CPPED1-targeting microRNA-371a-5p expression in human placenta associates with spontaneous delivery

RESEARCH ARTICLE CPPED1-targeting microRNA-371a-5p expression in human placenta associates with spontaneous delivery Ravindra Daddali 1,2 , Marja OjaniemiID 1,2 , Mikko Hallman 1,2 , Mika Ra ¨met 1,2,3 , Antti M. HaapalainenID 1,2 * 1PEDEGO Research Unit and Medical Research Center Oulu, University of Oulu, Oulu, Finland, 2Department of Children and Adolescents, Oulu University Hospital, Oulu, Finland, 3BioMediTech, Institute and Faculty of Medical and Life Sciences, University of Tampere, Tampere, Finland *[email protected] Abstract MicroRNAs (miRNAs) are important regulators of gene expression, and their expression is associated with many physiological conditions. Here, we investigated potential associations between expression levels of miRNAs in human placenta and the onset of spontaneous term birth. Using RNA sequencing, we identified 54 miRNAs differentially expressed during spontaneous term labor compared to elective term births. Expression levels of 23 miRNAs were upregulated, whereas 31 were downregulated at least 1.5-fold. The upregulated miRNA miR-371a-5p putatively targets CPPED1, expression of which decreases during spontaneous birth. We used a luciferase reporter–based assay to test whether a miR-371a5p mimic affected translation when it bound to the 30untranslated region of CPPED1. In this setting, the miR-371a-5p mimic resulted in lower luciferase activity, which suggests that miR-371a-5p regulates levels of CPPED1. In conclusion, inversely correlated levels of miR371a-5p and CPPED1 suggest a role for both in spontaneous delivery. Introduction Human full-term pregnancy lasts approximately 40 weeks. Known factors that affect the maintenance of pregnancy or initiation of labor include several steroid hormones, peptide hormones, and prostaglandins. Many of these are secreted by the placenta and have effects on implantation, immunomodulation of the mother, cervical remodeling and ripening, and uterine contractions. Maternal immunomodulation in particular is vital to the tolerance of antigens of paternal origin [1–4]. Increased corticotrophin-releasing hormone in maternal plasma and secretion of fetal fibronectin into cervicovaginal fluids are indicators of delivery [5,6]. However, the timing of human labor, particularly the changes at the molecular level resulting in labor, are not currently understood. Expression of miRNAs are associated with various cellular and physiological functions, including pregnancy, inflammation, infection and immunity, gamete formation, zygote formation, embryogenesis, and fetal development [7,8]. There are three pregnancy-specific PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 1 / 21 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Daddali R, Ojaniemi M, Hallman M, Ra¨met M, Haapalainen AM (2020) CPPED1targeting microRNA-371a-5p expression in human placenta associates with spontaneous delivery. PLoS ONE 15(6): e0234403. https://doi.org/ 10.1371/journal.pone.0234403 Editor: Tamas Zakar, John Hunter Hospital, AUSTRALIA Received: February 10, 2020 Accepted: May 24, 2020 Published: June 10, 2020 Copyright: ©2020 Daddali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: MicroRNAomics data are available from the NCBI Gene Expression Omnibus (accession number GSE140870). Funding: This work was supported by the Jane and Aatos Erkko Foundation (MH and MR), Sigrid Juse ´lius Foundation (MH), Competitive State Research Financing of the Expert Responsibility Area of Oulu University Hospital (MR), and Pediatric Research Foundation (AMH). Competing interests: The authors declare no competing interests. miRNA clusters: chromosome 19 miRNA (C19MC) and chromosome 14 miRNA (C14MC) clusters and the miR-371-3 cluster. The miRNAs of these clusters are mainly expressed by the placenta, and expression levels are associated with gestational age [9–11]. The placenta secretes a characteristic pattern of miRNAs into the maternal circulation. Some placenta-secreted miRNAs also traverse into the fetus [12]. Studies