DOI QR코드

DOI QR Code

Expressions of MicroRNA-150 and MicroRNA-424 Targeted to C-reactive Protein in Trophoblast Cell Line

영양막세포에서의 C-reactive protein 조절 microRNA-150과 microRNA-424 발현 분석

  • 김희성 (단국대학교 의과대학 제일병원 진단검사의학과)
  • Received : 2019.09.09
  • Accepted : 2019.11.04
  • Published : 2019.11.28

Abstract

Abnormalities of trophoblast due to early inflammation in pregnancy increase the expression of CRP and affect maternal-fetal interactions, leading to preterm birth and preeclampsia. However, biomarkers related to the regulation of CRP expression have not been found. In this study, miRNA associated with increased expression of CRP was identified and their expression was analyzed to reveal biomarkers involved in the regulation mechanism of trophoblast inflammation through miRNAs. miRNAs that were predicted to regulate CRP gene expression in miRNA databases (mirna, TargetScan, MicroCosm) were screened and HTR-8/SVneo cell lines were treated with LPS (20 ng/mL) to induce inflammatory responses in vitro, with selected miR-7, miR-150, miR-186 and miR-424. The expression was analyzed by qRT-PCR. As a result, expression of CRP was significantly increased in LPS-treated trophoblast (p<0.001) and miR-150 and miR-424 expression were significantly decreased (p<0.001). Thus, miR-150 and miR-424 are involved in the regulation of CRP expression in inflammatory-induced trophoblast and may be useful for the prenatal diagnosis of inflammatory obstetric diseases.

임신 초기 염증으로 인한 영양막세포의 기능 이상은 C-reactive protein (CRP)의 발현을 증가시켜 산모와 태아의 상호작용에 영향을 미침으로써, 조산 및 자간전증 등을 유발한다. 그러나, CRP 발현 조절과 관련된 생체표지자 발굴 및 개발은 미흡한 실정이다. 본 연구는 염증이 유발된 영양막세포에서 증가된 CRP 발현과 관련된 miRNA를 발굴 및 그 발현을 분석함으로써, miRNA를 통해 영양막세포 염증 조절 기전에 관여하는 생체표지자를 밝히고자 한다. miRNA 데이터베이스(mirna, TargetScan, MicroCosm)에서 공통적으로 CRP 유전자 발현을 조절할 것으로 예측되는 miR-7, miR-150, miR-186, 그리고 miR-424를 선별하여 HTR-8/SVneo에 LPS (20ng/mL)를 처리하여 in vitro 상에서 염증 반응을 유도하였다. 각각의 miRNAs의 발현을 qRT-PCR 방법으로 비교 분석하였다. 그 결과, LPS 처리된 영양막세포에서 CRP의 발현은 유의성 있게 증가되었다(p<0.001). miR-150와 miR-424는 발현이 유의성 있게 감소됨을 확인하였다(p<0.001). 따라서, 염증이 유도된 영양막세포에서의 CRP 발현을 조절하는 기전에 miR-150와 miR-424가 관여하는 것을 의미하며, 향후 염증성 산과질환의 산전 진단에 유용한 자료로 사용될 것으로 사료된다.

