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Serum Circulating MicroRNAs as Biomarkers of Osteoporotic Fracture

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Osteoporosis is a common skeletal disorder characterized by increased risk of bone fracture (BF) due to fragility. BFs, particularly hip fracture, are a major concern in health care because of the associated morbidity and mortality, mainly in the elderly. Lately the involvement of epigenetic mechanisms in the pathophysiology of many diseases has been recognized. In this context, the identification of microRNAs (miRNAs) specific to BF should represent a substantial step forward in diagnostics and therapeutics. The present study aimed to identify specific miRNAs in osteoporotic BF patients compared to those in osteoarthritic controls. In the profiling stage, total RNA was extracted from serum, two pools were prepared, and then retro-transcribed in triplicate. Levels of 179 serum miRNAs were analyzed by real-time PCR, and 42 of them showed significance (P < 0.05), and 12 passed the false discovery rate test for multiple comparisons. Six miRNAs were selected for the replication stage and individually analyzed in sera from 15 BF patients and 12 controls. Results showed that 3 miRNAs (miR-122-5p, miR-125b-5p, and miR-21-5p) were valuable upregulated biomarkers in BF with respect to controls and, significantly, their levels were not affected by hemolysis. For miR-21-5p, the difference detected between groups was independent of age (P = 0.005) and its levels correlated to those of CTx (r = 0.76; P < 0.00001), a marker of bone resorption. In conclusion, several miRNAs may be biomarkers of BF, particularly miR-21-5p. Further studies are needed in order to better characterize the levels of these miRNAs in other bone diseases and to elucidate the mechanism involved in the association of these three miRNAs with osteoporotic BF.

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  1. Kanis JA, McCloskey EV, Johansson H, Cooper C, Rizzoli R, Reginster JY (2013) European guidance for the diagnosis and management of osteoporosis in postmenopausal women. Osteoporos Int 24:23–57

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Kanis JA, Oden A, Johnell O, De Laet C, Jonsson B, Oglesby AK (2003) The components of excess mortality after hip fracture. Bone 32:468–473

    Article  CAS  PubMed  Google Scholar 

  3. Ralston SH, Uitterlinden AG (2010) Genetics of osteoporosis. Endocr Rev 31:629–662

    Article  CAS  PubMed  Google Scholar 

  4. Hsu YH, Kiel DP (2012) Clinical review: genome-wide association studies of skeletal phenotypes: what we have learned and where we are headed. J Clin Endocrinol Metab 97:E1958–E1977

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Guil S, Esteller M (2009) DNA methylomes, histone codes and miRNAs: tying it all together. Int J Biochem Cell Biol 41:87–95

    Article  CAS  PubMed  Google Scholar 

  6. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297

    Article  CAS  PubMed  Google Scholar 

  7. He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5:522–531

    Article  CAS  PubMed  Google Scholar 

  8. Brookes E, Shi Y (2014) Diverse epigenetic mechanisms of human disease. Annu Rev Genet 48:237–268

    Article  CAS  PubMed  Google Scholar 

  9. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, Aldler H, Rattan S, Keating M, Rai K, Rassenti L, Kipps T, Negrini M, Bullrich F, Croce CM (2002) Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 99:15524–15529

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Kosaka N, Iguchi H, Ochiya T (2010) Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis. Cancer Sci 101:2087–2092

    Article  CAS  PubMed  Google Scholar 

  11. Lian JB, Stein GS, van Wijnen AJ, Stein JL, Hassan MQ, Gaur T, Zhang Y (2012) MicroRNA control of bone formation and homeostasis. Nat Rev Endocrinol 8:212–227

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Gluer CC (1999) Monitoring skeletal changes by radiological techniques. J Bone Miner Res 14:1952–1962

    Article  CAS  PubMed  Google Scholar 

  13. Garnero P (2014) New developments in biological markers of bone metabolism in osteoporosis. Bone 66:46–55

    Article  CAS  PubMed  Google Scholar 

  14. Kirschner MB, Kao SC, Edelman JJ, Armstrong NJ, Vallely MP, van Zandwijk N, Reid G (2011) Haemolysis during sample preparation alters microRNA content of plasma. PLoS One. 6:e24145

