micrornas ( mirnas ) are an extensive family of small ( 1824 nucleotides ) , single - stranded non - coding rnas , which regulate gene expression in mammalian cells through binding to the seed sequences of the 3-untranslated region ( utr ) of target mrna sequences and mediate the degradation of mrna in the rna - induced silencing complex ( 1,2 ) . each mirna regulates numerous mrnas , and mirnas are involved in various cellular processes , including proliferation , differentiation , cell cycle , invasion and apoptosis ( 36 ) . the alterations in their expression levels may lead to human diseases , including cardiovascular disease and cancer ( 710 ) . the formation of bone by osteoblast cells and their primary functional activities involve a series of multiple signals , including bone morphogenic protein ( bmp ) , wnt ligands , notch ligands , hormones and growth factors , including transforming growth factor ( tgf ) and tumor necrosis factor ( tnf ) and cytokines . in addition to these factors , tissue - specific transcription factors and co - factors mediate the expression of genes for the biosynthesis and mineralization of bone matrix , and the remodeling and formation of bone ( 1115 ) . mesenchymal stem cells ( mscs ) are induced to differentiate into preosteoblasts , which are regulated by the signaling cascades initiated by the various signals , including cytokine stimuli . pre - osteoblasts differentiate into osteoblasts , which is controlled by gene expression affected by chromatin modifiers , transcription factors or mirna alterations ( 16 ) . osteoblasts produce bone matrix , which mineralizes into bone tissue , and osteoblasts are engulfed in the matrix during bone formation . experimental evidence shows that mirnas are critical for stem cell activities , particularly in the stemness maintenance or direct differentiation into lineage cells ( 7,8 ) . to date , several studies have examined the functions of mirnas in mscs ( 17,18 ) . certain mirnas have been shown to regulate the process of differentiation of mscs into different cell lineages using high - through gene expression microprofiling assays . this has led to the specific targets of these mirnas being determined , and the roles of mirnas are gradually being elucidated ( fig . hupkes m et al reported that the expression of mirna regulated the terminal differentiation of c2c12 myoblasts via lineage - specific changes . it was found that the overexpression of mir-378 enhances alkaline phosphatase ( alp ) activity , calcium deposition and the mrna expression levels of osteogenic marker genes in the presence of bmp2 ( 21 ) . mir-322 , a bmp-2-downregulated mirna , is a regulator of osteoblast differentiation . using gain - and loss - of - function experiments of mir-322 in the c2c12 mouse myoblast cell line , mc3t3-e1 mouse embryonic osteoblasts and primary murine bone marrow - derived mscs , the overexpression of mir-322 has been demonstrated to enhance the bmp-2 response , increasing the expression of osteoblast - specific transcription factor ( osx ) and other osteogenic genes . transducer of erbb2 ( tob2 ) has been identified as a target of mir-322 , as reporter assays revealed that mir-322 binds to the specific sequence in the 3-utr of tob2 . tob2 is a negative regulator of osteogenesis , which binds to and mediates the degradation of osx mrna . this demonstrates a novel molecular mechanism controlling osteogenesis through the specific mir-322/tob2 regulation of specific target mrnas ( 22 ) . mir-196a is involved in the proliferation and osteogenic differentiation of human adipose - derived stem cells ( hadscs ) . the overexpression of mir-196a inhibits hasc proliferation and enhances osteogenic differentiation by targeting the 3utr sequence of homeobox ( hox)c8 mrna . the expression of hoxc8 in c2c12 cells decreases the alkaline phosphatase activity induced by bmp-2 . the expression of hoxc8 is decreased during the osteogenic differentiation of hadscs , and concomitant with an increase in the level of mir-196a . thus , inhibition of mir-196a decreases the protein levels of hoxc8 in hadscs , and is accompanied by increased osteogenic differentiation . this indicates that mir-196a has a positive effect in osteogenic differentiation in hadscs by repressing hoxc8 ( 23 ) . table i. wnt/-catenin signaling has been well defined in the osteogenic differentiation of mscs ( 2427 ) . mir-346 promotes the osteogenic differentiation of human bone marrow mscs ( hbmscs ) by targeting glycogen synthase kinase-3 ( gsk-3 ) through binding to the 3-utr of its mrna . the decreased gsk-3 results in an increase of -catenin , which is translocated into the nucleus and activates the downstream genes of the wnt/-catenin pathway . therefore , mir-346 positively regulates the osteogenic differentiation of hbmscs through the wnt/-catenin pathway ( 2831 ) . the expression of mir-27 is increased during hfob1.19 cell ( human sv40 transfected osteoblast ) differentiation . the ectopic expression of mir-27 promotes the differentiation of hfob1.19 cells by directly targeting and inhibiting the gene expression of adenomatous polyposis coli ( apc ) . this inhibition of the expression of apc