Keywords

References

  1. R. B. Donker, J. F. Mouillet, T. Chu, C. A. Hubel, D. B. Stolz, A. E. Morelli, and Y. Sadovsky, "The expression profile of C19MC microRNAs in primary human trophoblast cells and exosomes," Mol Hum Reprod, Vol.18, No.8, pp.417-424, 2012. https://doi.org/10.1093/molehr/gas013
  2. E. R. Norwitz, "Defective implantation and placentation: laying the blueprint for pregnancy complications," Reprod Biomed Online, Vol.14, No.1, pp.101-109, 2007.
  3. L. Ji, J. Brkic, M. Liu, G. Fu, C. Peng, and Y. L. Wang, "Placental trophoblast cell differentiation: physiological regulation and pathological relevance to preeclampsia," Mol Aspects Med, Vol.34, No.5, pp.981-1023, 2013. https://doi.org/10.1016/j.mam.2012.12.008
  4. M. Halasz and J. Szekeres-Bartho, "The role of progesterone in implantation and trophoblast invasion," J Reprod Immunol, Vol.97, No.1, pp.43-50, 2013. https://doi.org/10.1016/j.jri.2012.10.011
  5. O. Genbacev, R. Joslin, C. H. Damsky, B. M. Polliotti, and S. J. Fisher, "Hypoxia alters early gestation human cytotrophoblast differentiation/ invasion in vitro and models the placental defects that occur in preeclampsia," J Clin Invest, Vol.97, No.2, pp.540-550, 1996. https://doi.org/10.1172/JCI118447
  6. B. M. Sibai, "Thrombophilia and severe preeclampsia: time to screen and treat in future pregnancies?," Hypertension, Vol.46, No.6, pp.1252-1253, 2005. https://doi.org/10.1161/01.hyp.0000188904.47575.7e
  7. I. Kushner and G. Feldmann, "Control of the acute phase response. Demonstration of C-reactive protein synthesis and secretion by hepatocytes during acute inflammation in the rabbit," J Exp Med, Vol.148, No.2, pp.466-477, 1978. https://doi.org/10.1084/jem.148.2.466
  8. S. Black, I. Kushner, and D. Samols, "C-reactive protein," Journal of Biological Chemistry, Vol.279, No.47, pp.48487-48490, 2004. https://doi.org/10.1074/jbc.R400025200
  9. M. Cai, G. K. Kolluru, and A. Ahmed, "Small Molecule, Big Prospects: MicroRNA in Pregnancy and Its Complications," J Pregnancy, Vol.2017, p.6972732, 2017.
  10. E. N. Kim, B. H. Yoon, E. J. Jeon, J. B. Lee, J. S. Hong, J. Y. Lee, D. Hwang, K. C. Kim, J. S. Kim, and C. J. Kim, "Placental deposition of C-reactive protein is a common feature of human pregnancy," Placenta, Vol.36, No.6, pp.704-707, 2015. https://doi.org/10.1016/j.placenta.2015.03.006
  11. C. W. Redman, G. P. Sacks, and I. L. Sargent, "Preeclampsia: an excessive maternal inflammatory response to pregnancy," Am J Obstet Gynecol, Vol.180, No.2, Pt.1, pp.499-506, 1999. https://doi.org/10.1016/S0002-9378(99)70239-5
  12. M. T. Gervasi, T. Chaiworapongsa, P. Pacora, N. Naccasha, B. H. Yoon, E. Maymon, and R. Romero, "Phenotypic and metabolic characteristics of monocytes and granulocytes in preeclampsia," Am J Obstet Gynecol, Vol.185, No.4, pp.792-797, 2001. https://doi.org/10.1067/mob.2001.117311
  13. C. W. Park, B. H. Yoon, J. S. Park, and J. K. Jun, "An elevated maternal serum C-reactive protein in the context of intra-amniotic inflammation is an indicator that the development of amnionitis, an intense fetal and AF inflammatory response are likely in patients with preterm labor: clinical implications," J Matern Fetal Neonatal Med, Vol.26, No.9, pp.847-853, 2013. https://doi.org/10.3109/14767058.2013.783806
