MicroRNA-29b Promote Osteogenic Differentiation Of Human Adipose-Derived Mesenchymal Stem Cells Via The PTEN/AKT/?-catenin Signaling Pathway

BackgroundHuman adipose-derived mesenchymal stem cells(h ADSCs)are self-renewing and multipotent cells which hold great promise in potential treatments for tissue regeneration.These cells differentiate along several committed phenotypes,including osteogenic lineages,in response to multiple environmental stimuli,using complex pathways regulated at both the transcriptional and posttranscriptional levels.However,the regulation of cellular osteogenic pathways is not fully elucidated.Investigation of the molecular mechanism of ADSC osteogenic differentiation could help explain the pathogenesis of such skeletal diseases as osteoporosis and lead to the development of possible regenerative therapies.ObjectivesTo investigate the effects of micro RNAs on the process of osteogenic differentiation of h ADSCs and explore the mechanism of miR-29 b in the regulation of osteogenic differentiation.Our findings provide novel insights into the mechanism underlying osteogenic differentiation and contribute to development of therapies for bone defect via targeting micro RNAs.Methods1.Adipose tissues of 3 samples were obtained by selective liposuction.hADSCs were then isolated,digested and cultured.HADSCs was subcultured into 6-well plate and cultured in osteoblast specific induction medium for 21 days to induce osteogenic differentiation.Osteogenic differentiation was detected by Alizarin Red staining,Alkaline phosphatase(ALP)staining.The expression of osteoblast related markers,Runx2 and bone sialic acid protein(BSP),osteopontin(OPN),osteopontin(ON)were detected by western blot and real-time fluorescence quantitative PCR(q RT-PCR).2.The micro RNA expression profile before and after the differentiation of h ADSCs was analyzed by microarray technique,and the micro RNAs that might be involved in the process were screened and the results of the chip were verified by real-time fluorescence quantitative PCR.3.The miR-29 b overexpression or low expression model was constructed by transfecting agmir-29 b or antagmir-29 b into h ADSCs,and the effects of miR-29 b on the differentiation of bone was examined to further confirm that the expression change of micro RNA was a possible mechanism for regulating h ADSCs osteogenic differentiation.4.The target gene of miR-29 b was predicted on the basis of previous studies,and the expression or low expression model was constructed by transfecting micro RNA mimics or inhibitor into h ADSCs,and the expression of protein of the target gene was detected to confirm the guesses.5.The expression of PTEN,phosphorylated Akt(p-Akt),p-GSK-3? and p-?-catenin was detected by Western blot.6.The PTEN overexpression model was constructed by using pc DNA plasmid.The h ADSCs,was co-transfected with agomiR-29 b and pc DNA-PTEN.Then,alizarin red staining and alkaline phosphatase staining,Western blot and q RT-PCR were used to observe the effects of PTEN overexpression on the function of miR-29 b in h ADSCs osteogenic differentiation.Results1.The results showed that the activity of ALP and the deposition of calcium salt in the early-forming bone index of the h ADSCs under the induction of the bone-specific induction medium were increased,and the expression of the bone markers Runx2,BSP,OPN and OCN were increased,and the promoting effect was time-dependent.2.The results of microarray analysis showed that totally 24 micro RNAs up-regulated whileas 14 down-regulated in comparison with non-differentiated cells.miR-29b-3p(miR-29b)was one of the most significantly upregulated micro RNAs,and we selected it for further study.q RT-PCR results confirmed that in the h ADSCs differentiation,the expression of miR-29 b was significantly up-regulated and this promoting effect was time-dependent.3.By alizarin red staining,ALP,detection of Runx2,BSP,OPN and OCN expression,we found that overexpression of miR-29 b promoted the activity of ALP and calcium salt deposition in stem cell early osteogenesis,and the osteogenesis index Runx2,BSP,OPN and OCN,indicating overexpression of miR-29 b promoted h ADSCs' osteogenic differentiation.On the other hand,silencing the expression of miR-29 b inhibited the differentiation process.4.Bioinformatics and double-luciferase assays show that PTEN is the target of miR-29 b.Overexpression of miR-29 b inhibits its expression while silencing of miR-29 b promotes its expression.Further studies show that PTEN is down-regulated during the differentiation of h ADSCs,and this inhibitory effect is time-dependent.5.The results of Western blot showed that the expression of phosphorylated Akt(p-Akt),p-GSK-3? and p-?-catenin in h ADSCs was significantly improved in h ADSCs,suggesting that the expression of miR-29 b in h ADSCs could activate the signal pathway of PTEN/AKT/?-catenin in h ADSCs.6.Further studies have shown that the expression of PTEN can reverse the promoting effects of miR-29 b in the process of h ADSCs osteogenic differentiation.Conclusions1.The expression of miR-29 b in h ADSCs is up-regulated in the process of bone differentiation,and its promoting effect is time-dependent,and the overexpression of miR-29 b can promote the h ADSCs osteogenic differentiation.2.miR-29 b promotes hADSCs osteogenic differentiation by targeting PTEN.3.The interaction of the miR-29 b with the PTEN/AKT/?-catenin signaling pathway constitutes a control loop that is involved in the regulation of osteogenic differentiation of h ADSCs.