have suggested roles for placental miRNAs in both pregnancy and pregnancy-related complications. In particular, C19MC and C14MC miRNAs in the maternal circulation may serve as indicators of preeclampsia and other pregnancy complications [10,13]. Perturbations in placental miRNA and changes in cervical miRNA expression levels at different time points during pregnancy are associated with preeclampsia and preterm birth [14–19]. Recent studies have also demonstrated that miRNA concentration changes are potent biomarkers for predicting preterm labor [15,20–22]. We showed previously that expression levels of calcineurin like phosphoesterase domain containing protein 1 (CPPED1) are associated with timing of birth and that spontaneous term placentas have decreased levels of CPPED1 compared to elective term placentas of deliveries without signs or symptoms of labor [23]. Additionally, expression of CPPED1 is decreased in invasive bladder cancer and overexpression of CPPED1 delays progression of the cell cycle [24]. Furthermore, CPPED1 knockdown improves insulin-stimulated glucose uptake in adipocytes [25] and there are type 2 diabetes–associated loci in the CPPED1 promotor region [26]. Recent studies characterized the antitumor role of CPPED1 by suppressing IL6 expression and secretion levels in cancer patients by activating the STAT3 pathway [27]. CPPED1 dephosphorylates phospho-Ser473 of AKT serine/threonine kinase 1 (AKT1) [24]. AKT1 is part of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, a conserved intracellular signalling pathway that plays a central role in cellular quiescence, proliferation, cancer, placental development, and fetal growth [28,29]. The PI3K reaction product phosphatidylinositol(3,4,5) triphosphate (PIP3) binds to and recruits pleckstrin homology (PH) domain–containing proteins such as 3-phosphoinositide dependent protein kinase 1 (PDK1), PDK2, and AKT1 to the cell membrane, which activates AKT1 [30]. AKT1 is inactivated by dephosphorylation of two key residues: Thr308 by protein phosphatase 2A (PP2A) and Ser473 by CPPED1 or PH domain and leucine-rich repeat protein phosphatase (PHLPP) [31,32]. FOXO1 transcription factor is an important downstream target of AKT1 [33]. Phosphorylation of FOXO1 by AKT1 results in cytosolic localization of FOXO1 and changes in the transcription of specific genes [34,35]. In the nucleus, FOXO1 and progesterone receptor B are located in the same transcriptional complex [36–38]. Progesterone, the ligand of progesterone receptor, is important for uterine quiescence and, consequently, maintaining pregnancy [2]. In the present study, we performed comparative miRNA expression profiling of placentas from spontaneous term deliveries and elective term births. We found variations in miRNA expression profiles during spontaneous term labor. By comparing the miRNAome data with our previously published human placental proteome [23], we identified a putative labor-associated miRNA:protein pair: miR-371a-5p:CPPED1. MiR-371a-5p is part of the pregnancy-specific miR-371-3 cluster. We found evidence suggesting that miR-371a-5p regulates CPPED1 mRNA during spontaneous delivery. Materials and methods Placental tissue samples Human placental tissue samples were collected at Oulu University Hospital as described previously [39] and included samples from the basal and chorionic plates of the placenta. The ethics committee of Oulu University approved the study, and all mothers provided written informed consent. All experiments were performed in accordance with relevant guidelines and PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 2 / 21 regulations. Mothers who delivered electively by caesarean section had no signs and symptoms of labor. Consequently, for our miRNAomic analyses we could use these elective term deliveries as a control to compare with spontaneous term labors to identify changes associated with the initiation of spontaneous labor. We also performed qPCR analysis of CPPED1 expression in spontaneous preterm versus spontaneous term labor to investigate whether expression changes were