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miRNA and Asymmetric siRNA: Small RNAs with Large Effects on Bone Metabolism
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrinology and mineral metabolism.ORCID iD: 0000-0002-6243-0167
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1156
Keyword [en]
miRNA, cp-siRNA, RNAi, bone, Osteoblast, Sequencing, Differential expression, Wnt pathway
National Category
Basic Medicine
Research subject
Biology with specialization in Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-264451ISBN: 978-91-554-9396-7 (print)OAI: oai:DiVA.org:uu-264451DiVA: diva2:865482
Public defence
2015-12-16, Enghoffsalen, Ingång 50, Akademiska Sjukhuset, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-11-24 Created: 2015-10-12 Last updated: 2016-01-13
List of papers
1. Global miRNA expression and correlation with mRNA levels in primary human bone cells
Open this publication in new window or tab >>Global miRNA expression and correlation with mRNA levels in primary human bone cells
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2015 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 21, no 8, 1433-1443 p.Article in journal (Refereed) Published
Abstract [en]

MicroRNAs (miRNAs) are important post-transcriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. The aim of this study was to investigate miRNA-mRNA interactions that may be relevant for bone metabolism by assessing correlations and interindividual variability in miRNA levels as well as global correlations between miRNA and mRNA levels in a large cohort of primary human osteoblasts (HOBs) obtained during orthopedic surgery in otherwise healthy individuals. We identified differential expression (DE) of 24 miRNAs, and found 9 miRNAs exhibiting DE between males and females. We identified hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b and their target genes as important modulators of bone metabolism. Further, we used an integrated analysis of global miRNA-mRNA correlations, mRNA-expression profiling, DE, bioinformatics analysis, and functional studies to identify novel target genes for miRNAs with the potential to regulate osteoblast differentiation and extracellular matrix production. Functional studies by overexpression and knockdown of miRNAs showed that, the differentially expressed miRNAs hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b target genes highly relevant to bone metabolism, e.g., collagen, type I, alpha 1 (COL1A1), osteonectin (SPARC), Runt-related transcription factor 2 (RUNX2), osteocalcin (BGLAP), and frizzled-related protein (FRZB). These miRNAs orchestrate the activities of key regulators of osteoblast differentiation and extracellular matrix proteins by their convergent action on target genes and pathways to control the skeletal gene expression.

Keyword
miRNA, mRNA, osteoblasts, interindividual variation, differential expression
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-260282 (URN)10.1261/rna.049148.114 (DOI)000358016000005 ()26078267 (PubMedID)
Funder
Swedish Research Council
Available from: 2015-08-21 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
2. Second generation sequencing of microRNA in Human Bone Cells treated with Parathyroid Hormone or Dexamethasone
Open this publication in new window or tab >>Second generation sequencing of microRNA in Human Bone Cells treated with Parathyroid Hormone or Dexamethasone
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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.

Keyword
miRNA; osteoblasts; RNA sequencing; differential expression
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-264440 (URN)10.1016/j.bone.2015.12.053 (DOI)000370914600021 ()26748295 (PubMedID)
Funder
Swedish Research Council, 2009-2852
Available from: 2015-10-12 Created: 2015-10-12 Last updated: 2017-12-01Bibliographically approved
3. miR-203 and miR-320 regulate Bone Morphogenetic Protein-2-induced osteoblast differentiation by targeting Distal-less Homeobox 5 (Dlx5)
Open this publication in new window or tab >>miR-203 and miR-320 regulate Bone Morphogenetic Protein-2-induced osteoblast differentiation by targeting Distal-less Homeobox 5 (Dlx5)
2017 (English)In: Genes, ISSN 2073-4425, E-ISSN 2073-4425, Vol. 8, no 1, E4Article in journal (Refereed) Published
Abstract [en]

MicroRNAs (miRNAs) are a family of small, non-coding RNAs (17–24 nucleotides), which regulate gene expression either by the degradation of the target mRNAs or inhibiting the translation of genes. Recent studies have indicated that miRNA plays an important role in regulating osteoblast differentiation. In this study, we identified miR-203 and miR-320b as important miRNAs modulating osteoblast differentiation. We identified Dlx5 as potential common target by prediction algorithms and confirmed this by knock-down and over expression of the miRNAs and assessing Dlx5 at mRNA and protein levels and specificity was verified by luciferase reporter assays. We examined the effect of miR-203 and miR-320b on osteoblast differentiation by transfecting with pre- and anti-miRs. Over-expression of miR-203 and miR-320b inhibited osteoblast differentiation, whereas inhibition of miR-203 and miR-320b stimulated alkaline phosphatase activity and matrix mineralization. We show that miR-203 and miR-320b negatively regulate BMP-2-induced osteoblast differentiation by suppressing Dlx5, which in turn suppresses the downstream osteogenic master transcription factor Runx2 and Osx and together they suppress osteoblast differentiation. Taken together, we propose a role for miR-203 and miR-320b in modulating bone metabolism.

National Category
Clinical Medicine
Identifiers
urn:nbn:se:uu:diva-264442 (URN)10.3390/genes8010004 (DOI)
Available from: 2015-10-12 Created: 2015-10-12 Last updated: 2017-12-01Bibliographically approved
4. Endosmolytic cell penetrating siRNA for carrier free transfection
Open this publication in new window or tab >>Endosmolytic cell penetrating siRNA for carrier free transfection
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(English)Manuscript (preprint) (Other academic)
National Category
Clinical Medicine
Identifiers
urn:nbn:se:uu:diva-264443 (URN)
Available from: 2015-10-12 Created: 2015-10-12 Last updated: 2015-11-24

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