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MicroRNAs as antiphospholipid syndrome biomarkers
Figure 2. Interaction network of microRNAs identified potential mRNA targets involved in clinical features of antiphospholipid syndrome. Using microRNA Target Filter of QIAGEN’s Ingenuity Pathway Analysis (IPA, QIAGEN Redwood City, CA, USA, www.qiagen.com/ingenuity), the software generated a network including the selected microRNAs (miRNAs or miR) and their mRNA targets, filtered by coronary artery disease, thrombosis, abortion and cerebrovascular dysfunction. Only targets experimentally observed and predicted with high confidence are shown and related by direct interactions to their specific miRNA regulators.
Target gene prediction and integrated analysis by Ingenuity Pathway Analysis
The altered miRNAs were further analyzed to obtain informa- tion about biological functions, pathways and networks by using the web-based bioinformatics tool QIAGEN’s Ingenuity Pathway Analysis (IPA; Ingenuity Systems, http://www.INGENUITY.com). For this purpose, all differentially regulated miRNAs and fold changes were imported into IPA20 (Online Supplementary Appendix).
Details of purification of IgG, in vitro exposure of monocytes and endothelial cells to aPL antibodies, and the statistical analysis are available in the Online Supplementary Appendix.
Results
Differentially expressed miRNAs in the plasma of APS patients and HDs
In the discovery phase (exploratory cohort), we identi- fied 39 miRNAs that were differentially expressed between APS patients and HDs (cut off: 1.7-fold change), including 19 up-regulated and 20 down-regulated (Figure 1A). Using the IPA software, the functional analysis of the altered miRNAs in APS patients showed that a large num- ber of them had validated and putative target mRNAs mainly involved in connective tissue disorders, inflamma- tory response, reproductive system disease, CVD or skele- tal and muscular disorders (Figure 1C).
Bioinformatic identification and analysis of deregulated miRNAs related to the pathophysiology of APS and analysis of potential protein targets
In silico studies were performed to identify the altered miRNAs that might have as potential targets a number of genes/proteins involved in the development of clinical manifestations related to APS, such as coronary artery dis- ease, thrombosis, abortion, and cerebrovascular dysfunc- tion. IPA identified 11 altered miRNAs as the main regula- tors of proteins involved in the pathology of APS, includ- ing miRNA 34a-5p, 15a-5p, 145a-5p, 133b-3p, 124-3p, 206, 20a-5p, 19b-3p, 210-3p, 296-5p and 374a-5p. This set of 11 miRNAs included, among others, the top 5 up-regu- lated miRNAs and 3 out of the top 5 down-regulated miRNAs in the PCR-array. The expression levels of the 11 selected miRNAs were analyzed in all study subjects by RT-PCR (Figure 1B). MiR-124 and miR-34a were found increased in APS patients in relation to healthy donors, while miR-20a, miR-19b and miR145a were found reduced. The remaining microRNAs were also found to be altered, showing a trend to either increase or reduction as observed in the discovery phase, thus validating the data obtained by PCR-array.
We further developed a network that defined the inter- action between miRNA-mRNA targets (Figure 2). Key pro- teins involved in the pathophysiology of APS, and identi-
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