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Tumor Biology

, Volume 37, Issue 7, pp 9875–9886 | Cite as

Expression and mechanisms of long non-coding RNA genes MEG3 and ANRIL in gallbladder cancer

  • Bo Liu
  • Er-Dong Shen
  • Ming-Mei Liao
  • Yong-Bin Hu
  • Kai Wu
  • Pu Yang
  • Lin Zhou
  • Wei-Dong Chen
Original Article

Abstract

The objective of this study was to investigate the expression, proliferation, and apoptosis function of long-chain non-coding RNA maternally expressed gene 3 (MEG3) and antisense non-coding RNA at the INK4 locus (ANRIL) in gallbladder cancer (GBC) tissues. GBC tissues and adjacent normal samples were collected from 84 patients from January 2008 to June 2010. Empty vector, pcDNA-MEG3, and pcDNA-ANRIL vectors were transfected into GBC-SD and QBC939 cells. An MTT assay, real-time quantitative polymerase chain reaction (RT-qPCR), flow cytometry, Western blotting, and immunohistochemistry were applied. The effects of MEG3 and ANRIL were also verified in mice. Compared with normal tissues, the expression of MEG3 was significantly lower in GBC tissues, whereas the expression of ANRIL was significantly higher (both P < 0.05). The overexpression of MEG3 and underexpression of ANRIL were significantly associated with GBC prognosis (both P < 0.05). The expressions of MEG3 and ANRIL were higher in pcDNA-MEG3 and pcDNA-ANRIL-transfected cells than in empty vector-transfected cells in vitro (both P < 0.05). Most of the pcDNA-MEG3-transfected cells were in the G0-G1 phase, which showed reduced cell activity and clone counts and increased p53 and decreased cyclin D1, whereas the pcDNA-ANRIL-transfected cells were mostly in the S phase and showed contrasting behavior. Mice injected with pcDNA-MEG3-transfected cells had smaller and lighter tumors, decreased ki-67 levels, and increased caspase 3 levels, whereas those injected with pcDNA-ANRIL showed contrasting results (all P < 0.05). MEG3 can inhibit the proliferation of GBC cells and promote apoptosis, whereas ANRIL can improve the proliferation of gallbladder cells and inhibit apoptosis. Collectively, our results suggest that therapeutic strategies directed toward upregulating MEG3 and downregulating ANRIL may be clinically relevant for the inhibition of GBC deterioration.

Keywords

Gallbladder cancer MEG3 ANRIL Proliferation Apoptosis 

Notes

Acknowledgments

This project was supported by the National Natural Science Foundation of China (No: 81302075). We would like to acknowledge the reviewers for their helpful comments on this paper.

Compliance with ethical standards

All in vivo studies were carried out in strict accordance with the recommendations of the Animal Experiment Association.

Ethics approval and consent to participate

The study was approved by the Institute Review Board of Xiangya No. 2 Hospital of Central South University and fulfilled the ethical principles outlined in the Declaration of Helsinki. All patients and their family members provided written informed consent.

