Tumor Biology

, Volume 36, Issue 5, pp 3735–3743 | Cite as

Construction of a plasmid for overexpression of human circadian gene period2 and its biological activity in osteosarcoma cells

  • An-yuan Cheng
  • Yan Zhang
  • Hong-jun Mei
  • Shuo Fang
  • Peng Ji
  • Jian Yang
  • Ling Yu
  • Wei-chun Guo
Research Article
First Online:
Received:
Accepted:

Abstract

Beyond their established role in the mammalian circadian clock, recent studies have confirmed that the circadian genes have been implicated in tumor onset and progression. Currently, the biological effects of circadian genes on osteosarcoma cells’ proliferation and migration are not well understood. Period2 (Per2) is one of the core circadian genes that act as master regulators of development and is frequently dysregulated in several cancers. However, the effects of human Per2 (hPer2) on the biological behavior of osteosarcoma cells are rarely reported. In the present study, to address the expression of hPer2 in osteosarcoma cells, the pEGFP-N1-hPer2 eukaryotic expression vector was constructed and transfected into cultured MG63 cells using Lipofectamine™ 2000. The overexpression of hPer2 in MG63 cells was verified by qRT-PCR and Western blotting, respectively. Finally, we investigated the effects of hPer2 protein overexpression on MG63 cells’ viability, cycle, apoptosis, and invasive ability. In conclusion, the recombinant pEGFP-N1-hPer2 plasmid had been constructed successfully and expressed effectively in MG63 cells. Furthermore, results also showed that the viability, proliferation, and invasive abilities were suppressed, and the apoptosis was enhanced in MG63 cells. This preliminary study provides ground work for further research on the roles of circadian gene hPer2 in osteosarcoma cells MG63 and would offer promise for the development of novel therapeutic strategies in the treatment of osteosarcoma.

Keywords

Biological rhythm Circadian gene Per2 Osteosarcoma MG63 Proliferation Cell cycle Invasion 
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Notes

Acknowledgments

We thank the Central Laboratory of the First Affiliated Hospital of Wuhan University. We also want to thank Ji-Shuang Zhu for his assistance in primer designing and Dr. Ling Yu for his careful review of the manuscript.

Conflicts of interest

None.