associated with the length of pregnancy. MiRNAomes of human placentas The miRNAomic study included placental samples from the basal plate (n= 6 per group) collected after either spontaneous term (gestational age from 40 weeks + 0 days to 41 weeks + 3 days) or elective caesarean term (gestational age from 38 weeks + 0 days to 42 weeks + 0 days) birth. Births with pregnancyor labor-associated complications such as placental abruption, polyhydramnios, and preeclampsia were excluded. MiRNAs were isolated with the NucleoSpin miRNA kit (Macherey-Nagel). RNA sample quality was good, as determined with the Fragment Analyzer (Advanced Analytical Technologies). MiRNA library preparation and HiSeq 2500 sequencing were done at the Finnish Functional Genomics Centre in Finland. Sequencing data were analyzed by the Bioinformatics Unit at the Turku Centre for Biotechnology and Biocenter Finland. A fold-change of 1.5 and pvalue of 0.05 were set as threshold values when filtering differentially expressed miRNAs. Quantitative PCR of hsa-miR-371a-5p MicroRNAs were isolated from human placentas with the NucleoSpin miRNA kit (MachereyNagel), and RNA quality was determined with the Agilent 2100 Bionalyzer system at the Biocenter Oulu Sequencing Center in Finland. First-strand cDNA synthesis was done with the miRCURY LNA RT Kit (Qiagen). The following samples were included in qRT-PCR: spontaneous term birth (n= 19) and elective term birth (n= 14). Gestational ages were 38 weeks + 6 days to 41 weeks + 6 days for spontaneous term birth and 38 weeks + 0 days to 42 weeks + 0 days for elective term birth. qPCR was done with the miRCURY LNA miRNA PCR Assay kit (Qiagen) on a LightCycler196 (Roche). Validated and optimized PCR primers hsa-miR371a-5p (YP00204493) and hsa-miR-103a-3p (YP00204063) were obtained from Qiagen. hsamiR-103a-3p mRNA was used as a reference gene. All samples were measured in triplicate and normalized in accordance with the ΔΔC t method. Melting curve analysis and agarose gel electrophoresis indicated a single and specific PCR product. Statistical analyses were done with SPSS Statistics 20.0 (IBM Corporation). Significant differences in expression levels were identified by nonparametric Mann–Whitney Utest. Quantitative PCR of CPPED1 qRT-PCR analysis included samples of the following placentas: basal plate of spontaneous preterm birth (n= 20), chorionic plate of spontaneous preterm birth (n= 20), basal plate of elective preterm birth (n= 34), chorionic plate of elective preterm birth (n= 33), basal plate of spontaneous term birth (n= 22), chorionic plate of spontaneous term birth (n= 19), basal plate of elective term birth (n= 14), and chorionic plate of elective term birth (n= 14). Gestational ages were 25 weeks + 2 days to 36 weeks + 6 days for spontaneous preterm labor, 25 weeks + 1 day to 36 weeks + 6 days for elective preterm birth, 38 weeks + 6 days to 41 weeks + 6 days for spontaneous term birth, and 38 weeks + 1 day to 42 weeks + 0 days for elective term birth. Only the spontaneous preterm and elective preterm groups contained cases of preeclampsia and intra-amniotic inflammation (Fig 1). RNeasy Micro Kit (Qiagen) was used for RNA isolation. cDNA synthesis and quantitative PCR (qPCR) were done as described PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 3 / 21 previously [23,39]. Briefly, qPCR was done as an intron-spanning assay with the LightCycler196 (Roche). Cytochrome c1 (CYC1) mRNA was used as a reference gene. All samples were measured in triplicate and normalized in accordance with the ΔΔC t method. Statistical analyses were conducted with SPSS Statistics 20.0 (IBM Corporation). Significant differences in expression levels were identified by nonparametric Kruskall–Wallis test, in which significance values were adjusted by the Bonferroni correction for multiple tests. Cloning of 30untranslated region (UTR) of CPPED1 into pmirGLO vector The 30UTR of CPPED1 was amplified by PCR with two primer pairs: 50-cacttgctagcCG CTCCTTCCCGTTCCCG-30and 50-catctgtcgacTAATTTACAAAGAAAAGAGATTTAAT AGACTC-30for the full-length 30UTR (WT), and 