  14. R. A. Howman, A. K. Charles, A. Jacques, D. A. Doherty, K. Simmer, T. Strunk, P. C. Richmond, C. H. Cole, and D. P. Burgner, "Inflammatory and haematological markers in the maternal, umbilical cord and infant circulation in histological chorioamnionitis," PLoS One, Vol.7, No.12, p.e51836, 2012. https://doi.org/10.1371/journal.pone.0051836
  15. F. Mirzaie, F. Rahimi-Shorbaf, and A. H. Kazeronie, "Association of maternal serum C-reactive protein levels with severity of preeclampsia," Acta Medica Iranica, pp.293-296, 2009.
  16. I. Brosens, R. Pijnenborg, L. Vercruysse, and R. Romero, "The "Great Obstetrical Syndromes" are associated with disorders of deep placentation," Am J Obstet Gynecol, Vol.204, No.3, pp.193-201, 2011. https://doi.org/10.1016/j.ajog.2010.08.009
  17. R. Romero, J. P. Kusanovic, T. Chaiworapongsa, and S. S. Hassan. "Placental bed disorders in preterm labor, preterm PROM, spontaneous abortion and abruptio placentae," Best Pract Res Clin Obstet Gynaecol, Vol.25, No.3, pp.313-327, 2011. https://doi.org/10.1016/j.bpobgyn.2011.02.006
  18. S. Lee and S. Vasudevan, "Post-transcriptional stimulation of gene expression by microRNAs," Adv Exp Med Biol, Vol.768, pp.97-126, 2013. https://doi.org/10.1007/978-1-4614-5107-5_7
  19. D. P. Bartel, "MicroRNAs: target recognition and regulatory functions," Cell, Vol.136, No.2, pp.215-233, 2009. https://doi.org/10.1016/j.cell.2009.01.002
  20. P. Zhou, W. Xu, X. Peng, Z. Luo, Q. Xing, X. Chen, C. Hou, W. Liang, J. Zhou, X. Wu, Z. Songyang, and S. Jiang, "Large-scale screens of miRNA-mRNA interactions unveiled that the 3'UTR of a gene is targeted by multiple miRNAs," PLoS One, Vol.8, No.7, p.e68204, 2013. https://doi.org/10.1371/journal.pone.0068204
  21. Y. Huang, X. J. Shen, Q. Zou, S. P. Wang, S. M. Tang, and G. Z. Zhang, "Biological functions of microRNAs: a review," J Physiol Biochem, Vol.67, No.1, pp.129-139, 2011. https://doi.org/10.1007/s13105-010-0050-6
  22. M. S. Ebert and P. A. Sharp, "Roles for microRNAs in conferring robustness to biological processes," Cell, Vol.149, No.3, pp.515-524, 2012. https://doi.org/10.1016/j.cell.2012.04.005
  23. A. Markou, Y. Liang, and E. Lianidou, "Prognostic, therapeutic and diagnostic potential of microRNAs in non-small cell lung cancer," Clin Chem Lab Med, Vol.49, No.10, pp.1591-1603, 2011. https://doi.org/10.1515/CCLM.2011.661
  24. L. Mulrane, R. Klinger, S. F. McGee, W. M. Gallagher, and D. P. O'Connor, "microRNAs: a new class of breast cancer biomarkers," Expert Rev Mol Diagn, Vol.14, No.3, pp.347-363, 2014. https://doi.org/10.1586/14737159.2014.901153
  25. C. C. Pritchard, E. Kroh, B. Wood, J. D. Arroyo, K. J. Dougherty, M. M. Miyaji, J. F. Tait, and M. Tewari, "Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies," Cancer Prev Res (Phila), Vol.5, No.3, pp.492-497, 2012. https://doi.org/10.1158/1940-6207.CAPR-11-0370
  26. H. Schwarzenbach, N. Nishida, G. A. Calin, and K. Pantel, "Clinical relevance of circulating cell-free microRNAs in cancer," Nat Rev Clin Oncol, Vol.11, No.3, pp.145-156, 2014. https://doi.org/10.1038/nrclinonc.2014.5
