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  • br Materials and methods br Data analysis

    2018-10-24


    Materials and methods
    Data analysis All results are presented as means±SEM. Comparisons between groups were analyzed using t-tests (two-sided). Differences with p values of less than 0.05 were considered significant. The p values were corrected for multiple testing procedures and to control type I error rates.
    Results
    Discussion Osteogenesis and adipogenesis of MSCs maintain a homeostasis under physiological conditions. Once the homeostasis of osteogenic and adipogenic differentiation of MSCs are disrupted, disorders such as osteoporosis may occur. But what factors are involved and how they orchestrate to regulate the specification of cell fate remain elusive. Recently, Scheideler et al. (2008) found that gene repression to be most prevalent prior to commitment in both lineages, and computational analysis suggested that gene repression before commitment of MSCs is mediated by miRNAs. MiR-17 family is composed of six miRNAs (miR-17-5p, -20a, -106a, -20b, 106b, and -93) which are derived from miR-17-92 cluster and its two paralogues (Mendell, 2008), and have same ‘seed sequence’ that has been verified as an important element for mRNA targeting (Lim et al., 2003; Ye et al., 2008). Recently, several members of the miR-17 family have been identified to be specifically expressed in either undifferentiated or differentiating embryonic stem GSK461364 (Houbaviy et al., 2003; Suh et al., 2004; Tang et al., 2006). These miRNAs were also found to play an important role in early mammalian development, hematopoietic and adipocytic differentiation (Foshay and Gallicano, 2009; Garzon et al., 2006; Ventura et al., 2008; Wang et al., 2008). More recently, miR-20a was reported to be a regulator of osteogenic differentiation of human MSCs by co-regulating BMP signaling (Zhang et al., 2011c). Our data showed that other two important members of miR-17 family, miR-17-5p and miR-106a, can suppress osteogenic differentiation and promote adipogenic differentiation of hADSCs. To elucidate the mechanisms underlying the actions of miR-17-5p and miR-106a on hADSCs differentiation, we detected the expression of osteoblastic and adipocytic related genes during hADSCs differentiation and found that the expression level of BMP2, TAZ and MSX2 mRNA peaked after 4days of osteogenic induction and then gradually decreased, which is consistent with the recent finding that BMP2 was involved in the earliest steps of osteogenesis (Tsuji et al., 2006). These expression patterns were inversely correlated with those of miR-17-5p and miR-106a during hADSCs differentiation. As a member of TGFβ/BMPs pathway, BMP2 has been shown to be involved in osteogenesis, fracture healing, and bone formation (Cohen, 2002). BMP2 can determine the differentiation process of MSCs through activating Runx2-dependent gene transcription and repressing PPARγ-dependent gene transcription (Cheng et al., 2003; Foshay and Gallicano, 2009; Hong et al., 2005; Peng et al., 2004). Based on bioinformatics analysis that indicated BMP2 is a potential target of miR-17-5p and miR-106a, we overexpressed miR-17-5p and miR-106a and found that upregulation of either miR-17-5p or miR-106a decreased BMP2 expression at protein level. In addition, inhibition of either miR-17-5p or miR-106a increased BMP2 expression, strongly suggesting that BMP2 is a direct target gene of miR-17-5p and miR-106a during osteogenic and adipogenic differentiation of hADSCs. Then, we demonstrated that both miR-17-5p and miR-106a directly targeted the 3′UTR region of BMP2 by luciferase reporter assay. Recently, miRNAs with similar ‘seed sequence’ have been reported to regulate a similar set of genes and have overlapping functions (Foshay and Gallicano, 2009; Ventura et al., 2008). In this study, we found that although miR-106a and miR-17-5p possess the same ‘seed sequence’, miR-106a mimic was less effective than miR-17-5p mimic in downregulating BMP2 in luciferase activity assay suggesting that nucleotide sequences other than ‘seed sequence’ may contribute to the difference of targeting efficiency in the same miRNA family. This might be one of the reasons that different members of miR-17 family showed different efficiency in regulating osteogenic and adipogenic differentiation. For instance, miR-17-5p can more effectively regulate adipogenesis and osteogenesis than miR-106a.