leads to the accumulation of -catenin , which is a key protein activating wnt signaling . mir-218 positively regulates the osteogenesis of hadsc , directly targeting secreted frizzled - related protein 2 and dickkopf wnt signaling pathway inhibitor 2 , thus enhancing wnt/-catenin signaling progression . mimics of the wnt/-catenin signal increase the expression of mir-218 , which forms a positive feedback loop to promote osteogenesis ( 33 ) . the activation of notch signaling in apical papilla stem cells ( scaps ) inhibits cell differentiation . high expression levels of mir-34a inhibits notch signaling by directly targeting the 3utr of notch2 and hes1 mrna then suppressing the binding of notch2 , and hes1 , then the inhibition of notch signaling pathway , resulting in the expression of runt - related transcription factor 2 ( runx2 ) and osx , and enhancing the expression of osteocalcin ( ocn ) . mir-34a promotes osteogenesis in scaps through notch signaling by targeting notch 2 and hes1 ( 39 ) . mir-10a mimics significantly repress luciferase activity by the direct binding to the 3-utr of kruppel - like factor 4 ( klf4 ) . the inhibition of klf4 in hbmscs increases cell differentiation . therefore , mir-10a enhances the differentiation capability of hbmscs through the repression of klf4 ( 40 ) . mir-21 has been confirmed to promote the osteogenic differentiation of mouse bone marrow cells by targeting sprouty homolog 1 ( spry1 ) , negatively regulating the osteogenic differentiation of mscs . inhibition of the tnf- signaling pathway evidently improves bone formation and down - regulates spry1 expression , which suggests that mir-21 contributes towards bone formation through targeting spry1 in mscs ( 41 ) . mir-96 and mir-199a were both up - regulated during osteogenic induction of human bone marrow derived mscs . they may function through transcription factor sry - box 9 , and fatty acid binding protein 4 , however , the detailed molecular mechanism remains poorly understood ( 42 ) . mir-22 inhibits the expression of histone deacetylase 6 ( hdac6 ) by binding to the similar sequence of the 3-utr ( 43 ) . mir-29b is understood to promote osteogenesis by supressing the negative regulators , including histone deacetylase 4 , tgf-3 , activin a receptor ( type iia ) , catenin beta interacting protein 1 and dual specificity phosphatase 2 , during the induction of osteogenic differentiation . these anti - osteogenic factors negatively modulate extracellular matrix excretion by differentiated osteoblasts and bone formation ( 44,45 ) . hupkes m et al reported that the expression of mirna regulated the terminal differentiation of c2c12 myoblasts via lineage - specific changes . it was found that the overexpression of mir-378 enhances alkaline phosphatase ( alp ) activity , calcium deposition and the mrna expression levels of osteogenic marker genes in the presence of bmp2 ( 21 ) . mir-322 , a bmp-2-downregulated mirna , is a regulator of osteoblast differentiation . using gain - and loss - of - function experiments of mir-322 in the c2c12 mouse myoblast cell line , mc3t3-e1 mouse embryonic osteoblasts and primary murine bone marrow - derived mscs , the overexpression of mir-322 has been demonstrated to enhance the bmp-2 response , increasing the expression of osteoblast - specific transcription factor ( osx ) and other osteogenic genes . transducer of erbb2 ( tob2 ) has been identified as a target of mir-322 , as reporter assays revealed that mir-322 binds to the specific sequence in the 3-utr of tob2 . tob2 is a negative regulator of osteogenesis , which binds to and mediates the degradation of osx mrna . this demonstrates a novel molecular mechanism controlling osteogenesis through the specific mir-322/tob2 regulation of specific target mrnas ( 22 ) . mir-196a is involved in the proliferation and osteogenic differentiation of human adipose - derived stem cells ( hadscs ) . the overexpression of mir-196a inhibits hasc proliferation and enhances osteogenic differentiation by targeting the 3utr sequence of homeobox ( hox)c8 mrna . the expression of hoxc8 in c2c12 cells decreases the alkaline phosphatase activity induced by bmp-2 . the expression of hoxc8 is decreased during the osteogenic differentiation of hadscs , and concomitant with an increase in the level of mir-196a . thus , inhibition of mir-196a decreases the protein levels of hoxc8 in hadscs , and is accompanied by increased osteogenic differentiation . this indicates that mir-196a has a positive effect in osteogenic differentiation in hadscs by repressing hoxc8 ( 23 ) . wnt/-catenin signaling has been well defined in the osteogenic differentiation of mscs ( 2427 ) . mir-346 promotes the osteogenic differentiation of human bone marrow mscs ( hbmscs ) by targeting glycogen synthase kinase-3 ( gsk-3 ) through binding to the 3-utr of its mrna . the decreased gsk-3 results in an increase of -catenin , which is translocated into the nucleus and activates the downstream genes of the wnt/-catenin pathway . therefore , mir-346 positively regulates the osteogenic differentiation of hbmscs through the wnt/-catenin pathway ( 2831 ) . the expression of mir-27 is increased during hfob1.19 cell ( human sv40 transfected osteoblast ) differentiation . the ectopic expression of mir-27 promotes the differentiation of hfob1.19 cells by directly targeting and inhibiting the gene expression of adenomatous polyposis coli ( apc ) . this inhibition of the expression of apc leads to the accumulation of -catenin , which is a key protein activating wnt signaling . mir-218 positively regulates the osteogenesis of hadsc , directly targeting secreted frizzled - related protein 2 and dickkopf wnt signaling pathway inhibitor 2 , thus enhancing wnt/-catenin signaling progression . mimics of the wnt/-catenin signal increase the expression of mir-218 , which forms a positive feedback loop to promote osteogenesis ( 33 ) . the activation of notch signaling in apical papilla stem cells ( scaps ) inhibits cell differentiation . high expression levels of mir-34a inhibits notch signaling by directly targeting the 3utr of notch2 and hes1 mrna then suppressing the binding of notch2 , and hes1 , then the inhibition of notch signaling pathway , resulting in the expression of runt - related transcription factor 2 ( runx2 ) and osx , and enhancing the expression of osteocalcin ( ocn ) . mir-34a promotes osteogenesis in scaps through notch signaling by targeting notch 2 and hes1 ( 39 ) . mir-10a mimics significantly repress luciferase activity by the direct binding to the 3-utr of kruppel - like factor 4 ( klf4 ) . the inhibition of klf4 in hbmscs increases cell differentiation . therefore , mir-10a enhances the differentiation capability of hbmscs through the repression of klf4 ( 40 ) . mir-21 has been confirmed to promote the osteogenic differentiation of mouse bone marrow cells by targeting sprouty homolog 1 ( spry1 ) , negatively regulating the osteogenic differentiation of mscs . inhibition of the tnf- signaling pathway evidently improves bone formation and down - regulates spry1 expression , which suggests that mir-21 contributes towards bone formation through targeting spry1 in mscs ( 41 ) . mir-96 and mir-199a were both up - regulated during osteogenic induction of human bone marrow derived mscs . they may function through transcription factor sry - box 9 , and fatty acid binding protein 4 , however , the detailed molecular mechanism remains poorly understood ( 42 ) . mir-22 inhibits the expression of histone deacetylase 6 ( hdac6 ) by binding to the similar sequence of the 3-utr ( 43 ) . mir-29b is understood to promote osteogenesis by supressing the negative regulators , including histone deacetylase 4 , tgf-3 , activin a receptor ( type iia ) , catenin beta interacting protein 1 and dual specificity phosphatase 2 , during the induction of osteogenic differentiation . these anti - osteogenic factors negatively modulate extracellular matrix excretion by differentiated osteoblasts and bone formation ( 44,45 ) . several mirnas directly target transcription factors , including osteopotin ( opn ) , ocn and runx2 , or signaling molecules , which promote the osteogenesis of mscs and inhibit osteogenic differentiation ( fig . 3 and table i ) . mir-140 - 5p inhibits the osteogenic differentiation in hmscs by directly blocking bmp2 , and consequently blocking bmp signaling components and critical regulators ( 46 ) . the overexpression of mir-31 represses the osteogenesis of hmscs by directly targeting special at - rich sequence - binding protein 2 ( satb2 ) , the knockdown of satb2 by specific sirna against satb2 inhibits osteogenic differentiation ( 47 ) . baglio et al found an inverse trend in mirna - target expression during osteogenic differentiation between the levels of mir-31 and osx . the inhibition of mir-31 leads to an increase in the endogenous expression of osx ( 48 ) . deng et al showed that the expression of mir-31 decreased progressively in bmsc cultures during differentiation . the upregulation of mir-31 significantly reduces the expression levels of osteogenic transcription factors , opn , bone sialoprotein , osx and ocn . these results suggest that an mir-31/satb2 axis is involved in the osteogenic differentiation of bmscs ( 49 ) . the overexpression of mir-30e stimulates adipocyte formation and inhibits osteoblast differentiation from marrow stromal cells . low - density lipoprotein receptor - related protein 6 ( lrp6 ) is one of the critical co - receptors for wnts . mir-30 has been reported to target the lrp6 directly and inhibit the expression of lrp6 expression . thus , mir-30e also controls osteogenesis in periodontal ligament stem cell ( pdlscs ) by targeting lrp6 and affecting canonical wnt/-catenin signaling ( 50 ) . there is also evidence that mir-30 family members negatively regulate bmp-2-induced osteoblast differentiation by targeting small mothers against decapentaplegic ( smad)1 and runx2 . as smad1 and runx2 are key positive transcription factors in osteogenic differentiation , mir-30 family members negatively regulate the osteogenic differentiation through smad1 and runx2 ( 51 ) . liu et al ( 52 ) elucidated that transcription factor 3 ( tcf3 ) enhances osteogenesis in pdlscs . furthermore , the overexpression of tcf3 blocks the effect of mir-17 on the modulation of wnt signaling ( 52 ) . inflammation promotes the