Table 2.
miRNAs related to bone fracture risk in postmenopausal OP.
| Study | Study design | Biomarker source | Sample handling | Quantification platform | Evaluated miRNA | Normalization strategy | Reported miRNA biomarker | Potential target gene | AUC-Sensitivity (%)-Specificity (%) | Limits |
|---|---|---|---|---|---|---|---|---|---|---|
| (Garmilla-Ezquerra et al., 2015) | Discovery cohort: 8 women with OP hip fracture, 8 women with severe hip OA without OP fractures (control group) Replication cohort: 19 women with OP hip fracture, 19 women with severe hip OA without OP fractures (control group) |
Bone specimens | Trabecular bone cylinders obtained from central part of femoral head using a trephine. Fragments cut into small pieces, washed with PBS, snap-frozen in liquid nitrogen, and stored at -70°C | Discovery stage: TaqMan array human miRNA A + B cards v3 Replication stage: TaqMan RT-qPCR |
Discovery stage: 760 miRNAs tested Replication stage: miR-187, miR-193a-3p, miR-214, miR-518f, miR-636, and miR-210 |
NormFinder and GeNorm programs identified miR-222 and let-7b as most stable normalizators. | ↑ miR-518f in OP fractures group vs. control group↑ miR-187 in control group vs. OP fractures group | IGFBP1, DKK1, WISP1, CTNNBIP1 (identified for miR-518f using microRNA.org, mirbase.org, and targetscan.org prediction algorithms but not validated by in vitro experiments) | / | Small sample size; OA patients as control group; no validation of the identified target genes; no information about the stem-loop arm of miRNA origin; no ROC analysis. |
| (De-Ugarte et al., 2015) | Discovery cohort: 6 PM OP women and 6 PM OA women (control group) both with femoral neck fracture Replication cohort: 7 PM OP women and 6 PM OA women (control group) both with femoral neck fracture |
Fresh bone specimens | Bone fragments from femoral neck transcervical region reduced to small pieces, washed three times with PBS, and stored at -80°C | Discovery stage: miRCURY LNA™ microRNA Array performed by Exiqon Services Replication stage: RT-qPCR performed by Exiqon Services |
Discovery stage: 1932 miRNAs tested Replication stage: miR-675-5p, miR-30c-1-3p, miR-483-5p, miR-542-5p, miR-142-3p, miR-223-3p, miR-32-3p, and miR-320a |
Discovery stage: Lowess (Locally Weighted Scatterplot Smoothing) global regression algorithm. Replication stage: average of miR-let-7e-5p expression in each sample |
↑ miR-320a and miR-483-5p in OP fractures vs. control group | ARPP-19, BMP3 and 6, BMPR1A, CAMTA1, DNER, ESRRG, IGF1, IGF1R, IL6R, JAK2, PPARGC1A, LEPR, MAPK1, MCL, NR3C1, PDGFD, PTGER3, RARG, RXRA, SGK, SP1, SRF, TFR1 (identified for miR-320a using PicTar, TargetScan Human, miRDB, MiRanda, DIANA-TarBase, and miRTarBase database)SRF and MAPK3 (identified for miR-483-5p using mirTArBase) | / | Small sample size; OA patients as control group; no validation of the identified target genes; no ROC analysis |
| (Seeliger et al., 2014) | Discovery cohort: 10 OP (7 women and 3 men) and 10 non-OP (10 women) as control group, both with femoral neck or pertrochanteric fracture Replication cohort: 30 OP women and 30 non-OP women (control group), both with femoral neck or pertrochanteric fracture |
Discovery stage: cell-free serum Replication stage: cell-free serum and bone tissue |
/ | Screening: human Serum & Plasma miRNA PCR Array MIHS-106Z Validation: SYBR RT-qPCR |
