTL2 imprinted domain in human neoplasia: analysis of neuroblastoma, phaeochromocytoma and Wilms’ tumour. Br J Cancer. 92:1574–1580. 2005.PubMed/NCBI
9	Zhou Y, Zhong Y, Wang Y, et al: Activation of p53 by MEG3 non-coding RNA. J Biol Chem. 282:24731–24742. 2007. View Article : Google Scholar : PubMed/NCBI
10	Braconi C, Kogure T, Valeri N, et al: microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer. Oncogene. 30:4750–4756. 2011. View Article : Google Scholar : PubMed/NCBI
11	Wang P, Ren Z and Sun P: Overexpression of the long non-coding RNA MEG3 impairs in vitro glioma cell proliferation. J Cell Biochem. 113:1868–1874. 2012. View Article : Google Scholar : PubMed/NCBI
12	Benetatos L, Hatzimichael E, Dasoula A, et al: CpG methylation analysis of the MEG3 and SNRPN imprinted genes in acute myeloid leukemia and myelodysplastic syndromes. Leuk Res. 34:148–153. 2010. View Article : Google Scholar : PubMed/NCBI
13	Feng S, Cong S, Zhang X, et al: MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells. Nucleic Acids Res. 39:6669–6678. 2011. View Article : Google Scholar : PubMed/NCBI
14	Anwar SL, Krech T, Hasemeier B, et al: Loss of imprinting and allelic switching at the DLK1-MEG3 locus in human hepatocellular carcinoma. PLoS One. 7:e494622012. View Article : Google Scholar : PubMed/NCBI
15	Benetatos L, Vartholomatos G and Hatzimichael E: MEG3 imprinted gene contribution in tumorigenesis. Int J Cancer. 129:773–779. 2011. View Article : Google Scholar : PubMed/NCBI
16	Kagami M, O’Sullivan MJ, Green AJ, et al: The IG-DMR and the MEG3-DMR at human chromosome 14q32.2: hierarchical interaction and distinct functional properties as imprinting control centers. PLoS Genet. 6:e10009922010.PubMed/NCBI
17	Jones PA and Baylin SB: The fundamental role of epigenetic events in cancer. Nat Rev Genet. 3:415–428. 2002.PubMed/NCBI
18	Baylin SB and Ohm JE: Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction? Nat Rev Cancer. 6:107–116. 2006. View Article : Google Scholar : PubMed/NCBI
19	Yoon JH, Dammann R and Pfeifer GP: Hypermethylation of the CpG island of the RASSF1A gene in ovarian and renal cell carcinomas. Int J Cancer. 94:212–217. 2001.PubMed/NCBI
20	Ibanez de Caceres I, Battagli C, Esteller M, et al: Tumor cell-specific BRCA1 and RASSF1A hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients. Cancer Res. 64:6476–6481. 2004.
21	Chan KY, Ozçelik H, Cheung AN, Ngan HY and Khoo US: Epigenetic factors controlling the BRCA1 and BRCA2 genes in sporadic ovarian cancer. Cancer Res. 62:4151–4156. 2002.PubMed/NCBI
22	Kim T, Veronese A, Pichiorri F, et al: p53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1 and ZEB2. J Exp Med. 208:875–883. 2011. View Article : Google Scholar : PubMed/NCBI
23	Huarte M, Guttman M, Feldser D, et al: A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell. 142:409–419. 2010. View Article : Google Scholar : PubMed/NCBI
24	Brooks CL and Gu W: p53 regulation by ubiquitin. FEBS Lett. 585:2803–2809. 2011. View Article : Google Scholar : PubMed/NCBI
25	Staub J, Chien J, Pan Y, et al: Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance. Oncogene. 26:4969–4978. 2007. View Article : Google Scholar : PubMed/NCBI