Background MicroRNAs have a functional role in numerous physiological and pathological processes by regulating the expression of their target genes. A number of miRNAs have been involved in the pathogenesis of antiphospholipid sindrome (APS). However, the miRNA-mRNA regulatory network involved in the regulation of the proatherothrombotic status in APS patients is far from being fully understood.
Objectives To identify and characterize novel regulatory miRNA/mRNA/protein networks related to atherothrombosis in monocytes from APS.
Methods Twenty one APS patients and 27 healthy donors were included in the study. A multi-step approach combining microarray miRNA (Nanostring technology) and mRNA expression profile (44K, Agilent, one color) and bioinformatics analysis was adopted to identify the APS specific miRNA-mRNA regulatory networks. Target genes of the differentially expressed miRNAs were identified by using the Igenuity Pathway Analysis Software (IPA). To recognize the putative miRNA-mRNA target pairs and interaction networks, the subsets of miRNAs and mRNAs were characterized by the miRNA Target Filter tool of IPA. The resulting identified interactions were validated by RT-PCR and western blot on the whole cohort of APS patients and by functional analyses using miRNA overexpression experiments in monocytes purified from APS patients.
Results Comparative analysis of the mRNA profiles showed significantly different expressions of 519 genes in APS monocytes (352 up-regulated and 167 down-regulated). Functional analysis by using IPA showed that about 33% of altered genes were involved in inflammation and cardiovascular disease (CVD). Comparative analysis of the miRNA profiles showed significantly different expressions of 22 miRNAs in APS monocytes (3 up-regulated and 19 down-regulated). Functional IPA analysis showed that miRNAs altered were mainly related to connective tissue disorders, inflammatory response and reproductive system disease. A total of 56 genes were inversely correlated, and predicted as CVD-related target genes of 19 differentially expressed miRNAs. An interaction network of those genes and miRNAs differentially expressed in APS monocytes were also identified. Interestingly, a few number of miRNAs (i.e. miR-30b, miR-145, miR-199, miR-17, miR-26a) regulate idependently multiple gene targets belonging to the cluster of genes related to inflammation and atherogenesis, (i.e. VEGF, TGFβ, LDLR, PPARD, IL1, IL2, IL12, VCAM1, ITGA, CACNB2, MMP3). Transfection assays validated regulatory relationships between those miRNAS and a number of targets at both RNA and protein levels.
Conclusions Our system biology approach introduced and nominated 1) important deregulated miRNAs involved in atherothrombosis in monocytes from APS; 2) Significant and confident deregulated mRNAs which were anti-correlatively targeted by deregulated miRNAs; and 3) dysregulated proteins in association with deregulated miRNA-mRNAs interactions. These results introduce a possible mechanism of function of deregulated miRNAs and mRNAs in atherothrombosis-related APS that could be used as potential therapeutic targets.
Acknowledgement Supported by FIS (PI01333/2015) and CTS-7940.
Disclosure of Interest None declared
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