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. 2020 Apr 11;10(4):589. doi: 10.3390/biom10040589

Table 2.

Circulating miRNAs in OP fracture.

Study Population Variables in Pre-Analytical Phase:
- Sample matrix
- Centrifugation conditions
- Storage conditions		 Quantification Method
- Target miRNAs
- Analytical method
- Applied normalization strategy		 - Validated miRNAs
- AUC; sensitivity; specificity		 Reference
Screening Screening
Screening: OP (3 men + 7 women) and non-OP (10 women) both with pertrochanteric or femoral neck fracture
Validation: OP (30 women) and non-OP (30 women) both with pertrochanteric or femoral neck fracture		 - Serum
- n.d.
- n.d.		 - 83 miRNAs
- Human Serum & Plasma miRNA PCR Array MIHS-106Z
- snRNU6 and SNORD96a 		- miR-637, miR-223-3p, miR-148a-3p, miR-125b-5p, miR-124-3p, miR-122a-5p, miR-100-5p, miR-93, miR-27a-3p, miR-25-3p, miR-24-3p, miR-23a-3p, and miR-21-5p
- RT-qPCR
- snRNU6 and SNORD96a 		- ↑ miR-148a, miR-125b, miR-124a, miR-122a, miR-100, miR-93, miR-24, miR-23a, and miR-21 in fractured OP vs. fractured non-OP
- 0.61; 62.5%; 62.3% (miR-148a),
0.76; 76.4%; 75.0% (miR-125b),
0.69; 61.4%; 61.0% (miR-124a),
0.77; 74.1%; 72.1% (miR-122a),
0.69; 62.9%; 61.7% (miR-100),
0.68; 69.0%; 68.3% (miR-93),
0.63; 60.3%; 60.4% (miR-24),
0.63; 57.4%; 56.7% (miR-23a),
0.63; 61.3%; 61.7% (miR-21)		 [87]
OP with femoral neck fracture (7 PM women + 7 men), and non-OP (7 PM women + 7 men) - Serum, left at RT for 30 min
- 1900g, 10 min
- −80 °C		 - miR-148a-3p, miR-125b-5p, miR-124-3p, miR-122-5p, miR-100-5p, miR-93-5p, miR-24-3p, miR-23a-3p, and miR-21-5p
- RT-qPCR
- SNORD96a 		- ↑ miR-148a-3p, miR-125b-5p, miR-124-3p, miR-122-5p, miR-100-5p, miR-93-5p, miR-24-3p, miR-23a-3p, and miR-21-5p in PM women with OP fracture vs. non-OP PM women;
↑ miR-148a-3p, miR-124-3p, miR-122-5p, miR-100-5p, miR-93-5p, miR-24-3p, miR-23a-3p, and miR-21-5p in men with OP fracture vs. non-OP men
- n.d.		 [88]
Screening: Caucasian women with hip OA (n = 5) or with OP subcapital hip fracture (n = 8)
Validation: Caucasian women with hip OA (n = 12) or with OP subcapital hip fracture (n = 15)		 - Serum
- n.d.
