Table 2.

MiRNAs identified in osteogenic differentiation of stem cells, target mRNA, effect of miRNA modulation on osteogenesis and potential use of identified miRNAs to enhance osteogenic differentiation

MiRNA	Expression during osteogenesis	mRNA targets of miRNA and mRNA function in osteogenesis	Reported effect of miRNA modulation on osteogenesis	Potential use of miRNA in inducing osteogenesis
miR-23a	miR-23a was reported to be down-regulated during osteogenic differentiation of human BMSCs (hBMSCs) [81].	miR-23a was demonstrated to directly target the 3’UTR of LRP5; an essential component of the Wnt signalling pathway. miR-23a is likely to be down-regulated during osteogenesis, enabling derepression of LRP5 expression, subsequently enabling Wnt signalling to direct osteogenesis [81].	Overexpression of miR-23a, using miR-23a mimic, resulted in the down-regulation of osteogenic differentiation, evidenced by the down-regulation of ALP, OPN, RUNX2 and IBSP mRNA. Inhibition of endogenous miR-23a, using anti-miR-23a, resulted in enhancement of osteogenic differentiation, evidenced by the up-regulation of ALP, OPN, RUNX2 and IBSP mRNA [81].	Decrease endogenous miR-23a levels with a miR-23a inhibitor.
miR-29a	miR-29a was reported to be up-regulated during the osteogenic differentiation of hFOB1.19 cells [82].	miR-29a was demonstrated to directly target the 3’UTRs of negative regulators of Wnt signalling: Dkk1, Kremen2 and sFRP2 [82]. miR-29a is likely to be up-regulated during osteogenesis to inhibit the negative regulators of Wnt signalling, indirectly promoting osteogenic differentiation.	Inhibition of endogenous miR-29a during osteogenic differentiation of hFOB1.19 cells, using miR-29a inhibitor, resulted in down-regulation of osteogenic differentiation, evidenced by down-regulation of OCN and ALP mRNA. Overexpression of miR-29a, using miR-29a mimic, resulted in the up-regulation of OCN mRNA [82].	Increase miR-29a levels with a miR-29a mimic.
miR-34a	miR-34a was reported to be up-regulated during osteogenic differentiation of hBMSC; however, miR-34a was found to negatively regulate differentiation [83••].	miR-34a was observed to target JAG1 and cell cycle regulators CDK4 and CDK6 [83••].	Inhibition of endogenous miR-34a, using anti-miR-34a, was found to enhance osteogenic differentiation. hBMSCs transfected with anti-miR34a were subcutaneously implanted in mice, with the support of a scaffold. In this in vivo model of bone regeneration, an increase in bone formation was observed in mice which had received miR-34a silenced hBMSCs [83••].	Decrease endogenous miR-34a levels with a miR-34a inhibitor.
miR-138	miR-138 was reported to be down-regulated during osteogenic differentiation of human MSCs (hMSCs) [84].	miR-138 was demonstrated to directly target the 3’UTR of PTK2, which encodes focal adhesion kinase (FAK) [84]. During osteoblast differentiation is it thought that Grb2-Sos-Ras pathway is activated by FAK inducing ERK1/2, and subsequently downstream genes associated with osteogenesis [85].	Inhibition of endogenous miR-138 in hMSCs, using anti-miR-138, resulted in enhanced osteogenic differentiation as measured by an increase in OCN and ALP mRNA levels and matrix mineralisation. Overexpression of miR-138, using pre-miR-138, was found to reduce osteogenic differentiation. miR-138 silenced hMSCs loaded onto a scaffold and implanted subcutaneously in mice, resulted in an increase in bone formation and up-regulation OCN and ALP mRNA [84].	Decrease endogenous miR-138 levels with a miR-138 inhibitor.