Screening: 83 miRNAs tested Validation: miR-21-5p, miR-23-3p, miR-24-3p, miR-25-3p, miR-27a-3p, miR-93, miR-100-5p, miR-122a-5p, miR-124-3p, miR-125b-5p, miR-148a-3p, miR-223-3p, and miR-637 |
Average of SNORD96a and snRNU6 | ↑ miR-21, miR-23a, miR-24, miR-93, miR-100, miR-122a, miR-124a, miR-125b, and miR-148a in OP fracture serum vs. controls↑ miR-21, miR-23a, miR-24, miR-25, miR-100, and miR-125b in bone tissue from OP fracture patients vs. control | PDCD4, cFos (miR-21); RUNX2 (miR-23a/miR-24-2/miR-27a complex); OSX (miR-93); BMPR2 (miR-100); VCAN (miR-124a); RANKL (miR-148a)(identified from previous papers but not validated in this paper) | 0.63-61.3-61.7 (miR-21), 0.63-57.4-56.7 (miR-23a), 0.63-60.3-60.4 (miR-24), 0.68-69.0-68.3 (miR-93), 0.69-62.9-61.7 (miR-100), 0.77-74.1-72.1 (miR-122a), 0.69-61.4-61.0 (miR-124a), 0.76-76.4-75.0 (miR-125b), 0.61-62.5-62.3 (miR-148a) for OP fracture vs. non-OP | Small sample size; no validation of the target genes. |
| (Panach et al., 2015) | Discovery stage: 8 Caucasian women with OP subcapital hip fracture and 5 with severe OA of hip requiring surgery (control group)Replication stage: 15 Caucasian women with OP subcapital hip fracture and 12 with severe OA of hip requiring surgery (control group) | Cell-free serum | Serum samples obtained from fasting blood stored at -80°C | Discovery stage: miRCURY LNA Universal RT microRNA PCR, Serum/Plasma Focus microRNA PCR Panel Replication stage: Exiqon LNA RT-qPCR |
Screening: 179 miRNAs tested Validation: miR-143-3p, miR-122-5p, miR-125b-5p, miR-210, miR-21-5p, and miR-34a-5p |
GeNorm identified miR-93-5p | ↑ miR-122-5p, miR-125b-5p, and miR-21-5p in OP fracture vs. control group | / | 0.87 (miR-122-5p), 0.76 (miR-125-5p), and 0.87 (miR-21-5p) for OP fracture vs. control group | Small sample size, OA patients as control group; no evaluation of the target genes |
| (Weilner et al., 2015) | Discovery stage: 7 PM Caucasian women with femoral neck OP fracture and 7 PM women without femoral fracture (control group)Replication stage: 12 PM Caucasian women with femoral neck OP fracture and 11 PM women without femoral fracture (control group) | Cell-free serum | Serum obtained from blood samples centrifugied at RT and 2000g for 15 min, after incubation at RT for 30 min, and stored at -80°C | Screening: Exiqon serum/plasma focus panels Validation: RT-qPCR |
Screening: 175 miRNAs tested Validation: miR-10a-5p, miR-10b-5p, miR-22-3p, miR 133b, miR-328-3p, and let-7g-5p |
Normalization of Cp-values based on average Cp of the detected miRNAs | ↓ miR-22-3p, miR-328-3p, and let-7g-5p in OP fracture serum vs. control group | / | / | Small sample size; no evaluation of the target genes; the mean age of patients recruited for the discovery and validation study was significantly different (71 years and 80 years, respectively); no ROC analysis |
| (Kocijan et al., 2016) | 10 women with PM OP low trauma fracture and 11 healthy PM women without low-trauma fracture | Cell-free serum | Fasting blood samples immediately centrifuged and serum stored a -80°C | SYBR Green RT-qPCR | 187 miRNAs tested | Global mean | ↑ miR-152-3p, miR-335-5p, miR-320a and↓ let-7b-5p, miR-7-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-29b-3p, miR-30e-5p, miR-93-5p, miR-140-5p, miR-215-5p, miR-186-5p, miR-324-3p, miR-365a-3p, miR-378a-5p, miR-532-5p, and miR-550a-3p in fractured group