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36

    Article  PubMed Central  PubMed  Google Scholar 

  16. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3:0034.1–0034.11

    Article  Google Scholar 

  17. Blondal T, Jensby NS, Baker A, Andreasen D, Mouritzen P, Wrang TM, Dahlsveen IK (2013) Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods 59:S1–S6

    Article  CAS  PubMed  Google Scholar 

  18. Krzeszinski JY, Wei W, Huynh H, Jin Z, Wang X, Chang TC, Xie XJ, He L, Mangala LS, Lopez-Berestein G, Sood AK, Mendell JT, Wan Y (2014) miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2. Nature 512:431–435

    Article  CAS  PubMed  Google Scholar 

  19. Seibel MJ (2005) Biochemical markers of bone turnover: part I: biochemistry and variability. Clin Biochem Rev 26:97–122

    PubMed Central  PubMed  Google Scholar 

  20. Cortez MA, Calin GA (2009) MicroRNA identification in plasma and serum: a new tool to diagnose and monitor diseases. Expert Opin Biol Ther 9:703–711

    Article  CAS  PubMed  Google Scholar 

  21. Seeliger C, Karpinski K, Haug AT, Vester H, Schmitt A, Bauer JS, van Griensven M (2014) Five freely circulating miRNAs and bone tissue miRNAs are associated with osteoporotic fractures. J Bone Miner Res 29:1718–1728

    Article  CAS  PubMed  Google Scholar 

  22. Garmilla-Ezquerra P, Sanudo C, Delgado-Calle J, Perez-Nunez MI, Sumillera M, Riancho JA (2015) Analysis of the bone microRNome in osteoporotic fractures. Calcif Tissue Int 96:30–37

    Article  CAS  PubMed  Google Scholar 

  23. Mizuno Y, Tokuzawa Y, Ninomiya Y, Yagi K, Yatsuka-Kanesaki Y, Suda T, Fukuda T, Katagiri T, Kondoh Y, Amemiya T, Tashiro H, Okazaki Y (2009) miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b. FEBS Lett 583:2263–2268

    Article  CAS  PubMed  Google Scholar 

  24. Li H, Wang Z, Fu Q, Zhang J (2014) Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients. Biomarkers 19:553–556

    Article  CAS  PubMed  Google Scholar 

  25. Sugatani T, Hruska KA (2013) Down-regulation of miR-21 biogenesis by estrogen action contributes to osteoclastic apoptosis. J Cell Biochem 114:1217–1222

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Mizuno Y, Yagi K, Tokuzawa Y, Kanesaki-Yatsuka Y, Suda T, Katagiri T, Fukuda T, Maruyama M, Okuda A, Amemiya T, Kondoh Y, Tashiro H, Okazaki Y (2008) miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun 368:267–272

    Article  CAS  PubMed  Google Scholar 

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The authors are indebted to Mrs. R. Aliaga and Dr. V. Ballester for their technical assistance in management of the patients and biochemical assays. Likewise, the authors are indebted to Drs. A. López Castel and JM Fernández-Costa, both from Valentia Biopharma (Valencia, Spain) for their excellent technical assistance regarding miRNA profiling and validation experiments. This work was supported by grant PI12/02582 from the Fondo de Investigación Sanitaria (FIS, Madrid, Spain), including funds from EU’s FEDER Program. Layla Panach is a predoctoral fellow from the Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario).

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Correspondence to Miguel Ángel García-Pérez.

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Human and Animal Rights and Informed Consent

The study was conducted according to the Helsinki Declaration of 2000 and was approved by the Clinical Research Ethics Committee of the Institute of Health Research, INCLIVA. All participants in the study read and signed an informed consent according to the regulations of INCLIVA.

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Panach, L., Mifsut, D., Tarín, J.J. et al. Serum Circulating MicroRNAs as Biomarkers of Osteoporotic Fracture. Calcif Tissue Int 97, 495–505 (2015).

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