Altered Expression of Circulating MicroRNA in Plasma of Patients with Primary Osteoarthritis and <i>In Silico</i> Analysis of Their Pathways

<div><p>Objective</p><p>To analyze a set of circulating microRNA (miRNA) in plasma from patients with primary Osteoarthritis (OA) and describe the biological significance of altered miRNA in OA based on an <i>in silico</i> analysis of their target genes.</p><p>Methods</p><p>miRNA expression was analyzed using TaqMan Low Density Arrays and independent assays. The search for potential messenger RNA (mRNA) targets of the differentially expressed miRNA was performed by means of the miRWalk and miRecords database; we conducted the biological relevance of the predicted miRNA targets by pathway analysis with the Reactome and DAVID databases.</p><p>Results</p><p>We measured the expression of 380 miRNA in OA; 12 miRNA were overexpressed under the OA condition (<i>p</i> value, ≤0.05; fold change, >2). These results were validated by the detection of some selected miRNA by quantitative PCR (qPCR). <i>In silico</i> analysis showed that target messenger RNA (mRNA) were potentially regulated by these miRNA, including genes such as <i>SMAD1</i>, <i>IL-1B</i>, <i>COL3A</i>, <i>VEGFA</i>, and <i>FGFR1</i>, important in chondrocyte maintenance and differentiation. Some metabolic pathways affected by the miRNA: mRNA ratio are signaling Bone morphogenetic proteins (BMP), Platelet-derived growth factor (PDGF), and Nerve growth factor (NGF), these latter two involved in the process of pain.</p><p>Conclusions</p><p>We identified 12 miRNA in the plasma of patients with primary OA. Specific miRNA that are altered in the disease could be released into plasma, either due to cartilage damage or to an inherent cellular mechanism. Several miRNA could regulate genes and pathways related with development of the disease; eight of these circulating miRNA are described, to our knowledge, for first time in OA.</p></div

Transcripts targeted of microRNAs up-regulated in samples Osteoarthritis (OA).

<p>(A) Tabulation of the number of transcripts targeted of 10 miRNA; (B) Tabulation of the number of predicted targets for 150 genes involved in OA; (C) Predicted targets that have been confirmed previously in other entities and OA; (D) Percentage of predicted target genes involved in OA.</p

<b>Table.1</b> Characteristics of the study sample.

<p>TLDA: TaqMan profiling Low Density Array; OA: Osteoarthritis; BMI: Body mass index.</p

MNDA expression status in two different microarray experiments also validate our finding [81].

<p>MNDA expression status in two different microarray experiments also validate our finding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042678#pone.0042678-EMBL3" target="_blank">[81]</a>.</p

Cancer related deregulated pathways.

<p>Statistical enrichment of deregulated pathways within our differential gene-sets include canonical cancer pathways such as DNA damage repair, AMPK and RAS. Molecules marked with a red star correspond to differentially expressed genes in the cancer/control contrast.</p

Pathways associated with microRNA (miRNA) up-regulated in Osteoarthritis (OA).

<p>Enriched biological processes among the 12 up-regulated miRNA. Red shows the e-value as more significant, while less significance is denoted in blue.</p

Barcode validation results for differential over-expression of <i>MEF2C</i>, <i>SMAD3</i> and <i>POU2AF1</i> within two platforms for several gene-probes in different breast cancer tissues [60], [78].

<p>Barcode validation results for differential over-expression of <i>MEF2C</i>, <i>SMAD3</i> and <i>POU2AF1</i> within two platforms for several gene-probes in different breast cancer tissues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042678#pone.0042678-Barcode1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042678#pone.0042678-McCall1" target="_blank">[78]</a>.</p

GEO [29] identifier and references for the Microarray experiments used here, first column is GEO key ID, second and third columns are the corresponding number of samples cases/controls resp.

<p>Fourth column is the reference entry.</p

Profiling of plasma MicroRNA (miRNA) levels in patients with Osteoarthritis (OA).

<p>(A) Microarray analysis for miRNA levels was performed using RNA extracts from plasma of healthy subjects as control and patients with OA of different OA disease stages; (B) Unsupervised hierarchical clustering of the differentially expressed miRNA. Bright green: down-regulation; black, no change; bright red: up-regulation.</p

Metabolism related deregulated pathways.

<p>Statistical enrichment of deregulated pathways within our differential gene-sets include metabolic pathways. For instance, both branches of the cholesterol biosynthesis pathway are affected. Molecules marked with a red star correspond to differentially expressed genes in the cancer/control contrast.</p