References

  1. 1.
    Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res. 2009;152:3–13.CrossRefPubMedGoogle Scholar
  2. 2.
    Admassi D. Osteosarcoma of medial cuniform bone. Ethiop Med J. 2009;47:305–8.PubMedGoogle Scholar
  3. 3.
    Yu H, Meng X, Wu J, Pan C, Ying X, Zhou Y, et al. Cryptochrome1 overexpression correlates with tumor progression and poor prognosis in patients with colorectal cancer. PLoS One. 2013;8(4):e61679.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Relles D, Sendecki J, Chipitsyna G, Hyslop T, Yeo CJ, Arafat HA. Circadian gene expression and clinicopathologic correlates in pancreatic cancer. J Gastrointest Surg. 2013;17(3):443–50.CrossRefPubMedGoogle Scholar
  5. 5.
    Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;(3):1101–8. (VIP paper 003 Roles of microRNA-206 in Osteosarcoma Pathogenesis and progression).Google Scholar
  6. 6.
    Galat A. Peptidylprolyl cis/trans isomerases (immunophilins): biological diversity—targets—functions. Curr Top Med Chem. 2003;3:1315–47.CrossRefPubMedGoogle Scholar
  7. 7.
    Lu KP, Suizu F, Zhou XZ, Finn G, Lam P, Wulf G. Targeting carcinogenesis: a role for the prolyl isomerase Pin1? Mol Carcinog. 2006;45:397–402.CrossRefPubMedGoogle Scholar
  8. 8.
    Martin JW, Squire JA, Zielenska M. The genetics of osteosarcoma. Sarcoma. 2012;13:627254.Google Scholar
  9. 9.
    Yamamoto N, Tsuchiya H. Chemotherapy for osteosarcoma-where does it come from? What is it? Where is it going? Expert Opin Pharmacother. 2013;14(16):2183–93.CrossRefPubMedGoogle Scholar
  10. 10.
    Luetke A, Meyers PA, Lewis I, Juergens H. Osteosarcoma treatment—where do we stand? A state of the art review. Cancer Treat Rev. 2014;40(4):523–32.CrossRefPubMedGoogle Scholar
  11. 11.
    Yang J, Zhang W. New molecular insights into osteosarcoma targeted therapy. Curr Opin Oncol. 2013;25(4):398–406.CrossRefPubMedGoogle Scholar
  12. 12.
    Kelleher FC, Rao A, Maguire A. Circadian molecular clocks and cancer. Cancer Lett. 2014;342(1):9–18.CrossRefPubMedGoogle Scholar
  13. 13.
    Ripperger JA, Albrecht U. The circadian clock component PERIOD2: from molecular to cerebral functions. Prog Brain Res. 2012;199:233–45.CrossRefPubMedGoogle Scholar
  14. 14.
    Partch CL, Green CB, Takahashi JS. Molecular architecture of the mammalian circadian clock. Trends Cell Biol. 2014;24(2):90–9.CrossRefPubMedGoogle Scholar
  15. 15.
    Greene MW. Circadian rhythms and tumor growth. Cancer Lett. 2012;318(2):115–23.CrossRefPubMedGoogle Scholar
  16. 16.
    Beckett M, Roden LC. Mechanisms by which circadian rhythm disruption may lead to cancer. S Afr J Sci. 2009;105(11/12):415–20.Google Scholar
  17. 17.
    Ishida N. Circadian clock, cancer and lipid metabolism. Neurosci Res. 2007;57:483–90.CrossRefPubMedGoogle Scholar
  18. 18.
    Chen ST, Choo KB, Hou MF, Yeh KT, Kuo SJ, Chang JG. Deregulated expression of the PER1, PER2 and PER3 genes in breast cancers. Carcinogenesis. 2005;26(7):1241–6.CrossRefPubMedGoogle Scholar
  19. 19.
    Lin YM, Chang JH, Yeh KT, Yang MY, Liu TC, Lin SF, et al. Disturbance of circadian gene expression in hepatocellular carcinoma. Mol Carcinog. 2008;47:925–33.CrossRefPubMedGoogle Scholar
  20. 20.
    Halmos T, Suba I. Physiological and pathophysiological role of the circadian clock system. Orv Hetil. 2012;153(35):1370–9.CrossRefPubMedGoogle Scholar
  21. 21.
    Bollinger T, Schibler U. Circadian rhythms—from genes to physiology and disease. Swiss Med Wkly. 2014;144:w13984.PubMedGoogle Scholar
  22. 22.
    Štorcelová M, Vicián M, Reis R, Zeman M, Herichová I. Expression of cell cycle regulatory factors hus1, gadd45a, rb1, cdkn2a and mre11a correlates with expression of clock gene per2 in human colorectal carcinoma tissue. Mol Biol Rep. 2013;40(11):6351–61.CrossRefPubMedGoogle Scholar
  23. 23.
    Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, Wong WH, et al. Extensive and divergent circadian gene expression in liver and heart. Nature. 2002;417:78–83.CrossRefPubMedGoogle Scholar
  24. 24.
    Badiu C. Genetic clock of biologic rhythms. J Cell Mol Med. 2003;7:408–16.CrossRefPubMedGoogle Scholar
  25. 25.
    Gery S, Komatsu N, Baldjyan L, Yu A, Koo D. The circadian gene per1 plays an important role in cell growth and DNA damage control in human cancer cells. Mol Cell. 2006;22:375–82.CrossRefPubMedGoogle Scholar
  26. 26.
    Gréchez-Cassiau A, Rayet B, Guillaumond F, Teboul M, Delaunay F. The circadian clock component BMAL1 is a critical regulator of p21WAF1/ CIP1expression and hepatocyte proliferation. Biol Chem. 2008;(283):4535–42.Google Scholar
  27. 27.
    Mullenders J, Fabius AW, Madiredjo M, Bernards R, Beijersbergen RL. A large scale shRNA barcode screen identifies the circadian clock component ARNTL as putative regulator of the p53 tumor suppressor pathway. PLoS One. 2009;4:e4798.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Atkinson G, Jones H, Ainslie PN. Circadian variation in the circulatory responses to exercise: relevance to the morning peaks in strokes and cardiac events. Eur J Appl Physiol. 2010;108(1):15–29.CrossRefPubMedGoogle Scholar