  27. S. S. Luo, O. Ishibashi, G. Ishikawa, T. Ishikawa, A. Katayama, T. Mishima, T. Takizawa, T. Shigihara, T. Goto, A. Izumi, A. Ohkuchi, S. Matsubara, T. Takeshita, and T. Takizawa, "Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes," Biol Reprod, Vol.81, No.4, pp.717-729, 2009. https://doi.org/10.1095/biolreprod.108.075481
  28. D. M. Morales-Prieto, S. Ospina-Prieto, A. Schmidt, W. Chaiwangyen, and U. R. Markert. "Elsevier Trophoblast Research Award Lecture: origin, evolution and future of placenta miRNAs," Placenta Vol.35, pp.S39-45, 2014. https://doi.org/10.1016/j.placenta.2013.11.017
  29. P. Sethupathy, M. Megraw, and A. G. Hatzigeorgiou, "A guide through present computational approaches for the identification of mammalian microRNA targets," Nature Methods, Vol.3, No.11, pp.881-886, 2006. https://doi.org/10.1038/nmeth954
  30. V. Agarwal, G. W. Bell, J. W. Nam, and D. P. Bartel, "Predicting effective microRNA target sites in mammalian mRNAs," Elife, Vol.4, 2015.
  31. B. P. Lewis, C. B. Burge, and D. P. Bartel, "Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets," Cell, Vol.120, No.1, pp.15-20, 2005. https://doi.org/10.1016/j.cell.2004.12.035
  32. C. H. Graham, T. S. Hawley, R. G. Hawley, J. R. MacDougall, R. S. Kerbel, N. Khoo, and P. K. Lala, "Establishment and characterization of first trimester human trophoblast cells with extended lifespan," Exp Cell Res, Vol.206, No.2, pp.204-211, 1993. https://doi.org/10.1006/excr.1993.1139
  33. G. Sacks, L. Seyani, S. Lavery, and G. Trew, "Maternal C-reactive protein levels are raised at 4 weeks gestation," Human reproduction, Vol.19, No.4, pp.1025-1030, 2004. https://doi.org/10.1093/humrep/deh179
  34. E. Teran, C. Escudero, W. Moya, M. Flores, P. Vallance, and P. Lopez-Jaramillo, "Elevated C-reactive protein and pro-inflammatory cytokines in Andean women with preeclampsia," Int J Gynaecol Obstet, Vol.75, No.3, pp.243-249, 2001. https://doi.org/10.1016/S0020-7292(01)00499-4
  35. B. H. Yoon, J. K. Jun, K. H. Park, H. C. Syn, R. Gomez, and R. Romero, "Serum C-reactive protein, white blood cell count, and amniotic fluid white blood cell count in women with preterm premature rupture of membranes," Obstet Gynecol, Vol.88, No.6, pp.1034-1040, 1996. https://doi.org/10.1016/S0029-7844(96)00339-0
  36. G. B. Hvilsom, P. Thorsen, B. Jeune, and L. S. Bakketeig, "C-reactive protein: a serological marker for preterm delivery?," Acta Obstet Gynecol Scand, Vol.81, No.5, pp.424-429, 2002. https://doi.org/10.1080/j.1600-0412.2002.810509.x
  37. F. Ramaekers, A. Huysmans, G. Schaart, O. Moesker, and P. Vooijs, "Tissue distribution of keratin 7 as monitored by a monoclonal antibody," Exp Cell Res, Vol.170, No.1, pp.235-249, 1987. https://doi.org/10.1016/0014-4827(87)90133-9
  38. R. Apps, L. Gardner, and A. Moffett, "A critical look at HLA-G," Trends Immunol, Vol.29, No.7, pp.313-321, 2008. https://doi.org/10.1016/j.it.2008.02.012
  39. L. A. Cole, "Hyperglycosylated hCG, a review," Placenta, Vol.31, No.8, pp.653-664, 2010. https://doi.org/10.1016/j.placenta.2010.06.005
