Identification of reference miRNAs in plasma useful for the study of oestrogen-responsive miRNAs associated with acquired Protein S deficiency in pregnancy

BMC Research Notes, Jul 2017

Background Accumulating evidence indicate that circulating microRNAs (miRNAs) are useful independent non-invasive biomarkers, with unique miRNA signatures defined for various pathophysiological conditions. However, there are no established universal housekeeping miRNAs for the normalisation of miRNAs in body fluids. We have previously identified an oestrogen-responsive miRNA, miR-494, in regulating the anticoagulant, Protein S, in HuH-7 liver cells. Moreover, increased thrombotic risk associated with elevated circulating oestrogen levels is frequently observed in pregnant women and oral contraceptive users. In order to identify other oestrogen-responsive miRNAs, including miR-494, that may be indicative of increased thrombotic risk in plasma, we used nanoString analysis to identify robust and stable endogenous reference miRNAs for the study of oestrogen-responsive miRNAs in plasma. Results We compared the plasma miRNA expression profile of individuals with: (1) Low circulating oestrogens (healthy men and non-pregnant women not taking oral contraceptives), (2) High circulating synthetic oestrogens, (women taking oral contraceptives) and (3) High circulating natural oestrogens (pregnant females >14 weeks gestation). From the nanoString analyses, 11 candidate reference miRNAs which exhibited high counts and not significantly differentially expressed between groups were selected for validation using realtime quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (DDPCR) in pooled plasma samples, and the stability of their expression evaluated using NormFinder and BestKeeper algorithms. Four miRNAs (miR-25-5p, miR-188-5p, miR-222-3p and miR-520f) demonstrated detectable stable expression between groups and were further analysed by RT-qPCR in individual plasma samples, where miR-188-5p and miR-222-3p expression were identified as a stable pair of reference genes. The miRNA reference panel consisting of synthetic spike-ins cel-miR-39 and ath-miR159a, and reference miRNAs, miR-188-5p and miR-222-3p was useful in evaluating fold-change of the pregnancy-associated miRNA, miR-141-3p, between groups. Conclusion The miRNA reference panel will be useful for normalising qPCR data comparing miRNA expression between men and women, non-pregnant and pregnant females, and the potential effects of endogenous and synthetic oestrogens on plasma miRNA expression.

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Identification of reference miRNAs in plasma useful for the study of oestrogen-responsive miRNAs associated with acquired Protein S deficiency in pregnancy

Tay et al. BMC Res Notes Identification of reference miRNAs in plasma useful for the study of oestrogen-responsive miRNAs associated with acquired Protein S deficiency in pregnancy J. W. Tay 0 2 I. James 1 Q. W. Hughes 0 2 J. Y. Tiao 0 2 R. I. Baker 0 2 0 Western Australian Centre for Thrombosis and Haemostasis, Murdoch University , Murdoch , Australia 1 Institute for Immunology and Infectious Diseases, Murdoch University , Murdoch , Australia 2 Perth Blood Institute , Nedlands , Australia Background: Accumulating evidence indicate that circulating microRNAs (miRNAs) are useful independent noninvasive biomarkers, with unique miRNA signatures defined for various pathophysiological conditions. However, there are no established universal housekeeping miRNAs for the normalisation of miRNAs in body fluids. We have previously identified an oestrogen-responsive miRNA, miR-494, in regulating the anticoagulant, Protein S, in HuH-7 liver cells. Moreover, increased thrombotic risk associated with elevated circulating oestrogen levels is frequently observed in pregnant women and oral contraceptive users. In order to identify other oestrogen-responsive miRNAs, including miR-494, that may be indicative of increased thrombotic risk in plasma, we used nanoString analysis to identify robust and stable endogenous reference miRNAs for the study of oestrogen-responsive miRNAs in plasma. Results: We compared the plasma miRNA expression profile of individuals with: (1) Low circulating oestrogens (healthy men and non-pregnant women not taking oral contraceptives), (2) High circulating synthetic oestrogens, (women taking oral contraceptives) and (3) High circulating natural oestrogens (pregnant females >14 weeks gestation). From the nanoString analyses, 11 candidate reference miRNAs which exhibited high counts and not significantly differentially expressed between groups were selected for validation using realtime quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (DDPCR) in pooled plasma samples, and the stability of their expression evaluated using NormFinder and BestKeeper algorithms. Four miRNAs (miR-25-5p, miR-188-5p, miR-222-3p and miR520f ) demonstrated detectable stable expression between groups and were further analysed by RT-qPCR in individual plasma samples, where miR-188-5p and miR-222-3p expression were identified as a stable pair of reference genes. The miRNA reference panel consisting of synthetic spike-ins cel-miR-39 and ath-miR159a, and reference miRNAs, miR-188-5p and miR-222-3p was useful in evaluating fold-change of the pregnancy-associated miRNA, miR-141-3p, between groups. Conclusion: The miRNA reference panel will be useful for normalising qPCR data comparing miRNA expression between men and women, non-pregnant and pregnant females, and the potential effects of endogenous and synthetic oestrogens on plasma miRNA expression. Background MiRNAs are short, non-coding RNA species approximately 22 nucleotides in length that function to downregulate the expression of its target genes by binding to specific sequences in the 3′ untranslated region (UTR) of target mRNAs leading to the destabilisation of the mRNA or translational repression. Since their discovery in 1993 [ 1, 2 ], the role of miRNAs in regulating key cellular processes and their involvement in various pathophysiological conditions, particularly in solid tumours are well studied, and have been extensively reviewed [ 3–5 ]. It has also been demonstrated that miRNA profiles for human tumours are superior to mRNA markers, where poorly differentiated tumours could be successfully classified using miRNA expression profiles, whereas mRNA profiles were highly inaccurate when applied to the same tissue samples [6]. A comparison of miRNA and gene expression in frozen breast cancer tissue samples and their paired formalin-fixed paraffin-embedded tissue samples obtained during primary surgical resection showed that expression of miRNAs remains robust even in samples with degraded total RNA, clearly demonstrating the stability of miRNAs in compromised samples in comparison to mRNA markers [ 7 ]. The value of miRNAs as useful biomarkers was elevated when plasma and serum were found to be rich in miRNAs that appeared to be protected from RNase degradation [ 8, 9 ], and remained highly stable at room temperature and under adverse conditions such as multiple freeze–thaw cycles [ 10, 11 ]. A large variety of miRNAs have been detected in various fractions of circulating blood [ 12 ] such as erythrocytes [ 13 ], anucleate platelets [ 14 ], apoptotic bodies [ 15 ] and packaged in circulating microparticles shed from platelets and all other cells in the body [ 16 ]. As such, we hypothesise that circulating levels of tissue specific miRNAs can provide important information regarding the health status of target tissues, serving as useful non-invasive biomarkers. Due to significant protein and lipid variability between individual (...truncated)


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J. W. Tay, I. James, Q. W. Hughes, J. Y. Tiao, R. I. Baker. Identification of reference miRNAs in plasma useful for the study of oestrogen-responsive miRNAs associated with acquired Protein S deficiency in pregnancy, BMC Research Notes, 2017, pp. 312, Volume 10, Issue 1, DOI: 10.1186/s13104-017-2636-3