50-cacttgctagcCGCTCCTTCCCGT TCCCG-30and 50-catctgtcgacCAGTTCCGCATGGCTGGG-30for the 30UTR lacking the last 33 base pairs (Mut). Mismatches to the 30UTR region are indicated by lowercase, while introduced NheI and SalI sites at the 50and 30ends, respectively, of the PCR products are underlined. The PCR products WT (5087 bp) and Mut (5054 bp) were first subcloned into pJET1.2 blunt cloning vector (Thermo Scientific). Nucleotide sequences of the inserts were verified. The introduced NheI and SalI restriction sites allowed for release of the insert and ligation into the similarly digested pmirGLO vector (Promega), resulting in the full-length 30 UTR of CPPED1 (WT+pmirGLO) and a shorter variant of the 30UTR of CPPED1 (Mut+- pmirGLO) in the pmirGLO vector. Luciferase reporter assay HEK-293T cells were used in the luciferase reporter assay because of their high transfection efficiency. HEK-293T cells were cultured in DMEM (Invitrogen Life Technologies) Fig 1. CPPED1 mRNA levels in preterm and term placentas. Normalized expression levels of CPPED1 in human preterm and term placentas to compare spontaneous and elective deliveries. CPPED1 expression was decreased during spontaneous preterm birth in the chorionic plate (right panel) but not in the basal plate (left panel). At spontaneous term birth, CPPED1 mRNA levels decreased in both basal and chorionic plates. There was no difference in CPPED1 expression in placentas from preterm and term spontaneous and elective deliveries. Differences were analyzed with nonparametric Kruskall–Wallis test. Horizontal line denotes the median of each group. Statistically significant changes are indicated by one (p<0.05) and three (p<0.001) asterisks; NS, not significant. Preeclampsia and intra-amniotic inflammation cases are indicated by an open circle. https://doi.org/10.1371/journal.pone.0234403.g001 PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 4 / 21 supplemented with 10% fetal bovine serum (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 mg/ml streptomycin. Subculturing was performed with 0.05% trypsin/0.02% EDTA. Before transfection, cells were plated onto 96-well plates and allowed to reach approximately 90% confluence. On the day of transfection, HEK-293T cells were cotransfected with 100 ng of plasmid DNA (WT+pmirGLO or Mut+pmirGLO)–Lipofectamine3000 (Invitrogen Life Technologies) and miRNA (mimic or mimic negative control)-Lipofectamine3000 complexes, resulting in 10 nM miRNA mimic or negative control per well. MiRNA mimics were hsa-miR-371a-5p (MC12791, Ambion), hsa-miR-520d-5p (MC12934, Ambion), and hsa-miR524-5p (MC10753, Ambion). mirVana™miRNA Mimic, Negative Control #1 (Ambion) was used as a negative control. Transfections were performed in triplicate in two independent experiments. Cells were lysed 24 hours post-transfection, and luciferase reporter assays were performed with the Promega Dual-Luciferase Reporter Assay System (Promega). Firefly and Renilla luciferase activities were measured, and background signal was subtracted for each transfection. Firefly (experimental reporter) and Renilla (control reporter) luciferase ratios were calculated and compared with negative controls, which were set to 1.00. Significant differences were estimated with the nonparametric Mann–Whitney Utest (n= 6 per group). Results MiRNAomics of human placenta identifies miRNAs upand downregulated at spontaneous term labor MiRNAs regulate many physiological processes by affecting mRNA and protein levels [41]. To investigate whether expression levels of placental miRNAs are associated with spontaneous term labor, we characterized the miRNAomes of human placentas after spontaneous delivery (n= 6) and elective birth (n= 6). There were no signs or symptoms of labor in the cases of elective birth. miRNAomic comparisons of these two placental phenotypes revealed that 23 and 31 miRNAs were upand downregulated, respectively, during spontaneous term labor (Table 1). miR-323b-3p is a member of placenta-specific C14MC, while miR-373-3p, miR-371a-5p, miR371b-3p, miR-372-3p, and miR-372-5p are members of the placenta-specific