Conflicts of interest

None

References

  1. 1.
    Stinton LM, Shaffer EA. Epidemiology of gallbladder disease: cholelithiasis and cancer. Gut Liver. 2012;6(2):172–87.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lai CH, Lau WY. Gallbladder cancer—a comprehensive review. Surgeon. 2008;6(2):101–10.CrossRefPubMedGoogle Scholar
  3. 3.
    Hundal R, Shaffer EA. Gallbladder cancer: epidemiology and outcome. Clin Epidemiol. 2014;6:99–109.PubMedPubMedCentralGoogle Scholar
  4. 4.
    Rustagi T, Dasanu CA. Risk factors for gallbladder cancer and cholangiocarcinoma: similarities, differences and updates. J Gastrointest Cancer. 2012;43(2):137–47.CrossRefPubMedGoogle Scholar
  5. 5.
    Nath G, Gulati AK, Shukla VK. Role of bacteria in carcinogenesis, with special reference to carcinoma of the gallbladder. World J Gastroenterol. 2010;16(43):5395–404.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Zhu AX, Hong TS, Hezel AF, Kooby DA. Current management of gallbladder carcinoma. Oncologist. 2010;15(2):168–81.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Henley SJ, Weir HK, Jim MA, Watson M, Richardson LC. Gallbladder cancer incidence and mortality, United States 1999–2011. Cancer Epidemiol Biomarkers Prev. 2015;24(9):1319–26.CrossRefPubMedGoogle Scholar
  8. 8.
    Kanthan R, Senger JL, Ahmed S, Kanthan SC. Gallbladder cancer in the 21st century. J Oncol. 2015;2015:967472.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hong EK, Kim KK, Lee JN, Lee WK, Chung M, Kim YS, et al. Surgical outcome and prognostic factors in patients with gallbladder carcinoma. Korean J Hepatobiliary Pancreat Surg. 2014;18(4):129–37.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Li Z, Yu X, Shen J, Law PT, Chan MT, Wu WK. MicroRNA expression and its implications for diagnosis and therapy of gallbladder cancer. Oncotarget. 2015;6(16):13914–21.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ma MZ, Chu BF, Zhang Y, Weng MZ, Qin YY, Gong W, et al. Long non-coding RNA CCAT1 promotes gallbladder cancer development via negative modulation of miRNA-218-5p. Cell Death Dis. 2015;6:e1583.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ma MZ, Kong X, Weng MZ, Zhang MD, Qin YY, Gong W, et al. Long non-coding RNA-LET is a positive prognostic factor and exhibits tumor-suppressive activity in gallbladder cancer. Mol Carcinog. 2015;54(11):1397–406.CrossRefPubMedGoogle Scholar
  13. 13.
    Pang KC, Frith MC, Mattick JS. Rapid evolution of noncoding RNAs: lack of conservation does not mean lack of function. Trends Genet. 2006;22(1):1–5.CrossRefPubMedGoogle Scholar
  14. 14.
    Miyoshi N, Wagatsuma H, Wakana S, Shiroishi T, Nomura M, Aisaka K, 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 Cells. 2000;5(3):211–20.CrossRefPubMedGoogle Scholar
  15. 15.
    Li Y, Tang K, Zhou K, Wei Z, Zeng Z, He L, et al. Quantitative assessment of the effect of ABCA1 R219K polymorphism on the risk of coronary heart disease. Mol Biol Rep. 2012;39(2):1809–13.CrossRefPubMedGoogle Scholar
  16. 16.
    Qin R, Chen Z, Ding Y, Hao J, Hu J, Guo F. Long non-coding RNA MEG3 inhibits the proliferation of cervical carcinoma cells through the induction of cell cycle arrest and apoptosis. Neoplasma. 2013;60(5):486–92.CrossRefPubMedGoogle Scholar
  17. 17.
    Zhang X, Gejman R, Mahta A, Zhong Y, Rice KA, Zhou Y, et al. Maternally expressed gene 3, an imprinted noncoding RNA gene, is associated with meningioma pathogenesis and progression. Cancer Res. 2010;70(6):2350–8.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Jia LF, Wei SB, Gan YH, Guo Y, Gong K, Mitchelson K, et al. Expression, regulation and roles of miR-26a and MEG3 in tongue squamous cell carcinoma. Int J Cancer. 2014;135(10):2282–93.CrossRefPubMedGoogle Scholar
  19. 19.
