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Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma

ABSTRACT: We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%–65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%–95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18–55 years). In general, inter-individual variance of miRNA abundance was low or very low as indicated by coefficient of variation (CV) values of 0.6%–8.2%. miR-3665 and miR-1915-3p outperformed in this analysis (CVs: 0.6 and 2.4%, respectively). The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE209568 | GEO | 2022/07/25

REPOSITORIES: GEO

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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.

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Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma

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