miR-371-3 cluster (Table 1). The genes of the rest of the differentially regulated miRNAs are located in various different chromosomes. MiRNAs of C14MC are predominantly expressed during the first trimester of pregnancy, and C19MC members, together with members of the miR-371-3 cluster, are highly expressed toward the end of pregnancy [10,18]. Our data indicate that expression of selected miRNAs changes during the onset of labor. Functional annotation of miRNA targets shows enrichment of cancerand PI3K-AKT–related pathways A single miRNA can have hundreds of mRNA targets. To find out which pathways are enriched for targets of the miRNAs that we determined were upand downregulated (Table 1) during spontaneous labor, we obtained validated targets of the miRNAs from the MicroRNA-Target Database (miRTarBase) [42] (Table 2). These targets have been validated experimentally by reporter assay, western blot, or qPCR, resulting in strong evidence for the miRNA: mRNA pairing [42]. Next, we included all of the identified targets (Table 2) in an analysis performed with the DAVID functional annotation tool [43]. Within the list of identified KEGG terms, cancer-related pathways were overrepresented (Table 3). Moreover, functional annotation clustering revealed that the most significant cluster, with an enrichment score of 16.05, comprised hsa05200:Pathways in cancer, hsa05215:Prostate cancer, hsa04151:PI3K-Akt signaling pathway, and hsa04510:Focal adhesion (Table 3). Interestingly, the hsa04068:FoxO PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 5 / 21 Table 1. Upand downregulated human placenta miRNAs at spontaneous term labor. MiRNAs isolated from human placenta after spontaneous term labor were compared with miRNAs of placentas obtained from elective deliveries by caesarean section without signs or symptoms of labor. Thresholds used in filtering differentially expressed miRNAs were fold-change (FC) more or less than 1.5 and pvalue <0.05. MiRNAs were ranked based on both t-test pvalue and FC. In the average rank column, the value 1 is the strongest differentially expressed miRNA. Upand downregulated miRNAs are shown in red and green, respectively. Chromosomal locations of miRNAs belonging to either C14MC or miR-371-3 cluster are highlighted in orange. MicroRNA Average rank Fold-change pValue Chromosomal location hsa-miR-373-3p 2 2.61 0.001 19q13.42 hsa-miR-371a-5p 6 2.47 0.003 19q13.42 hsa-miR-371b-3p 7 2.34 0.003 19q13.42 hsa-miR-372-3p 11 2.16 0.007 19q13.42 hsa-miR-323b-3p 15 1.83 0.008 14q32.31 hsa-miR-760 23 1.70 0.007 1p22.1 hsa-miR-1254 26 1.63 0.007 NA hsa-miR-551a 28 1.67 0.009 1p36.32 hsa-miR-184 29 1.66 0.012 15q25.1 hsa-miR-6511b-3p 30 1.70 0.017 16p13.3 hsa-miR-4707-5p 38 1.60 0.022 14q11.2 hsa-miR-372-5p 39 1.62 0.026 19q13.42 hsa-miR-509-3p 40 1.62 0.025 Xq27.3 hsa-miR-6779-5p 44 1.54 0.018 17q12 hsa-miR-504-5p 46 1.68 0.040 Xq26.3 hsa-miR-99b-3p 48 1.49 0.014 19q13.41 hsa-miR-3140-3p 50 1.49 0.016 4q31.3 hsa-miR-133a-3p 52 1.52 0.021 18q11.2 hsa-miR-205-5p 54 1.60 0.036 1q32.2 hsa-miR-877-5p 59 1.49 0.023 6p21.33 hsa-miR-551b-5p 67 1.53 0.044 3q26.2 hsa-miR-449c-3p 71 1.46 0.033 5q11.2 hsa-miR-4665-3p 73 1.47 0.038 9p24.1 hsa-miR-4732-5p 1 -3.28 0.001 17q11.2 hsa-miR-6743-5p 4 -2.50 0.001 11p15.5 hsa-miR-1272 5 -2.86 0.005 15q22.31 hsa-miR-6765-3p 8 -2.35 0.004 14q32.33 hsa-miR-6872-3p 9 -2.08 0.004 3p21.31 hsa-miR-135a-5p 13 -4.79 0.026 3p21.2 hsa-miR-3144-3p 14 -1.97 0.009 6q22.31 hsa-miR-5100 16 -1.86 0.010 10q11.21 hsa-miR-6865-5p 17 -1.79 0.007 17p13.2 hsa-miR-501-5p 18 -1.99 0.013 Xp11.23 hsa-miR-5196-5p 19 -1.82 0.010 19q13.12 hsa-miR-2277-3p 20 -2.06 0.016 5q15 hsa-miR-1260a 21 -1.68 0.002 14q24.3 hsa-miR-1909-3p 22 -1.84 0.014 19p13.3 hsa-miR-4286 24 -1.70 0.011 8p23.1 hsa-let-7g-3p 27 -1.80 0.020 3p21.2 hsa-miR-1292-5p 34 -1.68 0.027 20p13 hsa-miR-4784 35 -1.63 0.026 2q21.1 hsa-miR-4304 37 -1.53 0.013 12q24.31 hsa-miR-3928-5p 41 -1.70 0.038 22q12.2 hsa-miR-4664-5p 42 -1.53 0.014 8q24.3 (Continued) PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 6 / 21 signaling pathway was also one of the targeted pathways. We