downregulation of mir-17 and the subsequent increased expression of smad ubiquitin regulatory factor 1 ( smurf1 ) , which is an important negative regulator of osteogenic differentiation in mscs by promoting the degradation of smurf1-mediated osteoblast - specific factors . smurf1 has been confirmed as a direct target of mir-17 in pdlscs using a luciferase reporter assay ( 52 ) . li h et al revealed that mir-17 - 5p and mir-106a have dual functions in hadsc fate , which can promote adipogenesis and inhibit osteogenesis . the inhibitory effects of mir-17 - 5p and mir-106a are due to directly targeting bmp2 , and subsequently decreased osteogenic taz , msx2 and runx2 protein levels ( 53 ) . during osteoblastic differentiation , the overexpression of estrogen - related receptor  ( err ) or mir-433 inhibits the expression levels of osteogenic marker genes including runx2 and alp . mir-433 directly targets three binding sites on the 3-utr of runx2 mrna , and decreases the levels of the runx2 transcript in c3h10t1/2 cells . anti - mir-433 recovers the err-suppressed expression of runx2 and activity of alp . err is able to upregulate the levels of mir-433 expression and further enhance the inhibitory role in osteogenic differentiation . this evidence demonstrates that mir-433 suppresses bmp2-induced osteogenic differentiation by targeting runx2 c3h10t1/2 cells ( 54 ) . mir-486 - 5p regulates the expression of silent information regulator 1 ( sirt1 ) , a major regulator of longevity and metabolic disorders . sirt1 also has an important role in the osteogenic process by targeting foxo3a and then upregulating the levels of runx2 promoter activity . mir-486 - 5p inhibits the expression of sirt1 through binding to the 3-utr region of sirt1 mrna ( 55 ) . the overexpression of mir-335 in hmscs derived from bone marrow , adipose tissue and articular cartilage inhibits their osteogenic and adipogenic potential . the expression of mir-335 in hmscs is upregulated by the activated canonical wnt signaling pathway . these results suggest that the downregulation of mir-335 is critical for the acquisition of msc phenotype differentiation into osteoblasts ( 56 ) . the overexpression of mir-135b downregulates the osteogenic differentiation of unrestricted somatic stem cells through targeting the key osteogenic factors , integrin - binding sialoprotein and osx , inhibiting the process of osteogenesis ( 57 ) . there is substantial evidence and experimental data confirming that mirnas have multi - dimensional roles in the induction of mscs into osteoblasts . mirnas function at all stages of osteoblast differentiation by inhibiting the negative regulators of signaling pathways operating in these cells ( 16 ) . they also have direct and indirect effects on phenotype development through the promotion or inhibition of positive or negative transcription factors in signaling pathways , which are involved in complex regulatory networks . this indicates an important technique , by which the network of osteogenic differentiation can be regulated through mirnas . nicotine can alter the expression of mirna and reduce human adult stem cell regenerative potential , demonstrating that mirnas function in the network ( 58,59 ) . currently , only a limited number of mirnas , identified through in vitro expression profiling of bone cells undergoing differentiation programs , have been characterized for their functional activity and known targets . numerous mirnas are associated with bone development , which can be utilized in new bone formation and the therapy of osteoporosis or bone malignant diseases . however , the crosstalk of multiple types of molecules in the regulatory network of the osteogenic differentiation process is not well - defined . lncrna and circular rnas which are emerging as novel regulators in the cellular processes , including osteogenesis , require further investigation ( 60 ) .
osteogenesis is a complex multi - step process involving the differentiation of mesenchymal stem cells ( mscs ) into osteoblast progenitor cells , preosteoblasts , osteoblasts and osteocytes , and the crosstalk between multiple cell types for the formation and remodeling of bone . 
 the signaling regulatory networks during osteogenesis include various components , including growth factors , transcription factors , micro ( mi)rnas and effectors , a number of which form feedback loops controlling the balance of osteogenic differentiation by positive or negative regulation . 
 mirnas have been found to be important regulators of osteogenic signaling pathways in multiple aspects and multiple signaling pathways . 
 the present review focusses on the progress in elucidating the role of mirna in the osteogenesis signaling networks of mscs as a substitute for bone implantation the the field of bone tissue engineering . 
 in particular , the review classifies which mirnas promote or suppress the osteogenic process , and summarizes which signaling pathway these mirnas are involved in . 
 improvements in knowledge of the characteristics of mirnas in osteogenesis provide an important step for their application in translational investigations of bone tissue engineering and bone disease .