  29. 29.
    Cermakian N, Boivin DB. The regulation of central and peripheral circadian clocks in humans. Obes Rev. 2009;10 Suppl 2:25–36.CrossRefPubMedGoogle Scholar
  30. 30.
    Hoffman AE, Yi CH, Zheng T, Stevens RG, Leaderer D, Zhang Y, et al. CLOCK in breast tumorigenesis: genetic, epigenetic, and transcriptional profiling analyses. Cancer Res. 2010;70:1459–68.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Mazzoccoli G, Panza A, Valvano MR, Palumbo O, Carella M, Pazienza V, et al. Clock gene expression levels and relationship with clinical and pathological features in colorectal cancer patients. Chronobiol Int. 2011;28(10):841–51.CrossRefPubMedGoogle Scholar
  32. 32.
    Gu X, Xing L, Shi G, Liu Z, Wang X, Qu Z, et al. The circadian mutation PER2(S662G) is linked to cell cycle progression and tumorigenesis. Cell Death Differ. 2012;19(3):397–405.CrossRefPubMedGoogle Scholar
  33. 33.
    Kolomeichuk SN, Gurov EV, Piskunova TS, Tyndyk ML, Anisimov VN. Expression of circadian Per1 and Per2 genes in the liver and breast tumor tissues of HER2/neu transgenic mice of different age. Bull Exp Biol Med. 2011;151(2):227–9.CrossRefPubMedGoogle Scholar
  34. 34.
    Karantanos T, Theodoropoulos G, Pektasides D, Gazouli M. Clock genes: their role in colorectal cancer. World J Gastroenterol. 2014;20(8):1986–92.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Rana S, Munawar M, Shahid A, Malik M, Ullah H, Fatima W, et al. Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia. Mol Biol Rep. 2014;41(1):95–103.CrossRefPubMedGoogle Scholar
  36. 36.
    Lengyel Z, Battyáni Z, Szekeres G, Csernus V, Nagy AD. Circadian clocks and tumor biology: what is to learn from human skin biopsies? Gen Comp Endocrinol. 2013;188:67–74.CrossRefPubMedGoogle Scholar
  37. 37.
    Koyomi M, Miyuki W, Yasuhiro H, Ishida N. Tumor growth suppression in vivo by overexpression of the circadian component, PER2. Genes Cells. 2010;15(4):351–8.CrossRefGoogle Scholar
  38. 38.
    Yang XM, Wood PA, Ansell C, Hrushesky WJM. Circadian time-dependent tumor suppressor function of period genes. Integr Cancer Ther. 2009;8(4):309–16.CrossRefPubMedGoogle Scholar
  39. 39.
    Chen ST, Choo KB, Hou MF, Yeh KT, Kuo SJ, Chang JG. Deregulated expression of the PER1, PER2 and PER3 gene in breast cancer. Oxford University Press. 2005;26(7):1241–6.Google Scholar
  40. 40.
    Gery S, Gombart AF, Yi WS, Koeffler C, Hofmann WK, Koeffler HP. Transcription profiling of C/EBP targets identifies Per2 as a gene implicated in myeloid leukemia. Blood. 2005;106(8):2827–36.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Laurence C, Tomoko K, Vincent L. The days and nights of cancer cells. Cancer Res. 2003;63(22):7545–52.Google Scholar
  42. 42.
    Filipski E. Host circadian clock as a control point in tumour progression. Natl Cancer Inst. 2002;94(9):690–7.CrossRefGoogle Scholar
  43. 43.
    Fu L, Pelicano H, Liu JS, Huang P, Lee CC. The circadian gene period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell. 2002;111(1):41–50.CrossRefPubMedGoogle Scholar
  44. 44.
    Gery S, Virk RK, Chumakov K, Yu A, Koeffler HP. The clock gene Per2 links the circadian system to the estrogen receptor. Oncogene. 2007;26(57):7916–20.CrossRefPubMedGoogle Scholar
  45. 45.
    Hua H, Wang Y, Wan C, Liu Y, Zhu B, Wang Z, et al. Inhibition of tumorigenesis by intratumoral delivery of the circadian gene mPer2 in C57BL/6 mice. Cancer Gene Ther. 2007;14(9):815–8.CrossRefPubMedGoogle Scholar
  46. 46.
    Hua H, Wang Y, Wan C, Liu Y, Zhu B, Yang C, et al. Circadian gene mPer2 overexpression induces cancer cell apoptosis. Cancer Sci. 2006;97(7):589–96.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Filipski E, King VM, Li XM, Granda TG, Mormont MC, Liu X. Host circadian clock as a control point in tumor progression. J Natl Cancer Inst. 2002;94:690–7.CrossRefPubMedGoogle Scholar
  48. 48.
    Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature. 2001;411:342–8.CrossRefPubMedGoogle Scholar
  49. 49.
    Sun CM, Huang SF, Zeng JM, Liu DB, Xiao Q, Tian WJ, et al. Per2 inhibits K562 leukemia cell growth in vitro and in vivo through cell cycle arrest and apoptosis induction. Pathol Oncol Res. 2010;16:403–11.CrossRefPubMedGoogle Scholar
  50. 50.
    Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature. 2000;408:307–10.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • An-yuan Cheng
    • 1
  • Yan Zhang
    • 2
  • Hong-jun Mei
    • 3
  • Shuo Fang
    • 1
  • Peng Ji
    • 1
  • Jian Yang
    • 1
  • Ling Yu
    • 1
  • Wei-chun Guo
    • 1
  1. 1.Department of OrthopedicRenmin Hospital of Wuhan UniversityWuhanPeople’s Republic of China
  2. 2.Department of CardiologyThe General Hospital of Fengfeng Group. Co, LtdHandanPeople’s Republic of China
  3. 3.Department of OrthopedicWuhan No.5 HospitalWuhanPeople’s Republic of China

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