Tumor Biology

, Volume 37, Issue 2, pp 2023–2035 | Cite as

Effect of silencing NEK2 on biological behaviors of HepG2 in human hepatoma cells and MAPK signal pathway

  • Mei-Xia Zhang
  • Xi-Ming Xu
  • Peng Zhang
  • Na-Na Han
  • Jun-Jian Deng
  • Ting-Ting Yu
  • Yuan-Yuan Gan
  • Xiao-Qin He
  • Zhi-Xiong Long
Original Article

Abstract

To investigate the expression level of NEK2 in 40 tissue specimens of primary liver cancer and to search for clues whether the effect of NEK2 depletion plays a role on biological behaviors of HepG2 cells and the relevant molecular mechanism are the objectives of this study. Real-time PCR and immunohistochemistry assessed expression level of NEK2 in specimens of cancerous tissues and carcinoma-adjacent tissues. The NEK2 expression level in HepG2, Huh7, SMMC, and 7402 cells was detected by real-time PCR and western blot to screen experimental cell line. To assess the expression levels of NEK2 mRNA and protein, an effective siRNA transfected into the HepG2 cells was designed. CCK8 and colony-forming assays were performed to verify short-term and long-term proliferative activities, respectively. Capacity of apoptosis and cell cycle changes were assessed by flow cytometry. Ability of transference and invasion was measured by Transwell Chambers. Western blot approach was used to determine the protein expression levels. There was significantly high expression level of NEK2 in cancerous tissues compared to adjacent tissues. The expression of NEK2 was higher in HepG2 cells than other cell lines. Real-time PCR and western blot shown there were obviously down-regulated NEK2 expression in the NEK2-siRNA group compared to control groups. The capacity of amplification and invasion was inhibited distinctly, and FCM revealed the apoptosis rate was increased and G1 phase was arrested in NEK2-siRNA group. Western blot indicated that low expression of NEK2 in HepG2 cells could increase the expression levels of Bax, caspase-3, P21, and TIMP-1, but significantly suppressed the c-myc, c-jun, Bcl-2, cyclinD1, CDK4, MMP2, and MMP9 expression levels and the phosphorylation levels of ERK, JNK, and P38 compared with the control groups. Our findings demonstrated that NEK2 could be a valuable carcinogenic factor and a promising therapeutic target for primary liver cancer; NEK2 may regulate proliferation, apoptosis, and other biological behaviors of HepG2 cells via MAPK signal pathway.

Keywords

NEK2 Small interfering RNA Proliferation Apoptosis MAPK 

Notes

Acknowledgments

Mei-Xia Zhang: Designed and performed research, statistical analysis, manuscript writing;

Xi-Ming Xu: Designed and performed research, manuscript writing;

Jun-Jian Deng, Na-Na Han: Performed research, statistical analysis;

Ting-Ting Yu, Yuan-Yuan Gan: Performed research, statistical analysis;

Xiao-Qin He, Peng Zhang, Zhi-Xiong Long: Statistical analysis.

Conflicts of interest

None

Funding

Natural Science Foundation of Hubei Province of China supports this work, Funding NO. 2012FKC143.