vs. control group | / | 0.962 (miR-152-3p), 0.959 (miR-30e-5p), 0.950 (miR-324-3p), 0.947(miR-140-5p), 0.944 (miR-19b-3p), 0.939 (miR-335-5p), 0.929 (miR-19a-3p), 0.909 (miR-550a-3p), 0.898 (miR-186-5p), 0.898 (miR-532-5p), 0.872 (miR-378a-5p), 0.870 (miR-320a), 0.879 (miR-93-5p), 0.857 (miR-16-5p), 0.853 (miR-215-5p), 0.852 (let-7b-5p), 0.824 (miR-7-5p), 0.838 (miR-29b-3p), and 0.809 (miR-365a-3p) for fracture group vs. control group | Small sample size; no evaluation of the target genes; arbitrary choice of the screened miRNAs |
| (Chen et al., 2017) | 30 PM Chinese women with OP and 30 PM Chinese women without OP (control group) both with hip fracture | Cell-free serum and bone tissues | Blood samples allowed to clot, centrifuged at 1500g, then serum isolated and stored. | Screening: Microarray Validation: TaqMan RT-qPCR |
Validation: miR-30, miR- 96, miR-125b, miR-4665-3p, and miR-5914 | snRNU6 | ↑ miR-125b, miR-30 and miR-5914 in serum and bone tissues from OP fracture vs. control group | / | 0.699 (miR-5914), 0.757 (miR-30), and 0.898 (miR-125b) for OP fracture vs. controls | Small sample size; no target genes evaluation; no information about the stem-loop arm of miRNA origin |
| (Yavropoulou et al., 2017) | 35 PM women with low bone mass without vertebral fractures, 35 with low bone mass and vertebral fractures, 30 HC | Cell-free serum | Blood samples collected in clot activator tubes, placed at RT for 10-60 min, centrifuged for 10 min at 1900g and 4°C. Serum samples centrifuged again for 10 min at 16000g and 4°C and frozen at -80°C | SYBR Green RT-qPCR | 14 miRNAs selected based on the existing literature: miR-21-5p, miR-23a-3p, miR-24-2-5p, miR-26a-5p, miR-29a, miR-33a-5p, miR-124-3p, miR-133a, miR-135b-5p, miR-214-3p, miR-218-5p, miR-335-3p, miR-422, and miR-2861 | Panel of SNORD95, SNORD96A, and snRNU6-2 | ↑ miR-124-3p, miR-2861, and ↓ miR-21-5p, miR-23a-3p, miR-29a-3p in OP vs. controls↓miR-21-5p in OP with vertebral fracture vs. OP without vertebral fracture | SPRY1, BMP3, DKK2, and SMAD7 (miR-21-5p); SATB2 and RUNX2 (miR-23a-3p); SATB2 and CALB1 (miR-24-2-5p); EPHA5, COL10A1, and COL19A1 (miR-26a-5p); DUSP2, COL3A1, COL5A3, and PTHLH (miR-29a); DKK2, WIF1, and OSTF1 (miR-33a-5p); HDAC5, NFATC1, and NFATC2, (miR-124-3p); ACVR1B, FOXO1, SIRT1, and SMAD5 (miR-135b-5p); ATP2A3, CTNNB1, and VDR (miR-214-3p); COL1A1, SFRP2, SOST, and EPHA5 (miR-218-5p); DKK1 and SPARC (miR-335-3p); HDAC5 (miR-2861)(Identified using miRBase, DIANA TOOLS, PicTar, miRDB, TargetScanHuman, miRGator, and microRNA database) | 0.66-66-71 (miR-21-5p) for OP with vertebral fracture vs. OP without vertebral fracture | Small sample size; no validation of the identified target genes |
| (Wang et al., 2018) | 45 OP patients, 15 non-OP (control group) both with femoral fracture | Cell-free serum and bone tissues | / | RT-qPCR | miR-7-5p, miR-24-3p, miR-27a-3p, miR-100, miR-125b, miR-128, miR-145-5p, miR-211-5p, miR-144-3p, and miR-122a | snRNU6 | ↑ miR-24-3p, 27a-3p, miR-100, miR-125b, miR-122a, miR-145, and ↓ miR-144-3p in serum from OP fracture vs. non-OP fracture↑ miR-24-3p, 27a-3p, miR-100, miR-125b, miR-128, miR-122a, and ↓ miR-144-3p in bone tissues form OP fracture vs. non-OP fracture | RANK (identified for miR-144-3p using TargetScan online software and validated by in vitro study) | / | Small sample size of the non-OP group; no ROC analysis |
