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Second generation sequencing of microRNA in Human Bone Cells treated with Parathyroid Hormone or Dexamethasone
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.ORCID iD: 0000-0002-6243-0167
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
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2016 (English)In: Bone, ISSN 8756-3282, E-ISSN 1873-2763, Vol. 84, 181-188 p.Article in journal (Refereed) Published
Abstract [en]

We investigated the impact of treatment with parathyroid hormone (PTH) and dexamethasone (DEX) for 2 and 24 h by RNA sequencing of miRNAs in primary human bone (HOB) cells. A total of 207 million reads were obtained, and normalized absolute expression retrieved for 373 most abundant miRNAs. In naive control cells, 7 miRNAs were differentially expressed (FDR < 0.05) between the two time points. Ten miRNAs exhibited differential expression (FDR < 0.05) across two time points and treatments after adjusting for expression in controls and were selected for downstream analyses. Results show significant effects on miRNA expression when comparing PTH with DEX at 2 h with even more pronounced effects at 24 h. Interestingly, several miRNAs exhibiting differences in expression are predicted to target genes involved in bone metabolism e.g. miR-30c2, miR-203 and miR-205 targeting RUNX2, and miR-320 targeting beta-catenin (CTNNB1) mRNA expression. CTNNB1 and RUNX2 levels were decreased after DEX treatment and increased after PTH treatment. Our analysis also identified 2 putative novel miRNAs in PTH and DEX treated cells at 24 h. RNA sequencing showed that PTH and DEX treatment affect miRNA expression in HOB cells and that regulated miRNAs in turn are correlated with expression levels of key genes involved in bone metabolism.

Place, publisher, year, edition, pages
2016. Vol. 84, 181-188 p.
Keyword [en]
miRNA; osteoblasts; RNA sequencing; differential expression
National Category
Endocrinology and Diabetes
URN: urn:nbn:se:uu:diva-264440DOI: 10.1016/j.bone.2015.12.053ISI: 000370914600021PubMedID: 26748295OAI: oai:DiVA.org:uu-264440DiVA: diva2:860435
Swedish Research Council, 2009-2852
Available from: 2015-10-12 Created: 2015-10-12 Last updated: 2016-04-05Bibliographically approved
In thesis
1. miRNA and Asymmetric siRNA: Small RNAs with Large Effects on Bone Metabolism
Open this publication in new window or tab >>miRNA and Asymmetric siRNA: Small RNAs with Large Effects on Bone Metabolism
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

RNA interference (RNAi) is a post-transcriptional gene silencing process elicited by double-stranded RNA, such as micro-RNA (miRNA) and small interfering RNA (siRNA). They are 18-25 nucleotide long, small non-coding RNAs acting as critical regulators in eukaryotic genome expression. They play an important role in regulating a wide range of biological processes such as cell cycle control, differentiation, aging and apoptosis. However, their role in supporting skeletal development and bone homeostasis is still poorly understood.

Osteoporotic fractures constitute a tremendous and growing problem in our ageing populations, with an annual incidence of approximately 60000 osteoporotic fractures in Sweden. Osteoporosis is referred as the “Silent epidemic” because bone loss is gradual and a basically symptomless development until a fracture occurs.

Results presented in this thesis provide a novel insight into crucial roles of   miRNAs in regulating bone homeostasis. The initial aim for the thesis was to perform global miRNA expression profiling in human bone cells, and to correlate these levels to global mRNA levels. We identified and functionally characterized several miRNAs that were differentially expressed and acted in important bone signaling pathways such as the Wnt and BMP pathways. These miRNAs included hsa-miR-29b, hsa-miR-30c2 and hsa-miR-125b, which we found targeting genes highly relevant to bone metabolism e.g. COL1A1, SPARC, RUNX2, BGLAP and FRZB.

Thereafter, the effect on the microRNAome upon external stimuli (e.g., Dexamethasone and Parathyroid hormone) was assessed by SOLiD sequencing. We observed a substantial difference in the expression of miRNAs between PTH and DEX treated cells. Understanding the changes in miRNAome in human bone cells under different conditions could provide new insight in bone remodeling, specifically differentiation and functional properties of osteoblasts.

Based on these studies, we furthermore identified Dlx5 as potential common target of miR-203 and miR-320b and these miRNAs negatively regulate BMP-2-induced osteoblast differentiation.

To activate the RNAi pathway, siRNA or miRNA molecules must be conveyed into the cytoplasm of target cells. Since challenges in cellular delivery of these small silencing RNA molecules so far have limited their clinical utility, we developed a new siRNA design that demonstrates a novel carrier-free cellular delivery. This development could potentially have a major impact in RNAi therapeutics.

In conclusion, this thesis provides novel insight of miRNAs that play a major role in the regulation of bone remodeling and differentiation and functional properties of osteoblasts. Our findings may have diagnostic and/or therapeutic implications in disorders of bone metabolism.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 60 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1156
miRNA, cp-siRNA, RNAi, bone, Osteoblast, Sequencing, Differential expression, Wnt pathway
National Category
Basic Medicine
Research subject
Biology with specialization in Molecular Biology
urn:nbn:se:uu:diva-264451 (URN)978-91-554-9396-7 (ISBN)
Public defence
2015-12-16, Enghoffsalen, Ingång 50, Akademiska Sjukhuset, Uppsala, 09:15 (English)
Available from: 2015-11-24 Created: 2015-10-12 Last updated: 2016-01-13

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Laxman, NavyaRubin, Carl-JohanMallmin, HansNilsson, OlleTellgren-Roth, ChristianKindmark, Andreas
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