References

  1. 1.
    El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132(7):2557–76.CrossRefPubMedGoogle Scholar
  2. 2.
    He J, Gu D, Wu X, Reynolds K, Duan X, Yao C, et al. Major causes of death among men and women in China. N Engl J Med. 2005;353(11):1124–34.CrossRefPubMedGoogle Scholar
  3. 3.
    Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer. 2001;94(2):153–6.CrossRefPubMedGoogle Scholar
  4. 4.
    Harvey K, Tapon N. The Salvador-Warts-Hippo pathway—an emerging tumour-suppressor network. Nat Rev Cancer. 2007;7(3):182–91.CrossRefPubMedGoogle Scholar
  5. 5.
    Hagiwara S, Kudo M, Nakatani T, Sakaguchi Y, Nagashima M, Fukuta N, et al. Combination therapy with PEG-IFN-alpha and 5-FU inhibits HepG2 tumour cell growth in nude mice by apoptosis of p53. Br J Cancer. 2007;97(11):1532–7.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Xie H, Ma H, Zhou D. Plasma HULC as a promising novel biomarker for the detection of hepatocellular carcinoma. BioMed Res Int. 2013;2013:136106.PubMedPubMedCentralGoogle Scholar
  7. 7.
    Zhang L, Yang F, Yuan JH, Yuan SX, Zhou WP, Huo XS, et al. Epigenetic activation of the MiR-200 family contributes to H19-mediated metastasis suppression in hepatocellular carcinoma. Carcinogenesis. 2013;34(3):577–86.CrossRefPubMedGoogle Scholar
  8. 8.
    Osmani SA, Pu RT, Morris NR. Mitotic induction and maintenance by overexpression of a G2-specific gene that encodes a potential protein kinase. Cell. 1988;53(2):237–44.CrossRefPubMedGoogle Scholar
  9. 9.
    Fry AM. The Nek2 protein kinase: a novel regulator of centrosome structure. Oncogene. 2002;21(40):6184–94.CrossRefPubMedGoogle Scholar
  10. 10.
    Hayward DG, Fry AM. Nek2 kinase in chromosome instability and cancer. Cancer Lett. 2006;237(2):155–66.CrossRefPubMedGoogle Scholar
  11. 11.
    Bowers AJ, Boylan JF. Nek8, a NIMA family kinase member, is overexpressed in primary human breast tumors. Gene. 2004;328:135–42.CrossRefPubMedGoogle Scholar
  12. 12.
    Tsunoda N, Kokuryo T, Oda K, Senga T, Yokoyama Y, Nagino M, et al. Nek2 as a novel molecular target for the treatment of breast carcinoma. Cancer Sci. 2009;100(1):111–6.CrossRefPubMedGoogle Scholar
  13. 13.
    Irving EA, Bamford M. Role of mitogen- and stress-activated kinases in ischemic injury. J Cereb Blood Flow Metab. 2002;22(6):631–47.CrossRefPubMedGoogle Scholar
  14. 14.
    Fry AM, Meraldi P, Nigg EA. A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators. EMBO J. 1998;17(2):470–81.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Fletcher L, Cerniglia GJ, Nigg EA, Yend TJ, Muschel RJ. Inhibition of centrosome separation after DNA damage: a role for Nek2. Radiat Res. 2004;162(2):128–35.CrossRefPubMedGoogle Scholar
  16. 16.
    Graf R. DdNek2, the first non-vertebrate homologue of human Nek2, is involved in the formation of microtubule-organizing centers. J Cell Sci. 2002;115(Pt 9):1919–29.PubMedGoogle Scholar
  17. 17.
    Uto K, Sagata N. Nek2B, a novel maternal form of Nek2 kinase, is essential for the assembly or maintenance of centrosomes in early Xenopus embryos. EMBO J. 2000;19(8):1816–26.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Prigent C, Glover DM, Giet R. Drosophila Nek2 protein kinase knockdown leads to centrosome maturation defects while overexpression causes centrosome fragmentation and cytokinesis failure. Exp Cell Res. 2005;303(1):1–13.PubMedGoogle Scholar
  19. 19.
    Hayward DG, Clarke RB, Faragher AJ, Pillai MR, Hagan IM, Fry AM. The centrosomal kinase Nek2 displays elevated levels of protein expression in human breast cancer. Cancer Res. 2004;64(20):7370–6.CrossRefPubMedGoogle Scholar
  20. 20.
    Marina M, Saavedra HI. Nek2 and Plk4: prognostic markers, drivers of breast tumorigenesis and drug resistance. Front Biosci. 2014;19:352–65.CrossRefGoogle Scholar
