한국산학기술학회논문지 (Journal of the Korea Academia-Industrial cooperation Society)

  • 제21권5호
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  • Pages.216-222
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  • 2020
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  • 1975-4701(pISSN)
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  • 2288-4688(eISSN)
한국산학기술학회 (The Korea Academia-Industrial cooperation Society)
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돼지에서 TRAF4 유전자 특성 및 Tight junction 관련 기능 분석

Characterization of TRAF4 mRNA and Functions related to tight junction in pig

  • 윤정희 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 황인설 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 황성수 (농촌진흥청 국외기술농업과) ;
  • 박미령 (농촌진흥청 국립축산과학원 동물바이오공학과)
  • Yun, Jeong-hee (Animal Biotechnolgy Division, National Institute of Animal Science, Rural Development Administration) ;
  • Hwang, In-Sul (Animal Biotechnolgy Division, National Institute of Animal Science, Rural Development Administration) ;
  • Hwang, Seongsoo (Division for KOPIA, Rural Development Administration) ;
  • Park, Mi-Ryung (Animal Biotechnolgy Division, National Institute of Animal Science, Rural Development Administration)
  • 투고 : 2020.03.13
  • 심사 : 2020.05.08
  • 발행 : 2020.05.31
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초록

Tumor necrosis factor receptor associated factor 4 (TRAF4)는 사람의 유방암에서 과발현 되며, 암세포전이, ROS 및 세포 극성 형성 등에 관여하는 것으로 알려져 있다. 그러나 돼지에서는 아직까지 그 기능과 특성에 대한 연구가 보고 된 바 없다. 따라서 본 연구에서는 돼지 TRAF4의 mRNA 전장서열을 분석하고, 그 기능과 특성을 알아보고자 수행되었다. TRAF4의 전장서열을 밝히기 위해 돼지 신장유래세포(pK15)에서 total RNA 추출하여 RACE (Rapid Amplification of cDNA ends) PCR을 수행하였다. 2,030 염기쌍의 mRNA 전장서열을 분석하였고, 470개의 아미노산으로 구성 되어 있는 것을 확인하였다. 사람과 쥐의 Homology를 분석한 결과 각각 93 % 그리고 90 %의 유사도를 가지며, 사람과는 8개, 쥐와는 12개의 아미노산 차이가 있음을 확인하였다. qPCR을 통하여 TRAF4, CLDN4, OCLN 그리고 TJP1의 발현을 분석한 결과 세포의 confluency 정도에 따라 발현이 다르게 나타남을 확인하였고, 세포가 40% 증식한 그룹 보다 60 %와 80 % 이상 증식 한 그룹에서 유의적으로 높게 나타났다. 또한 TRAF4의 기능을 확인하기 위하여 TRAF4 siRNA 처리 한 결과 TRAF4와 tight junction 관련 유전자가 낮게 발현됨을 관찰하였다. 따라서 사람과 마우스와 같이 돼지에서도 TRAF4가 발현되며, 세포-세포 간 중요한 역할을 하는 tight junction에 관여하는 것으로 사료된다.

Tumor necrosis factor receptor associated factor 4 (TRAF4) is found to be overexpressed in human breast cancer. It plays a role in cancer metastasis, production of reactive oxygen species, and cell polarity at membranes. The characteristics and functions of TRAF4 in pigs have not yet been identified. As the first step of research, the mRNA sequence of TRAF4 in porcine cells has been determined. To obtain the full-length sequence, rapid amplification of cDNA ends (RACE) has been carried out. Upon cloning, 2,030 bp of nucleotides were found to encode 470 amino acids, and 8 and 12 amino acids were different from those of the human and mouse TRAF4, respectively. The coding region of porcine TRAF4 was shown to be 93% and 90% homologous to human and mouse TRAF4, respectively. qPCR was conducted to determine the relative expression level of TRAF4. TRAF4 expression in pK15 was enhanced by cell-cell contacts. The mRNA levels of CLDN4, OCLN, and TJP1 at 60% and 80% confluency were significantly higher than at 40% confluency. Further, TRAF4 and tight junction-related genes were down-regulated upon treatment with TRAF4 siRNA. Thus, TRAF4 may affect the function of tight junctions in pig.