  40. A. Chakrabarty, S. Tranguch, T. Daikoku, K. Jensen, H. Furneaux, and S. K. Dey, "MicroRNA regulation of cyclooxygenase-2 during embryo implantation," Proc Natl Acad Sci U S A, Vol.104, No.38, pp.15144-15149, 2007. https://doi.org/10.1073/pnas.0705917104
  41. M. Bidarimath, K. Khalaj, J. M. Wessels, and C. Tayade, "MicroRNAs, immune cells and pregnancy," Cell Mol Immunol, Vol.11, No.6, pp.538-547, 2014. https://doi.org/10.1038/cmi.2014.45
  42. L. Gan, Z. Liu, M. Wei, Y. Chen, X. Yang, L. Chen, and X. Xiao, "MiR-210 and miR-155 as potential diagnostic markers for pre-eclampsia pregnancies," Medicine (Baltimore), Vol.96, No.28, p.e7515, 2017. https://doi.org/10.1097/MD.0000000000007515
  43. K. R. Bounds, V. L. Chiasson, L. J. Pan, S. Gupta, and P. Chatterjee, "MicroRNAs: new players in the pathobiology of preeclampsia," Frontiers in cardiovascular medicine, Vol.4, p.60, 2017. https://doi.org/10.3389/fcvm.2017.00060
  44. S. Monticelli, K. M. Ansel, C. Xiao, N. D. Socci, A. M. Krichevsky, T. H. Thai, N. Rajewsky, D. S. Marks, C. Sander, K. Rajewsky, A. Rao, and K. S. Kosik, "MicroRNA profiling of the murine hematopoietic system," Genome Biol, Vol.6, No.8, p.R71, 2005. https://doi.org/10.1186/gb-2005-6-8-r71
  45. B. Zhou, S. Wang, C. Mayr, D. P. Bartel, and H. F. Lodish, "miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely," Proc Natl Acad Sci U S A, Vol.104, No.17, pp.7080-7085, 2007. https://doi.org/10.1073/pnas.0702409104
  46. C. Xiao, D. P. Calado, G. Galler, T. H. Thai, H. C. Patterson, J. Wang, N. Rajewsky, T. P. Bender, and K. Rajewsky, "MiR-150 Controls B Cell Differentiation by Targeting the Transcription Factor c-Myb," Cell, Vol.165, No.4, p.1027, 2016. https://doi.org/10.1016/j.cell.2016.04.056
  47. Y. He, X. Jiang, and J. Chen, "The role of miR-150 in normal and malignant hematopoiesis," Oncogene, Vol.33, No.30, pp.3887-3893, 2014. https://doi.org/10.1038/onc.2013.346
  48. R. Garzon and C. M. Croce, "MicroRNAs in normal and malignant hematopoiesis," Curr Opin Hematol, Vol.15, No.4, pp.352-358, 2008. https://doi.org/10.1097/MOH.0b013e328303e15d
  49. N. Luo, W. Garvey, D. Wang, and Y. Fu, "MicroRNA-150 regulates lipid metabolism and inflammatory response," J Metab Syndr, Vol.2, No.2, 2013.
  50. J. F. Mouillet, T. Chu, C. A. Hubel, D. M. Nelson, W. T. Parks, and Y. Sadovsky, "The levels of hypoxia-regulated microRNAs in plasma of pregnant women with fetal growth restriction," Placenta, Vol.31, No.9, pp.781-784, 2010. https://doi.org/10.1016/j.placenta.2010.07.001
  51. G. Ghosh, I. V. Subramanian, N. Adhikari, X. Zhang, H. P. Joshi, D. Basi, Y. S. Chandrashekhar, J. L. Hall, S. Roy, Y. Zeng, and S. Ramakrishnan, "Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-alpha isoforms and promotes angiogenesis," J Clin Invest, Vol.120, No.11, pp.4141-4154, 2010. https://doi.org/10.1172/JCI42980
  52. K. Zhang, F. Song, X. Lu, W. Chen, C. Huang, L. Li, D. Liang, S. Cao, and H. Dai, "MicroRNA-322 inhibits inflammatory cytokine expression and promotes cell proliferation in LPS-stimulated murine macrophages by targeting NF-${\kappa}B1$ (p50)," Bioscience reports, Vol.37, No.1, p.BSR20160239, 2017. https://doi.org/10.1042/BSR20160239