| (Li et al., 2018) | 10 PM Chinese OP women with hip fracture and 10 HC | Cell-free serum | Blood samples allowed to clot then centrifuged at 1500g to obtain serum | TaqMan RT-qPCR | miR-133a | snRNU6 | ↑miR-133a in OP with fractures vs. HC | c-Fos, NFATc1, and TRAP for miR-133a identified by in vitro experiments | / | Small sample size; no ROC analysis |
ACVR1B, activin A receptor type 1B; ALPL, alkaline phosphatase; ANKH, ANKH inorganic pyrophosphate transport regulator; AR, androgen receptor; ARPP-19, cAMP-regulated phosphoprotein 19; ATF4, activating transcription factor 4; ATP2A3, sarcoplasmic/endoplasmic reticulum calcium ATPase 3; BMP2K, BMP2 inducible kinase; BMP3, bone morphogenetic protein 3; BMP6, bone morphogenetic protein 6; BMPR1A, bone morphogenetic protein receptor 1A; BMPR2, bone morphogenetic protein receptor type 2; CALB1, calbindin 1; CAMTA1, calmodulin binding transcription activator 1; CNR1, cannabinoid receptor 1; CNR2, cannabinoid receptor 2; COL10A1, collagen type X alpha 1 chain; COL19A1, collagen type XIX alpha 1 chain; COL1A1, collagen type I alpha 1 chain; COL3A1, collagen type III alpha 1 chain; COL5A3, collagen type V alpha 3 chain; CTNNB1, catenin beta 1, CTNNBIP1, catenin-interacting protein 1; DKK1, Dickkopf WNT signaling pathway inhibitor 1; DKK2, Dickkopf WNT signaling pathway inhibitor 2; DNER, delta and notch-like epidermal growth factor-related receptor; DUSP2, dual specificity phosphatase 2; EPHA5, EPH receptor A5; ESR1, estrogen receptor 1; ESRRG, estrogen related receptor gamma; FOXO1, Forkhead box O1, FSHB, follicle stimulating hormone subunit beta; HC, healthy controls; HDAC5, histone deacetylase 5; IGF1, insulin-like growth factor; IGF1R, insulin-like growth factor 1 receptor; IGFBP1, insulin-like growth factor binding protein 1; IL6R, interleukin 6 receptor, JAK2,Janus kinase 2; LEPR, leptin receptor; LRP6, LDL receptor related protein 6; MAPK1, mitogen-activated protein kinase 1; MAPK3, mitogen-activated protein kinase 3; MCL, myeloid cell leukemia; MSCs, mesenchymal stem cells; NFATC1, nuclear factor of activated T cells 1; NFATC2, nuclear factor of activated T cells 2; NR3C1, nuclear receptor subfamily 3 group C member 1; OA, osteoarthritis; OP, osteoporosis; OSTF1, osteoclast stimulating factor 1; OSX, osterix; PDCD4, programmed cell death 4; PDGFD, platelet-derived growth factor D; PM, postmenopausal; PPARGC1A, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PTGER3, prostaglandin E receptor 3; PTHLH, parathyroid hormone like hormone; RANKL, receptor activator of nuclear factor k B ligand; RARG, retinoic acid receptor gamma; RT, room temperature; RT-qPCR, real-time quantitative polymerase chain reaction; RUNX2, runt-related transcription factor 2; RXRA, retinoid X receptor alpha; SATB2, SATB homeobox 2; SFRP2, secreted frizzled related protein 2; SGK, serine/threonine protein-kinase; SIRT1, sirtuin 1; SMAD5, SMAD family member 5; SMAD7, SMAD family member 7; SOST, sclerostin; SPARC, secreted protein acidic and cysteine rich; SPRY1, protein sprouty homolog 1; SRF, serum response factor; T2DM, Type 2 diabetes mellitus; TFR1, transferrin receptor protein 1; TRAP, triiodothyronine receptor auxiliary protein; TSC22D3, TSC22 domain family member 3; VCAN, versican; VDR, vitamin D receptor; WIF1, WNT inhibitory factor 1;WISP1, WNT1-inducible-signaling pathway protein 1.