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참고문헌

  1. Regnier, Catherine H., Regis Masson, Valerie Kedinger, Julien Textoris, Isabelle Stoll, Marie-Pierre Chenard, Andree Dierich, Catherine Tomasetto, and Marie-Christine Rio. "Impaired Neural Tube Closure, Axial Skeleton Malformations, and Tracheal Ring Disruption in Traf4-Deficient Mice." Proceedings of the National Academy of Sciences, Vol. 99, No. 8, pp. 5585, 2002. DOI: https://doi.org/10.1073/pnas.052124799
  2. Tomasetto, C., C. Regnier, C. Moog-Lutz, M. G. Mattei, M. P. Chenard, R. Lidereau, P. Basset, and M. C. Rio. "Identification of Four Novel Human Genes Amplified and Overexpressed in Breast Carcinoma and Localized to the Q11-Q21.3 Region of Chromosome 17." Genomics, Vol. 28, No. 3, pp. 367-76, Aug. 1995. DOI: https://doi.org/10.1006/geno.1995.1163
  3. Arthur, J. F., Y. Shen, E. E. Gardiner, L. Coleman, D. Murphy, D. Kenny, R. K. Andrews, and M. C. Berndt. "Tnf Receptor-Associated Factor 4 (Traf4) Is a Novel Binding Partner of Glycoprotein Ib and Glycoprotein Vi in Human Platelets." J Thromb Haemost, Vol. 9, No. 1, pp. 163-72, Jan. 2011. DOI: https://doi.org/10.1111/j.1538-7836.2010.04091
  4. Kim, EunGi, Wanyeon Kim, Sungmin Lee, Jahyun Chun, JiHoon Kang, Gaeul Park, IkJoon Han, et al. "Traf4 Promotes Lung Cancer Aggressiveness by Modulating Tumor Microenvironment in Normal Fibroblasts." Scientific Reports, Vol. 7, No. 1, pp. 8923, Aug. 2017. DOI: https://doi.org/10.1038/s41598-017-09447-z
  5. Li, Wei, Cong Peng, Mee-Hyun Lee, DoYoung Lim, Feng Zhu, Yang Fu, Ge Yang, et al. "Traf4 Is a Critical Molecule for Akt Activation in Lung Cancer." [In eng]. Cancer research, Vol. 73, No. 23, pp.6938-50, 2013. DOI: https://doi.org/10.1158/0008-5472.CAN-13-0913
  6. Jura, N., N. F. Endres, K. Engel, S. Deindl, R. Das, M. H. Lamers, D. E. Wemmer, X. Zhang, and J. Kuriyan. "Mechanism for Activation of the Egf Receptor Catalytic Domain by the Juxtamembrane Segment." Cell, Vol. 137, No. 7, pp. 1293-307, Jun. 2009. DOI: https://doi.org/10.1016/j.cell.2009.04.025
  7. Regnier C. H, Tomasetto C, Moog-Lutz C, Chenard M-P, Wendling C, Basset P, Rio M-C., "Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma", The journal of biological chemistry, Vol. 270, No. 43, 1995. DOI: https://doi.org/10.1074/jbc.270.43.25715
  8. Kim, C. M., J. Y. Choi, E. A. Bhat, J. H. Jeong, Y. J. Son, S. Kim, and H. H. Park. "Crystal Structure of Traf1 Traf Domain and Its Implications in the Traf1-Mediated Intracellular Signaling Pathway." Sci Rep, 6, pp. 25526, May. 2016. DOI: https://doi.org/10.1038/srep25526
  9. Park, H. H. "Structure of Traf Family: Current Understanding of Receptor Recognition." Front Immunol, 9, pp. 1999, 2018. DOI: https://doi.org/10.3389/fimmu.2018.01999