  21. 21.
    Liu X, Gao Y, Lu Y, Zhang J, Li L, Yin F. Upregulation of NEK2 is associated with drug resistance in ovarian cancer. Oncol Rep. 2014;31(2):745–54.PubMedGoogle Scholar
  22. 22.
    Naro C, Barbagallo F, Chieffi P, Bourgeois CF, Paronetto MP, Sette C. The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival. Nucleic Acids Res. 2014;42(5):3218–27.CrossRefPubMedGoogle Scholar
  23. 23.
    Takahashi Y, Iwaya T, Sawada G, Kurashige J, Matsumura T, Uchi R, et al. Up-regulation of NEK2 by microRNA-128 methylation is associated with poor prognosis in colorectal cancer. Ann Surg Oncol. 2014;21(1):205–12.CrossRefPubMedGoogle Scholar
  24. 24.
    Zhou W, Yang Y, Xia J, Wang H, Salama ME, Xiong W, et al. NEK2 induces drug resistance mainly through activation of efflux drug pumps and is associated with poor prognosis in myeloma and other cancers. Cancer Cell. 2013;23(1):48–62.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Zhong W, Shen WF, Ning BF, Hu PF, Lin Y, Yue HY, et al. Inhibition of extracellular signal-regulated kinase 1 by adenovirus mediated small interfering RNA attenuates hepatic fibrosis in rats. Hepatology. 2009;50(5):1524–36.CrossRefPubMedGoogle Scholar
  26. 26.
    Tsuboi Y, Ichida T, Sugitani S, Genda T, Inayoshi J, Takamura M, et al. Overexpression of extracellular signal-regulated protein kinase and its correlation with proliferation in human hepatocellular carcinoma. Liver Int. 2004;24(5):432–6.CrossRefPubMedGoogle Scholar
  27. 27.
    Jia YL, Shi L, Zhou JN, Fu CJ, Chen L, Yuan HF, et al. Epimorphin promotes human hepatocellular carcinoma invasion and metastasis through activation of focal adhesion kinase/extracellular signal-regulated kinase/matrix metalloproteinase-9 axis. Hepatology. 2011;54(5):1808–18.CrossRefPubMedGoogle Scholar
  28. 28.
    Ning Z, Wang A, Liang J, Liu J, Zhou T, Yan Q, et al. Abnormal expression of Nek2 in pancreatic ductal adenocarcinoma: a novel marker for prognosis. Int J Clin Exp Pathol. 2014;7(5):2462–9.PubMedPubMedCentralGoogle Scholar
  29. 29.
    Raviv Z, Kalie E, Seger R. MEK5 and ERK5 are localized in the nuclei of resting as well as stimulated cells, while MEKK2 translocates from the cytosol to the nucleus upon stimulation. J Cell Sci. 2004;117(Pt 9):1773–84.CrossRefPubMedGoogle Scholar
  30. 30.
    Widmann C, Gibson S, Jarpe MB, Johnson GL. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev. 1999;79(1):143–80.PubMedGoogle Scholar
  31. 31.
    Sangiovanni A, Colombo E, Radaelli F, Bortoli A, Bovo G, Casiraghi MA, et al. Hepatocyte proliferation and risk of hepatocellular carcinoma in cirrhotic patients. Am J Gastroenterol. 2001;96(5):1575–80.CrossRefPubMedGoogle Scholar
  32. 32.
    Lou L, Ye W, Chen Y, Wu S, Jin L, He J, et al. Ardipusilloside inhibits survival, invasion and metastasis of human hepatocellular carcinoma cells. Phytomed. 2012;19(7):603–8.CrossRefGoogle Scholar
  33. 33.
    Chung TW, Lee YC, Kim CH. Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: involvement of invasive potential. FASEB J. 2004;18(10):1123–5.PubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Mei-Xia Zhang
    • 1
  • Xi-Ming Xu
    • 1
  • Peng Zhang
    • 2
  • Na-Na Han
    • 1
  • Jun-Jian Deng
    • 1
  • Ting-Ting Yu
    • 1
  • Yuan-Yuan Gan
    • 1
  • Xiao-Qin He
    • 1
  • Zhi-Xiong Long
    • 3
  1. 1.Department of Oncology, Renmin HospitalWuhan UniversityWuhanPeople’s Republic of China
  2. 2.Department of Urology, Zhongnan HospitalWuhan UniversityWuhanPeople’s Republic of China
  3. 3.Department of OncologyThe Fifth People’s HospitalWuhanPeople’s Republic of China

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