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Introduction Tissue engineering technology has become one of the most promising therapeutic approaches for bone regeneration in bone defects (Zou et al., 2011; Ye et al., 2011; Xiao et al., 2011). As a source of mesenchymal stem cells (MSCs), human adipose-derived stem cells (hASCs) are receiving more attention in bone tissue engineering (Bosnakovski et al., 2005; Zuk et al., 2002; Wang et al., 2011). However, the paucity of available information about the molecular pathways that govern the osteogenic differentiation of hASCs has hampered further development of hASC-based cell therapies. MicroRNAs (miRNAs) are a class of endogenously expressed, small non-coding RNA molecules that negatively regulate gene expression at the post-transcriptional level by abiraterone acetate pairing with the 3′ UTR of their target mRNAs (Thomas et al., 2010). They play vital roles in various biological processes, including the cell fate of embryonic stem cells, cell proliferation, apoptosis, differentiation, morphogenesis, carcinogenesis, and angiogenesis (Ambros, 2004; Hua et al., 2006; Xu et al., 2004). A single miRNA is often involved in several gene regulatory networks (Bartel, 2004; Krek et al., 2005), and overexpression or inhibition of miRNAs can regulate the endogenous expression of multiple growth factors simultaneously (Yau et al., 2012). Therefore, we hypothesized that the delivery of a desired miRNA may result in optimization of bone regeneration. Recent studies have reported that several miRNAs, such as miR-22, -100, -106a, -146a, and -148b, are involved in the osteogenic differentiation of stem cells (Cho et al., 2010; Huang et al., 2012; Li et al., 2013a; Liao et al., 2014; Qureshi et al., 2013; Zeng et al., 2012). However, further regulatory mechanisms of miRNAs in the osteogenesis of MSCs still await investigation. Our previous study showed that the inhibition of retinoblastoma binding protein 2 (RBP2) significantly improved the in vitro and in vivo osteogenic capacity of hASCs (Ge et al., 2011). Based on these data, we aimed to screen and select miRNAs that positively regulate the osteogenic differentiation of hASCs by targeting RBP2. Microarray analyses revealed that after osteogenic induction, 21 miRNAs were upregulated in hASCs (Zhang et al., 2012) and 51 miRNAs were upregulated in bone marrow-derived MSCs (BMSCs) (Gao et al., 2011), suggesting that 72 upregulated miRNAs had potential effects on the osteogenic differentiation of MSCs. Moreover, RNA22 prediction software indicated that 122 miRNAs might bind to the 3′ UTR of RBP2 mRNA. These two categories of miRNAs were combined and an intersection of five miRNAs was produced: miR-663, -34a, -26a, -17, and -155. The RNA22 prediction software predicted their corresponding folding energy (ΔG) was −14.00 kcal/mol, −16.8 kcal/mol, −12.50 kcal/mol, −13.20 kcal/mol, and −13.30 kcal/mol. According to the results predicted by RNA22 prediction software, miR-34a possessed the maximum likelihood for binding to the 3′ UTR of RBP2 mRNA (ΔG = −16.8 kcal/mol); therefore, we selected miR-34a for further investigation (Figure S1). NOTCH1 and CYCLIN D1 are direct target genes of miR-34a in tumor cells (Hermeking, 2010; Pang et al., 2010), and have effects on the proliferation and osteogenic differentiation of MSCs by regulating runt-related transcription factor 2 (RUNX2) (Engin et al., 2008), a key osteogenesis-associated transcription factor. Thus, NOTCH1 and CYCLIN D1 pathways were integrated into our hypothetical regulatory network of miR-34a. In this study, we investigated the functional roles of miR-34a in the osteogenic differentiation of hASCs both in vitro and in vivo, and explored whether miR-34a regulated this biological process through the RBP2/NOTCH1/CYCLIN D1 coregulatory network. Our study provided a better understanding of the role and mechanism of miR-34a in hASCs\' osteogenic differentiation and suggested that miR-34a could be a therapeutic target in future bone regeneration therapy, which will lead to advances in clinical bone tissue engineering.