    Wang P, Ren Z, Sun P. Overexpression of the long non-coding RNA MEG3 impairs in vitro glioma cell proliferation. J Cell Biochem. 2012;113(6):1868–74.CrossRefPubMedGoogle Scholar
  20. 20.
    Ying L, Huang Y, Chen H, Wang Y, Xia L, Chen Y, et al. Downregulated MEG3 activates autophagy and increases cell proliferation in bladder cancer. Mol Biosyst. 2013;9(3):407–11.CrossRefPubMedGoogle Scholar
  21. 21.
    Sun M, Xia R, Jin F, Xu T, Liu Z, De W, et al. Downregulated long noncoding RNA MEG3 is associated with poor prognosis and promotes cell proliferation in gastric cancer. Tumour Biol. 2014;35(2):1065–73.CrossRefPubMedGoogle Scholar
  22. 22.
    Anwar SL, Krech T, Hasemeier B, Schipper E, Schweitzer N, Vogel A, et al. Loss of imprinting and allelic switching at the DLK1-MEG3 locus in human hepatocellular carcinoma. PLoS One. 2012;7(11):e49462.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Pasmant E, Sabbagh A, Vidaud M, Bieche I. ANRIL, a long, noncoding RNA, is an unexpected major hotspot in GWAS. FASEB J. 2011;25(2):444–8.CrossRefPubMedGoogle Scholar
  24. 24.
    Broadbent HM, Peden JF, Lorkowski S, Goel A, Ongen H, Green F, et al. Susceptibility to coronary artery disease and diabetes is encoded by distinct, tightly linked SNPs in the ANRIL locus on chromosome 9p. Hum Mol Genet. 2008;17(6):806–14.CrossRefPubMedGoogle Scholar
  25. 25.
    Congrains A, Kamide K, Ohishi M, Rakugi H. ANRIL: molecular mechanisms and implications in human health. Int J Mol Sci. 2013;14(1):1278–92.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Yap KL, Li S, Munoz-Cabello AM, Raguz S, Zeng L, Mujtaba S, et al. Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol Cell. 2010;38(5):662–74.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Fong Y, Wagman L, Gonen M, Crawford J, Reed W, Swanson R, et al. Evidence-based gallbladder cancer staging: changing cancer staging by analysis of data from the national cancer database. Ann Surg. 2006;243(6):767–71. discussion 71-4.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    M PN. World Medical Association publishes the revised declaration of Helsinki. Natl Med J India. 2014;27(1):56.PubMedGoogle Scholar
  29. 29.
    Sheng X, Li J, Yang L, Chen Z, Zhao Q, Tan L, et al. Promoter hypermethylation influences the suppressive role of maternally expressed 3, a long non-coding RNA, in the development of epithelial ovarian cancer. Oncol Rep. 2014;32(1):277–85.PubMedGoogle Scholar
  30. 30.
    Yin DD, Liu ZJ, Zhang E, Kong R, Zhang ZH, Guo RH. Decreased expression of long noncoding RNA MEG3 affects cell proliferation and predicts a poor prognosis in patients with colorectal cancer. Tumour Biol. 2015;36(6):4851–9.CrossRefPubMedGoogle Scholar
  31. 31.
    He Y, Wu YT, Huang C, Meng XM, Ma TT, Wu BM, et al. Inhibitory effects of long noncoding RNA MEG3 on hepatic stellate cells activation and liver fibrogenesis. Biochim Biophys Acta. 2014;1842(11):2204–15.CrossRefPubMedGoogle Scholar
  32. 32.
    Peng W, Si S, Zhang Q, Li C, Zhao F, Wang F, et al. Long non-coding RNA MEG3 functions as a competing endogenous RNA to regulate gastric cancer progression. J Exp Clin Cancer Res. 2015;34:79.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Zhou Y, Zhang X, Klibanski A. MEG3 noncoding RNA: a tumor suppressor. J Mol Endocrinol. 2012;48(3):R45–53.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Gordon FE, Nutt CL, Cheunsuchon P, Nakayama Y, Provencher KA, Rice KA, et al. Increased expression of angiogenic genes in the brains of mouse meg3-null embryos. Endocrinology. 2010;151(6):2443–52.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Sun XN, Cao WG, Wang X, Wang Q, Gu BX, Yang QC, et al. Prognostic impact of vascular endothelial growth factor-a expression in resected gallbladder carcinoma. Tumour Biol. 2011;32(6):1183–90.CrossRefPubMedGoogle Scholar
  36. 36.