miR-146a	miR-146a was reported to be up-regulated in osteogenic diaphyseal cell populations isolated from the diaphysis of human foetal femora [72•].	miR-146a targets positive regulators of chondrogenesis, SMAD2 and SMAD3. Indirect promotion of osteogenic differentiation is suggested via miR-146a degradation of SMAD2 and SMAD3, during endochondral foetal skeletogenesis [72•].	miR-146a overexpression, using miR-146a mimic, resulted in down-regulation of SMAD2 and SMAD3 and an increase in RUNX2 mRNA, a marker of osteogenic differentiation [72•].	Increase miR-146a levels with a miR-146a mimic.
miR-218	miR-218 was reported to be up-regulated in MC3T3 cells [86] and hADSCs [87•] during osteogenic differentiation.	miR-218 was demonstrated to directly target the 3’UTR of negative regulators of Wnt signalling, SFRP2 and DKK1 [86].	Overexpression of miR-218 in murine BMSCs, using miR-210 lentivirus, resulted in enhanced osteogenic differentiation, evidenced by up-regulation of Runx2, Alp and Ocn mRNA [81]. Inhibition of miR-218 was found to reduce osteogenic differentiation of hADSCs [87•].	Increase miR-218 levels with a miR-218 mimic.
miR-346	miR-346 was reported to be up-regulated during osteogenic differentiation of hBMSCs [88].	miR-346 was demonstrated to directly target the 3’UTR of GSK-3β, a negative regulator of Wnt signalling. An increase in nuclear accumulation of β-catenin was observed during miR-346 overexpression, indicating an enhancement of Wnt signalling. miR-346 was suggested to promote osteogenesis through activation of the Wnt signalling pathway [88]. miR-346 is likely to be up-regulated during osteogenesis to inhibit the negative regulators of Wnt signalling, indirectly promoting osteogenic differentiation.	Overexpression of miR-346 in hBMSCs, using miR-346 mimic, resulted in enhanced osteogenic differentiation, evidenced by up-regulation of RUNX2, ALP and OPN mRNA and increased matrix mineralisation and ALP activity. Inhibition of endogenous miR-346, using anti-miR-346, resulted in the down-regulation osteogenic differentiation, evidenced by down-regulation of osteogenic marker mRNA expression, ALP activity and matrix mineralisation [88].	Increase miR-346 levels with a miR-346 mimic.
miR-637	miR-637 was reported to be down-regulated during osteogenic differentiation of hMSCs [89].	miR-637 was demonstrated to directly target the 3’UTR of OSX, which encodes osterix, a key transcription factor of osteoblasts. Down-regulation of miR-637 during osteogenesis results in osterix derepression, promoting osteogenic differentiation [89].	Overexpression of miR-637 in hMSCs, using a lentiviral- pre-miR-673 vector, down-regulated osteogenic differentiation, evidenced by a decrease in ALP activity and down-regulation of RUNX2 and BMP2 mRNA. Inhibition of endogenous miR637, using a lentiviral short-hairpin of pre-miR-637 vector, enhanced osteogenic differentiation, evidenced by an increase in ALP activity and up-regulation of RUNX2 and BMP2 mRNA [89].	Decrease endogenous miR-637 levels with a miR-637 inhibitor.
miR-2861	miR-2861 was reported to be up-regulated in BMP2-induced osteogenesis of murine BMSCs [90].	miR-2861 was demonstrated to directly target an amino acid coding sequence in Hdac4 mRNA [90]. miR-2861 is likely to be up-regulated during osteogenesis to suppress expression of the negative regulator of Runx2, hdac4.	Overexpression of miR-2861 in mice BMSCs, using pre-miR-2861, resulted in enhanced osteogenic differentiation, evidenced by increased ALP activity and osteocalcin secretion and up-regulation of Runx2. An in vivo model reported that injection of anti-miR-2861 in mice resulted in a reduction in femur bone mineral density and reduced osteoblast activity [90].	Increase miR-2861 levels with a miR-2861 mimic.