  10. Kim, C. M., Y. J. Son, S. Kim, S. Y. Kim, and H. H. Park. "Molecular Basis for Unique Specificity of Human Traf4 for Platelets Gpibbeta and Gpvi." Proc Natl Acad Sci U S A, Vol. 114, No. 43, pp. 11422-27, Oct. 2017. DOI: https://doi.org/10.1073/pnas.1708688114
  11. Marinis, J. M., J. E. Hutti, C. R. Homer, B. A. Cobb, L. C. Cantley, C. McDonald, and D. W. Abbott. "Ikappab Kinase Alpha Phosphorylation of Traf4 Downregulates Innate Immune Signaling." Mol Cell Biol, Vol. 32, No. 13, pp. 2479-89, Jul. 2012. DOI: https://doi.org/10.1128/MCB.00106-12
  12. Rousseau, A., A. G. McEwen, P. Poussin-Courmontagne, D. Rognan, Y. Nomine, M. C. Rio, C. Tomasetto, and F. Alpy. "Traf4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration." PLoS Biol, Vol. 11, No. 12 e1001726, Dec. 2013. DOI: https://doi.org/10.1371/journal.pbio.1001726
  13. Rousseau, A., L. P. Wilhelm, C. Tomasetto, and F. Alpy. "The Phosphoinositide-Binding Protein Traf4 Modulates Tight Junction Stability and Migration of Cancer Cells." Tissue Barriers, Vol. 2, No. 4, e975597, 2014. DOI: https://doi.org/10.4161/21688370.2014.975597
  14. Kedinger, V., F. Alpy, A. Baguet, M. Polette, I. Stoll, M. P. Chenard, C. Tomasetto, and M. C. Rio. "Tumor Necrosis Factor Receptor-Associated Factor 4 Is a Dynamic Tight Junction-Related Shuttle Protein Involved in Epithelium Homeostasis." PLoS One, Vol. 3, No. 10, e3518, 2008. DOI: https://doi.org/10.1371/journal.pone.0003518
  15. Niu, Fengfeng, Heng Ru, Wei Ding, Songying Ouyang, and Zhi-Jie Liu. "Structural Biology Study of Human Tnf Receptor Associated Factor 4 Traf Domain." Protein & Cell, Vol. 4, No. 9, pp. 687-94, Sep. 2013. DOI: https://doi.org/10.1007/s13238-013-3068-z
  16. Rousseau, A., M. C. Rio, and F. Alpy. "Traf4, at the Crossroad between Morphogenesis and Cancer." Cancers (Basel), Vol. 3, No. 2, pp. 2734-49, Jun. 2011. DOI: https://doi.org/10.3390/cancers3022734
  17. Masson, R., C. H. Regnier, M. P. Chenard, C. Wendling, M. G. Mattei, C. Tomasetto, and M. C. Rio. "Tumor Necrosis Factor Receptor Associated Factor 4 (Traf4) Expression Pattern During Mouse Development." Mech Dev, Vol. 71, No. 1-2, pp. 187-91, Feb. 1998. DOI: https://doi.org/10.1016/s0925-4773(97)00192-5
  18. Qiao, X., I. Roth, E. Feraille, and U. Hasler. "Different Effects of Zo-1, Zo-2 and Zo-3 Silencing on Kidney Collecting Duct Principal Cell Proliferation and Adhesion." Cell Cycle Vol. 13, No. 19, pp. 3059-75, 2014. DOI: https://doi.org/10.4161/15384101.2014.949091
  19. Amoozadeh, Y., S. Anwer, Q. Dan, S. Venugopal, Y. Shi, E. Branchard, E. Liedtke, et al. "Cell Confluence Regulates Claudin-2 Expression: Possible Role for Zo-1 and Rac." Am J Physiol Cell Physiol , Vol. 314, No. 3, C366-C78, Mar. 2018. DOI: https://doi.org/10.1152/ajpcell.00234.2017