Supplementary MaterialsFigure S1: Expression levels of miR-27b, miR-151 and miR-152 in human circulating monocytes from postmenopausal women with low and high BMD as shown by miRNA array. 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P?=?0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P?=?0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively Ataluren inhibitor database with miR-422a expression. Conclusions/Significance Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis. Introduction Osteoporosis is a common disease characterized by low bone mineral density (BMD) and low trauma fractures, mainly resulting from exceeding bone resorption by osteoclasts over bone formation by osteoblasts [1]. The immune system also has a strong association with bone metabolism [2], [3]. In particular, circulating monocytes are directly involved in osteoclastogenesis. They are precursors of osteoclasts [4] and also secrete osteoclastogenic factors such as IL-1 (interleukin-1), IL-6 and TNF- (tumor necrosis factor-alpha) [5].Human studies have also shown relationships between the expression levels of certain genes in circulating monocytes and osteoporosis: annexin A2 (ANXA2) [6], signal transducer and activator of transcription 1 (STAT1) [7], chemokine (C-C motif) receptor 3 (CCR3), histidine decarboxylase (HDC), and glucocorticoid receptor (GCR) [8]. MicroRNAs (miRNAs) are short (22 nt) non-coding RNA molecules that regulate gene expression usually by destabilizing mRNAs or by suppressing translation [9]. Many miRNAs are involved in osteoblastogenesis [10]C[15]. There are also some miRNAs that promote proliferation and differentiation of osteoclasts. MiR-21 downregulates PCD4 (programmed cell death 4) protein expression and induces osteoclastogenesis of major mouse BMMs (bone tissue marrow-derived monocyte/macrophage precursors) [16]. MiR-155 can induce osteoclast activity via putatively expected targeting of Dispatch (Src homology 2-including inositol phosphatase), a suppressor of osteoclastogenesis [17]. MiR-223 suppresses NF1-A (nuclear element 1-A) expression, which stimulates mouse osteoclast function and differentiation [18]. MiR-34c enhances osteoclastogenesis via post-transcriptional rules of genes involved in the Notch signaling pathway, such as Notch1, Notch2, and Jag1 [19]. MiR-378 is involved in the induction of osteoclast differentiation Ataluren inhibitor database since it is upregulated in mouse RAW264.7 cells that are differentiating into osteoclasts [20]. However, the role of miRNAs in human circulating monocytes in the etiology of osteoporosis remains largely unclear. A recent study found that miR-146a inhibited osteoclastogenesis in human circulating mononuclear cells [21]. Also, another and study found that miR-148a promotes osteoclastogenesis in human circulating mononuclear cells [22]. We previously found that miR-133a in circulating monocytes is upregulated in postmenopausal women with low BMD compared to postmenopausal women with high BMD, thus identifying miR-133a a potential biomarker associated with postmenopausal osteoporosis [23]. In this study, we intended to further identify other potential miRNA biomarkers in circulating monocytes for postmenopausal osteoporosis. Using microarray and qRT-PCR approaches, we found a significant increase of miR-422a expression in low BMD compared with high BMD subjects. Moreover, bioinformatic target gene analysis identified several possible target genes for miR-422a. Materials and Methods Human subjects and characteristics The Institutional Review Board at Creighton University approved the study, and all subjects signed informed-consent documents. Twenty postmenopausal Caucasian women were recruited from the Omaha, NE. Ten subjects had high MSK1 BMD (spine or hip Z score 0.84) and 10 had low BMD (spine or hip Z score ?0.84). The high and low BMD groups represent the top and bottom 20% of the BMD distributions of the age-, sex- and ethnicity-matched population. BMD (g/cm2) for the lumbar spine (L1-4) and total hip (femoral neck, trochanter and intertrochanteric region) were measured using a Hologic 4500A dual energy X-ray absorptiometry (DXA) scanner (Hologic Inc., Bedford, MA). The coefficient of variation (CV), which reflects the instrument’s precision, was 0.9% and 1.4% for the spine and hip BMD, respectively. The recruited women were considered postmenopausal if they had at least 12 months of no menses since the date of their last Ataluren inhibitor database menses. All subjects were 57C68 years of age. Detailed characteristics of the study subjects can be.