Serum Circulating MicroRNAs as Biomarkers of Osteoporotic Fracture

Abstract

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.

Identification of suitable reference gene and biomarkers of serum miRNAs for osteoporosis

“…Recently, the diagnostic potential of circulating miRNAs for postmenopausal osteoporosis has been investigated and some miRNAs were identified as potential biomarkers10121314. Because of the diverse study designs and models, more studies were required to establish circulating miRNAs biomarkers for osteoporosis with high specificity and sensitivity.…”

“…Weilner et al showed that miR-328-3p and let-7g-5p were down-regulated in the serum of patients with osteoporotic fracture and could modulate the osteogenic differentiation of human mesenchymal stem cells in vitro 13. Meanwhile, Panach et al identified three up-regulated miRNAs (miR-122-5p, miR-125b-5p and miR-21-5p) were valuable biomarkers for osteoporotic fracture14. Due to the diverse nature of study designs, the number and type of regulated miRNAs identified varies significantly9.…”

“…Wang et al identified snRNAU6, miR-92a and miR-16 and let-7a as internal reference gene group for qPCR normalization from osteogenesis imperfect patients16. Most of the results obtained by qPCR used different reference genes for normalization, such as miR-16, miR-93-5p in skeletal disease10121314. Although miR-93 had been defined as a plasma miRNA reference gene for tuberculosis17, it was also connected with osteoblast differentiation and bone mineralization18.…”

Epigenetic Mechanisms in Bone Biology and Osteoporosis: Can They Drive Therapeutic Choices?

“…These data were validated on a further 12 fractured women vs. 11 controls, confirming miR-22-3p, miR-328-3p, and let-7g-5p as differentially expressed in the presence of a fracture. Panach et al measured a panel of 179 miRNAs in the serum samples of eight osteoporotic women with fractures vs. five controls, and validated data on 15 patients with fractures vs. 12 controls [61]. They determined that miR-122-5p, miR-125b-5p, and miR-21-5p were upregulated in the presence of a fracture.…”

MicroRNA and Human Bone Health

“…MiR‐125b‐5p level was found to be inversely correlated with BMD at the femoral head, with an increased expression being observed in osteoblast and osteoclast from osteoporotic patients . When comparing the circulating miRNAs in patients with osteoporotic fracture with those in osteoarthritic controls, circulating miR‐125b‐5p was also significantly upregulated in the osteoporotic fracture group …”