- −80 °C		 - 179 miRNAs
- The miRCURY LNA Universal RT microRNA PCR, Serum/Plasma Focus microRNA PCR Panel
- A logarithmic transformation (log2)		 - miR-210, miR-143-3p, miR-125b-5p, miR-122-5p, miR-34a-5p, and miR-21-5p
- RT-qPCR
- miR-93-5p obtained from the GeNorm algorithm		 - ↑ miR-125b-5p, miR-122-5p, and miR-21-5p in fractured OP vs. control
- 0.76 (miR-125b-5p),
0.87 (miR-122-5p),
0.87 (miR-21-5p)		 [89]
Screening: PM Caucasian women with femoral neck OP fracture (n = 7) and without femoral fractures (n = 7)
Validation: PM Caucasian women with femoral neck OP fracture (n = 12) and without femoral fractures (n = 11) 		- Serum, left for 30 min at RT
- 2000g, RT, 15 min
- −80 °C		 - 175 miRNAs
- The Exiqon serum/plasma focus panels
- The average Cp of the detected miRNAs		 - miR-328-3p, miR 133b, miR-22-3p, miR-10a-5p, miR-10b-5p, and let-7g-5p
- RT-qPCR
- The average Cp of the detected miRNAs		 - ↓ miR-328-3p, miR-22-3p, and let-7g-5p in fractured OP vs. non-fractured
- n.d.		 [90]
PM Chinese women with OP (n = 30) and hip fractures or without OP (n = 30) - Serum
- 1500g
- n.d.		 - miR-5914, miR-4665-3p, miR-125b, miR-96, and miR-30
- RT-qPCR
- snRNU6		 - ↑ miR-5914, miR-125b, and miR-30 in fractured OP vs. controls
- 0.70 (miR-5914),
0.90 (miR-125b),
0.76 (miR-30)		 [91]
Healthy PM women (n = 30), PM women with low bone mass and vertebral fractures (n = 35) or with low bone mass but without vertebral fractures (n = 35) - Serum, left for 10–60 min at RT
- (i) 1900g, 4 °C, 10 min; (ii) 16,000g, 4 °C, 10 min
- −80 °C		 - miR-2861, miR-422a, miR-335-5p, miR-218-5p, miR-214-3p, miR-135b-5p, miR-133a-3p, miR-124-3p, miR-33a-5p, miR-29a-3p, miR-26a-5p, miR-24-2-5p, miR-23a-3p, and miR-21-5p
- RT-qPCR
- SNORD95, SNORD96A, and snRNU6-2		 - ↓ miR-21-5p in fractured OP vs. non-fractured OP;
↑ miR-2861, miR-124-3p, and ↓ miR-29a-3p, miR-23a-3p, miR-21-5p in OP vs. controls
- 0.66-66%-71% (miR-21-5p) for fractured OP		 [92]
OP (n = 45) and non-OP (n = 15) both with femoral fractures - Serum
- n.d.
- n.d.		 - miR-211-5p, miR-145-5p, miR-144-3p, miR-128, miR-125b, miR-122a, miR-100, miR-27a-3p, miR-24-3p, and miR-7-5p
- RT-qPCR
- snRNU6		 -↑ miR-145, miR-122a, miR-125b, miR-100, miR-27a-3p, miR-24-3p and ↓ miR-144-3p in fractured OP vs. fractured non-OP
- n.d.		 [93]
PM Chinese OP women (n = 10) with hip fractures and HC (n = 10) - Serum
- 1500g
- n.d.		 - miR-133a
- RT-qPCR
- snRNU6		 - ↑ miR-133a in fractured OP vs. HC
- n.d.		 [94]
PM women: HC (n = 42), with low BMD and no fractures (n = 39), with low BMD and vertebral fractures without OP treatment (n = 26), with low BMD and vertebral fractures receiving OP treatment (n = 19) - Serum
- n.d.
- −80 °C		 - miR-550a-3p, miR-532-3p, miR-486-3p, miR-451a, miR-375, miR-335-5p, miR-214-3p, miR-188-5p, miR-152-3p, miR-144-3p, miR-143-3p, miR-133b, miR-127-3p, miR-106b-5p, miR-96-5p, miR-30e-5p, miR-29b-3p, miR-23a-3p, miR-21-5p, miR-19b-3p
- RT-qPCR
- UniSp4		 - ↑ miR-532-3p, miR-486-3p, miR-375, miR-335-5p, miR-152-3p, miR-143-3p, miR-133b, miR-106b-5p, miR-30e-5p, miR-23a-3p, miR-21-5p, miR-19b-3p in low BMD+ vertebral fractures without OP treatment vs. HC;
↑ miR-532-3p, miR-375, miR-335-5p, miR-152-3p, miR-133b, miR-127-3p, miR-23a-3p, miR-21-5p, miR-19b-3p in low BMD+ vertebral fractures+OP treatment vs. HC;
↑ miR-550a-3p, miR-532-3p, miR-486-3p, miR-375, miR-335-5p, miR-214-3p, miR-152-3p, miR-143-3p, miR-127-3p, miR-106b-5p, miR-30e-5p, miR-23a-3p, miR-21-5p, miR-19b-3p in low BMD+ vertebral fractures without OP treatment vs. low BMD;
↑ miR-550a-3p, miR-532-3p, miR-486-3p, miR-375, miR-335-5p, miR-214-3p, miR-152-3p, miR-127-3p, miR-30e-5p, miR-23a-3p, miR-21-5p, miR-19b-3p in low BMD+ vertebral fractures+OP treatment vs. low BMD
- n.d.		 [95]
PM women (n = 10), premenopausal women (n = 10), and men (n = 16) with PM or idiopathic OP low traumatic fractures;
PM women (n = 11), premenopausal women (n = 12), and men (n = 16) without low-traumatic fractures 		- Serum
- n.d.
