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C. Perez-Sánchez et al.
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Figure 1. Antiphospholipid syndrome (APS) patients showed a specific circulating miRNAs profile related to clinical features of this autoimmune disorder. (A) To identify the changes that occurred in the expression levels of microRNAs (miR) in plasma from antiphospholipid syndrome versus controls, Human Serum & Plasma miRNA PCR-array (Qiagen) was performed in the study cohort. Expression levels of 19 miRNAs were found up-regulated in antiphospholipid syndrome, while 20 miRNAs were down-regulated. (B) Ingenuity Pathway Analysis (IPA) uncovered the main enriched biological functions and pathways in which these microRNAs are involved. The analysis included only the functions and pathways with average IPA score >2 [indicated as -log (P value)]. (C) Validation of selected miRNAs by RT-PCR in the whole cohort of APS patients and healthy donors. *P<0.05.
their modulation by autoantibodies, and their potential role as non-invasive biomarkers of disease features.
Methods
Patients
Ninety patients with primary APS and 42 healthy donors (HDs) were included in this study over a period of 24 months (see Online Supplementary Appendix). All experimental protocols were approved by the ethics committee of the Reina Sofia Hospital, Cordoba, Spain, and written informed consent was obtained. The characteristics of patients and HDs are shown in Table 1.
The adjusted global anti-phospholipid syndrome score (aGAPSS) was calculated for each APS patient, as previously described.12 Briefly, aGAPSS was calculated by adding the points corresponding to both the cardiovascular and thrombotic risk fac- tors, based on a linear transformation derived from the β regres- sion coefficient as follows: 3 for hyperlipidemia, 1 for arterial hypertension, 5 for aCL IgG/IgM, 4 for anti-β2GPI IgG/IgM, and 4 for LA.
Two additional cohorts of patients were further analyzed as dis- ease control, including 23 patients with thrombosis in the absence of an associated autoimmune disease [12 non-pregnant women and 11 men; mean age 44 (range: 21-73 years), including patients with objectively verified thrombotic events: 14 deep venous thrombosis and 9 thrombosis in intra-cerebral vessels], and 25 SLE patients without aPLs (Online Supplementary Table S1).
For details of blood sample collection and assessment of biolog-
ical parameters, B-Mode Ultrasound IMT and Ankle Brachial Index measurements see the Online Supplementary Appendix.
Isolation of miRNAs and analysis of miRNAs expression profiling
Total RNA, including the miRNA fraction, was extracted from both plasma and supernatants obtained from in vitro studies by using the QIAzol miRNeasy kit (Qiagen, Valencia, CA, USA) fol- lowing the manufacturer's instructions13 (Online Supplementary Appendix). To identify the changes that occurred in the expression levels of miRNAs in plasma from APS patients and HDs, a Human Serum & Plasma miRNA PCR-array (Qiagen) was performed (Online Supplementary Appendix) on an exploratory cohort (Online Supplementary Table S2).
Quantitative real-time PCR
A fixed volume of 3 μl of RNA solution from the 14 μl-eluate from RNA isolation of 200 μl plasma sample was used as input into the reverse transcription. Input RNA was reverse transcribed using the TaqMan miRNA Reverse Transcription kit and miRNA- specific stem-loop primers (Life Technologies, Madrid, Spain) (Online Supplementary Appendix). The expression levels of miRNAs were calculated by using 2-Ct and reciprocal ratios were performed [Ratio miR-A/miR-B = log (2-Ct miR-A/2-Ct miR-B)], as previously
2
described.14-19 Reciprocal ratios analysis is an approach that
bypasses the controversial issue of data normalization of miRNAs in plasma (self-normalization). Furthermore, miRNAs whose con- centrations are changed because of a pathology in opposite direc- tions can be effective in differentiating investigated populations.
haematologica | 2018; 103(5)