Osteoporosis is a devastating disease with an essential genetic component. GWAS have discovered genetic signals robustly associated with bone mineral density (BMD), but not the precise localization of effector genes. Here, we carry out physical and direct variant to gene mapping in human mesenchymal progenitor cell-derived osteoblasts employing a massively parallel, high resolution Capture C based method in order to simultaneously characterize the genome-wide interactions of all human promoters. By intersecting our Capture C and ATAC-seq data, we observe consistent contacts between candidate causal variants and putative target gene promoters in open chromatin for ~ 17% of the 273 BMD loci investigated. Knockdown of two novel implicated genes, ING3 at ‘CPED1-WNT16’ and EPDR1 at ‘STARD3NL’, inhibits osteoblastogenesis, while promoting adipogenesis. This approach therefore aids target discovery in osteoporosis, here on the example of two relevant genes involved in the fate determination of mesenchymal progenitors, and can be applied to other common genetic diseases.

μ-Opioid receptor (MOR) level is directly related to the function of opioid drugs, such as morphine and fentanyl. Although agonist treatment generally does not affect transcription of mor, previous studies suggest that morphine can affect the translation efficiency of MOR transcript via microRNAs (miRNAs). On the basis of miRNA microarray analyses of the hippocampal total RNA isolated from mice chronically treated with μ-opioid agonists, we found a miRNA (miR-339-3p) that was consistently and specifically increased by morphine (2-fold) and by fentanyl (3.8-fold). miR-339-3p bound to the MOR 3'-UTR and specifically suppressed reporter activity. Suppression was blunted by adding miR-339-3p inhibitor or mutating the miR-339-3p target site. In cells endogenously expressing MOR, miR-339-3p inhibited the production of MOR protein by destabilizing MOR mRNA. Up-regulation of miR-339-3p by fentanyl (EC(50)=0.75 nM) resulted from an increase in primary miRNA transcript. Mapping of the miR-339-3p primary RNA and its promoter revealed that the primary miR-339-3p was embedded in a noncoding 3'-UTR region of an unknown host gene and was coregulated by the host promoter. The identified promoter was activated by opioid agonist treatment (10 nM fentanyl or 10 μM morphine), a specific effect blocked by the opioid antagonist naloxone (10 μM). Taken together, these results suggest that miR-339-3p may serve as a negative feedback modulator of MOR signals by regulating intracellular MOR biosynthesis.

Background Bone accrual impacts lifelong skeletal health, but genetic discovery has been primarily limited to cross-sectional study designs and hampered by uncertainty about target effector genes. Here, we capture this dynamic phenotype by modeling longitudinal bone accrual across 11,000 bone scans in a cohort of healthy children and adolescents, followed by genome-wide association studies (GWAS) and variant-to-gene mapping with functional follow-up. Results We identify 40 loci, 35 not previously reported, with various degrees of supportive evidence, half residing in topological associated domains harboring known bone genes. Of several loci potentially associated with later-life fracture risk, a candidate SNP lookup provides the most compelling evidence for rs11195210 (SMC3). Variant-to-gene mapping combining ATAC-seq to assay open chromatin with high-resolution promoter-focused Capture C identifies contacts between GWAS loci and nearby gene promoters. siRNA knockdown of gene expression supports the putative effector gene at three specific loci in two osteoblast cell models. Finally, using CRISPR-Cas9 genome editing, we confirm that the immediate genomic region harboring the putative causal SNP influences PRPF38A expression, a location which is predicted to coincide with a set of binding sites for relevant transcription factors. Conclusions Using a new longitudinal approach, we expand the number of genetic loci putatively associated with pediatric bone gain. Functional follow-up in appropriate cell models finds novel candidate genes impacting bone accrual. Our data also raise the possibility that the cell fate decision between osteogenic and adipogenic lineages is important in normal bone accrual.

MicroRNAs (miRNA), a class of ~22-nucleotide RNA molecules, are important gene regulators that bind to the target sites of mRNAs to inhibit the gene expressions either through translational inhibition or mRNA destabilization. There are growing evidences that miRNAs have played several regulatory roles in opioid pharmacology. Like other research fields such as cancer biology, the area where numerous miRNAs are found to be involved in gene regulation, we assume that in opioid studies including research fields of drug additions and opioid receptor regulation, there may be more miRNAs waiting to be discovered. This review will summarize our current knowledge of miRNA functions on opioids biology and briefly describe future research directions of miRNAs related to opioids.

Osteoblast differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) can be induced by stimulation with canonical Notch ligand, Jagged1, or bone morphogenetic proteins (BMPs). However, it remains elusive how these two pathways lead to the same phenotypic outcome. Since Runx2 is regarded as a master regulator of osteoblastic differentiation, we targeted Runx2 with siRNA in hMSC. This abrogated both Jagged1 and BMP2 mediated osteoblastic differentiation, confirming the fundamental role for Runx2. However, while BMP stimulation increased Runx2 and downstream Osterix protein expression, Jagged1 treatment failed to upregulate either, suggesting that canonical Notch signals require basal Runx2 expression. To fully understand the transcriptomic profile of differentiating osteoblasts, RNA sequencing was performed in cells stimulated with BMP2 or Jagged1. There was common upregulation of ALPL and extracellular matrix genes, such as ACAN, HAS3, MCAM, and OLFML2B. Intriguingly, genes encoding components of Notch signaling (JAG1, HEY2, and HES4) were among the top 10 genes upregulated by both stimuli. Indeed, ALPL expression occurred concurrently with Notch activation and inhibiting Notch activity for up to 24 hours after BMP administration with DAPT (a gamma secretase inhibitor) completely abrogated hMSC osteoblastogenesis. Concordantly, RBPJ (recombination signal binding protein for immunoglobulin kappa J region, a critical downstream modulator of Notch signals) binding could be demonstrated within the ALPL and SP7 promoters. As such, siRNA-mediated ablation of RBPJ decreased BMP-mediated osteoblastogenesis. Finally, systemic Notch inhibition using diabenzazepine (DBZ) reduced BMP2-induced calvarial bone healing in mice supporting the critical regulatory role of Notch signaling in BMP-induced osteoblastogenesis.