Bone Abstracts

Introduction: Micro-RNAs (miRNAs) are important post-transcriptional regulators. By binding to complementary RNA strands, they affect mRNA levels and/or mRNA translation. We have previously identified ~90 miRNAs with significant expression levels, with a subset of miRNAs exhibiting interindividual and/or gender differences in expression. In the present project, we have investigated the impact of treatment of parathyroid hormone (PTH) and dexamethasone (DEXA) on global miRNA expression in primary human bone (HOB) cells by second generation sequencing.

Method: HOB cells were isolated from human trabecular bone collected from donors undergoing total hip replacement, and treated with either PTH or DEXA or left untreated for 2 and 24 h. Small RNA was isolated from these cells and cDNA synthesized. Second generation sequencing was performed using SOLiD4 on barcoded library constructs. Sequence reads were aligned to a scaffold consisting of all known miRNA sequences. Number of sequence reads mapping uniquely to each miRNA were counted. The value used for each miRNA was the number of reads per miRNA normalized to per million total reads.

Results: 207 million reads were obtained, and normalized absolute expression was retrieved for the 500 most abundant miRNAs. The 75 miRNAs that exhibited the highest mean expression across the four experiments per individual were taken forward. Results show significant differences in miRNA expression after 2 h, and even more differences after 24 h. Several miRNAs exhibiting significant differences in expression have predicted mRNA targets involved in bone metabolism, e.g. miR-197 targeting IGF and Wnt pathway members.

Conclusion: miRNA absolute expression data from second generation sequencing show that PTH and DEXA affect miRNA expression in HOB cells, and that these miRNAs in turn are correlated to expression levels of mRNAs known to affect bone metabolism.