    Zhou Y, Zhong Y, Wang Y, Zhang X, Batista DL, Gejman R, et al. Activation of p53 by MEG3 non-coding RNA. J Biol Chem. 2007;282(34):24731–42.CrossRefPubMedGoogle Scholar
  37. 37.
    Muller PA, Vousden KH. P53 mutations in cancer. Nat Cell Biol. 2013;15(1):2–8.CrossRefPubMedGoogle Scholar
  38. 38.
    Hermeking H. MicroRNAs in the p53 network: micromanagement of tumour suppression. Nat Rev Cancer. 2012;12(9):613–26.CrossRefPubMedGoogle Scholar
  39. 39.
    Feng Z, Levine AJ. The regulation of energy metabolism and the IGF-1/mTOR pathways by the p53 protein. Trends Cell Biol. 2010;20(7):427–34.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Brooks CL, Gu W. P53 regulation by ubiquitin. FEBS Lett. 2011;585(18):2803–9.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Chang HJ, Yoo BC, Kim SW, Lee BL, Kim WH. Significance of PML and p53 protein as molecular prognostic markers of gallbladder carcinomas. Pathol Oncol Res. 2007;13(4):326–35.CrossRefPubMedGoogle Scholar
  42. 42.
    Lin L, Gu ZT, Chen WH, Cao KJ. Increased expression of the long non-coding RNA ANRIL promotes lung cancer cell metastasis and correlates with poor prognosis. Diagn Pathol. 2015;10:14.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Nie FQ, Sun M, Yang JS, Xie M, Xu TP, Xia R, et al. Long noncoding RNA ANRIL promotes non-small cell lung cancer cell proliferation and inhibits apoptosis by silencing KLF2 and P21 expression. Mol Cancer Ther. 2015;14(1):268–77.CrossRefPubMedGoogle Scholar
  44. 44.
    Foroud T, Koller DL, Lai D, Sauerbeck L, Anderson C, Ko N, et al. Genome-wide association study of intracranial aneurysms confirms role of Anril and SOX17 in disease risk. Stroke. 2012;43(11):2846–52.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Matheu A, Maraver A, Collado M, Garcia-Cao I, Canamero M, Borras C, et al. Anti-aging activity of the Ink4/Arf locus. Aging Cell. 2009;8(2):152–61.CrossRefPubMedGoogle Scholar
  46. 46.
    Hirosue A, Ishihara K, Tokunaga K, Watanabe T, Saitoh N, Nakamoto M, et al. Quantitative assessment of higher-order chromatin structure of the INK4/ARF locus in human senescent cells. Aging Cell. 2012;11(3):553–6.CrossRefPubMedGoogle Scholar
  47. 47.
    Sato K, Nakagawa H, Tajima A, Yoshida K, Inoue I. ANRIL is implicated in the regulation of nucleus and potential transcriptional target of E2F1. Oncol Rep. 2010;24(3):701–7.PubMedGoogle Scholar
  48. 48.
    Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M, et al. Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15INK4B tumor suppressor gene. Oncogene. 2011;30(16):1956–62.CrossRefPubMedGoogle Scholar
  49. 49.
    Pai RK, Mojtahed K, Pai RK. Mutations in the RAS/RAF/MAP kinase pathway commonly occur in gallbladder adenomas but are uncommon in gallbladder adenocarcinomas. Appl Immunohistochem Mol Morphol. 2011;19(2):133–40.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Bo Liu
    • 1
  • Er-Dong Shen
    • 2
  • Ming-Mei Liao
    • 3
  • Yong-Bin Hu
    • 4
  • Kai Wu
    • 5
  • Pu Yang
    • 6
  • Lin Zhou
    • 7
  • Wei-Dong Chen
    • 1
  1. 1.Department of General Surgery, Xiangya No. 2 HospitalCentral South UniversityChangshaPeople’s Republic of China
  2. 2.Department of OncologyNo. 1 People’s Hospital of Yueyang CityYueyangPeople’s Republic of China
  3. 3.Department of General Surgery, Xiangya HospitalCentral South UniversityChangshaPeople’s Republic of China
  4. 4.Department of Pathology, Xiangya HospitalCentral South UniversityChangshaPeople’s Republic of China
  5. 5.Department of Physiatry, Xiangya HospitalCentral South UniversityChangshaPeople’s Republic of China
  6. 6.Department of Vascular Surgery, Xiangya HospitalCentral South UniversityChangshaPeople’s Republic of China
  7. 7.Department of Colorectal and Anal Surgery, Xiangya HospitalCentral South UniversityChangshaPeople’s Republic of China

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