Project description:We tested the hypothesis that a panel of placental mammal-specific miRNAs and their targets play important to establish receptivity to implantation and their dysregulated expression may be a feature in women with early pregnancy loss. Relative expression levels of miR-340-5p, −542-3p, and −671-5p all increased following treatment of Ishikawa cells with progesterone (10 μg/ml) for 24 hrs (p < 0.05). RNA sequencing of these P4-treated cells identified co-ordinate changes to 6,367 transcripts of which 1713 were predicted targets of miR-340-5p, 670 of miR-542-3p, and 618 of miR-671-5p. Quantitative proteomic analysis of Ishikawa cells transfected with mimic or inhibitor (48 hrs: n=3 biological replicates) for each of the P4-regulated miRNAs was carried out to identify targets of these miRNAs. Excluding off target effects, mir-340-5p mimic altered 1,369 proteins while inhibition changed expression of 376 proteins (p < 0.05) of which, 72 were common to both treatments. A total of 280 proteins were identified between predicted (mirDB) and confirmed (in vitro) targets. In total, 171 proteins predicted to be targets by mirDB were altered in vitro by treatment with miR-340-5p mimic or inhibitor and were also altered by treatment of endometrial epithelial cells with P4. In vitro targets of miR-542-3p identified 1,378 proteins altered by mimic while inhibition altered 975 a core of 200 proteins were changed by both. 100 protein targets were predicted and only 46 proteins were P4 regulated. miR-671-mimic altered 1,252 proteins with inhibition changing 492 proteins of which 97 were common to both, 95 were miDB predicted targets and 46 were also P4-regulated. All miRNAs were detected in endometrial biopsies taken from patients during the luteal phase of their cycle, irrespective of prior or future pregnancy outcomes Expression of mir-340-5p showed an overall increase in patients who had previously suffered a miscarriage and had a subsequent miscarriage, as compared to those who had infertility or previous miscarriage and subsequently went on to have a life birth outcome. The regulation of these miRNAs and their protein targets regulate the function of transport and secretion, and adhesion of the endometrial epithelia required for successful implantation in humans. Dysfunction of these miRNAs (and therefore the targets they regulate) may contribute to endometrial-derived recurrent pregnancy loss in women.

Project description:Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by persistent symptoms associated to the development of nasal polyps. To this day, the molecular mechanisms involved are still not well defined. However, it has been suggested that a sustained inflammation as allergy is involved in its onset. In this pilot study, we aimed to look into the effect of the allergic status of the patient and in their underlying mechanisms. To achieve this, we recruited 22 patients with CRSwNP and classified them in non-allergic and allergic using ImmunoCAP ISAC molecular diagnosis. Plasma samples were analyzed using liquid chromatography coupled to mass spectrometry (LC-MS). Subsequently, the identified changed metabolites from plasma that were commercially available were then analyzed by targeted analysis in some nasal polyps. Additionally, nasal polyp and mucosa tissue samples were examined for eosinophils and neutrophils. We found that 9 out of the 22 patients were sensitized to some aeroallergens (named as allergic). The other 13 patients had no sensitizations (non-allergic). Regarding metabolomics, we found that bilirubin, cortisol, lysophosphatidylcholines (LPCs) 16:0, 18:0 and 20:4 and lysophosphatidylinositol (LPI) 20:4, metabolites that are usually related to a sustained allergic inflammation, were unexpectedly increased in the plasma of non-allergic patients with CRSwNP compared to allergic patients with CRSwNP. LPC 16:0, LPC 18:0 and LPI 20:4 metabolites followed the same trend in the nasal polyp as they did in plasma. Comparison of nasal polyps with nasal mucosa tissue showed a significant increase in eosinophils (p < 0.001) and neutrophils (p < 0.01) in allergic patients with CRSwNP. There were also more eosinophils in the polyps of non-allergic patients with CRSwNP than in their nasal mucosa (p <0.01). The polyps from non-allergic patients with CRSwNP had less eosinophils than the polyps of allergic patients with CRSwNP (p < 0.05). Our data suggests that there is a systemic inflammatory response associated to CRSwNP in the absence of allergy, which could be accountable for the nasal polyp development. Allergic patients with CRSwNP presented a higher number of eosinophils located in nasal polyps suggesting that eosinophilia might be connected to the development of nasal polyps in these patients.

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Effects of anticholinergic agent on miRNAs profile and transcriptome in murine model of allergic rhinitis

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