- −80 °C		 - 187 miRNAs
- RT-qPCR
- Global mean		 - ↑ miR-335-5p, miR-320a, miR-152-3p and ↓ miR-550a-3p, miR-532-5p, miR-378a-5p, miR-365a-3p, miR-324-3p, miR-186-5p, miR-215-5p, miR-140-5p, miR-93-5p, miR-30e-5p, miR-29b-3p, miR-19a-3p, miR-19b-3p, miR-16-5p, miR-7-5p, and let-7b-5p in fractured groups vs. controls
- 0.94 (miR-335-5p), 0.87 (miR-320a), 0.96 (miR-152-3p),0.91 (miR-550a-3p), 0.90 (miR-532-5p), 0.87 (miR-378a-5p), 0.81 (miR-365a-3p), 0.95 (miR-324-3p), 0.90 (miR-186-5p), 0.85 (miR-215-5p), 0.95 (miR-140-5p), 0.88 (miR-93-5p), 0.96 (miR-30e-5p), 0.84 (miR-29b-3p), 0.93 (miR-19a-3p), 0.94 (miR-19b-3p), 0.86 (miR-16-5p), 0.82 (miR-7-5p), 0.85 (let-7b-5p) 		[97]
Non-OP (1 man + 11 women), osteopenia without fractures (9 men + 52 women), osteopenia with fractures (2 men + 13 women), OP without fractures (6 men + 27 women), and OP with fractures (2 men + 16 women) - Serum or plasma
- (i) 30 min, 2500g, RT; (ii) 30 min, 14,000g, 4 °C
- −80 °C		 - 370 miRNAs
- Serum and Plasma miRNA PCR arrays
- SNORD96A and RNU6-6P		 - 40 miRNAs
- RT-qPCR
- SNORD96A and RNU6-6P		 - ↓ plasma miR-4516 and serum miR-122-5p in OP vs. non-OP and osteopenia;
↓ plasma miR-4516 in OP with fracture;
- 0.75 (miR-122-5p+miR-4516 panel) for OP,
0.73; 71%; 62% (plasma miR-4516) for OP		 [99]
Screening: HC (4 women + 2 men), OP (5 women + 1 man) without vertebral fracture, OP (6 women) with vertebral fracture
Validation: HC (14 women + 10 men), OP (23 women + 1 man) without vertebral fracture, OP (23 women + 1 man) with vertebral fracture		 - Plasma
- n.d.
- n.d.		 - 384 miRNAs
- Microarray
- n.d.		 - miR-19b
- RT-qPCR
- snRNU6		 - ↓ miR-19b in OP with or without vertebral fracture vs. HC
- 0.95; 95.0%; 85.4% for fracture 0.93; 91.3%; 80.5% for non-fracture (miR-19b)		 [100]
T2DM women with (n = 20) and without (n = 20) fragility fractures since the T2DM onset; non-T2DM PM women with (n = 20) or without (n = 20) OP fragility fracture - Serum
- 2000g, 15 min
- n.d.		 - 375 miRNAs
- SYBR Green Low-density qPCR platform
- Cq values were computed using the second derivative maximum method 		- 10 candidate four-miRNA models for T2DM fracture status;
10 candidate four-miRNA models for OP fracture status
- AUC in a range of 0.92-0.97 for T2DM fracture; AUC in a range of 0.97-0.99 for OP fracture		 [101]

n.d.: not determined; AUC: Area Under the Curve; HC: Healthy Controls; OA: Osteoarthritis; OP: Osteoporosis; PM: Postmenopausal; RT: Room Temperature; RT-qPCR: Real-time quantitative polymerase chain reaction; T2DM: Type 2 diabetes mellitus. ↑ increased levels of the validated circulating miRNA; ↓: decreased levels of the validated circulating miRNA.