also separately analyzed the miRNA targets of upand downregulated miRNAs (Table 2) with the DAVID functional annotation tool; these results are shown in Table 4. Cancer-related pathways were also overrepresented in this analysis, and similar KEGG terms were obtained for the targets of both upand downregulated miRNAs (Table 4). Comparison of human placental proteomics and miRNAomics identifies CPPED1 as a common denominator We previously determined the proteomics of the human placenta [23]. In this prior study, we identified ten proteins that were upor downregulated in the placenta at spontaneous term delivery. None of these ten proteins were among the validated targets of the miRNAs (Table 2) that we identified in the placenta at spontaneous term delivery. To identify putative novel targets for the miRNAs (Table 1), we investigated whether there was any overlap between the proteomics [23] and computationally predicted targets of miRNAs of miRNAomics (Table 5). We found that our 54 miRNAs were computationally predicted to bind to 27,708 targets in total, based only on the 8mers/canonical sites found in the 30UTRs of the targets. We included these 27,708 predicted miRNA targets and ten proteins identified by proteomics (actin cytoplasmic 1 [ACTB], β-2-microglobulin, keratin type II cytoskeletal 8, keratin type II cytoskeletal 19, α2-macroglobulin, CPPED1, cytochrome b5, hemoglobin subunit γ-2, peroxiredoxin-2, and plasminogen activator inhibitor 2) in the comparison and identified only three miRNA:target pairs in the basal plate of the placenta: miR-371a-5p:CPPED1, miR-3614-3p:ACTB, and miR6872-3p:ACTB. During spontaneous term delivery, miR-371a-5p was upregulated (Table 1) but CPPED1 was downregulated [23], suggesting potential post-transcriptional regulation of CPPED1 by miR-371a-5p. By contrast, levels of miR-3614-3p and miR-6872-3p were downregulated and ACTB upregulated during spontaneous term delivery. CPPED1 mRNA levels are decreased and miR371a-5p levels increased during spontaneous delivery We previously found decreased amounts of CPPED1 in the basal and chorionic plates of the placenta at spontaneous term labor [23]. Levels of mRNA of CPPED1 decreased accordingly. We extended this study to preterm labor and performed quantitative PCR (qPCR) analysis of CPPED1 mRNA levels after spontaneous (n= 20) and elective caesarean (n= 33) preterm deliveries to determine whether CPPED1 levels are associated with the initiation of labor or gestational age. We measured CPPED1 mRNA levels separately for the basal and chorionic Table 1. (Continued) MicroRNA Average rank Fold-change pValue Chromosomal location hsa-miR-451a 45 -1.62 0.033 17q11.2 hsa-miR-6069 47 -1.46 0.008 22q12.3 hsa-miR-3614-3p 49 -1.52 0.018 17q22 hsa-miR-3117-3p 53 -1.54 0.026 1p31.3 hsa-miR-7850-5p 57 -1.48 0.020 19p13.3 hsa-miR-3922-5p 58 -1.55 0.036 12q23.3 hsa-miR-1908-3p 61 -1.60 0.044 11q12.2 hsa-miR-6763-5p 62 -1.58 0.042 12q24.33 hsa-miR-6071 63 -1.50 0.027 2p11.2 hsa-miR-7977 64 -1.46 0.022 3q26.32 https://doi.org/10.1371/journal.pone.0234403.t001 PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 7 / 21 plates of the placenta (Fig 1). Moreover, we performed qPCR analysis of miR-371a5-p levels in placentas from spontaneous (n= 19) and elective (n= 14) term birth to confirm the miRNAomics finding. miR-371a-5p levels were determined only for the basal plate (Fig 2). CPPED1 mRNA levels decreased 1.5-fold (p<0.001) in the chorionic plate of the placenta at spontaneous preterm birth. We did not observe this change in the basal plate of the placenta. At spontaneous term labor, CPPED1 mRNA levels decreased in both the basal (1.2-fold, p= 0.04) and chorionic (1.5-fold, p<0.001) plates (Fig 1). However, there was no difference in basal or chorionic plate levels of CPPED1 mRNA between spontaneous preterm and spontaneous term deliveries, or between elective preterm and elective term deliveries. This suggests that CPPED1 mRNA levels do not vary in accordance with gestational age; rather, they decrease at the onset of spontaneous labor regardless of gestational age. MiR-371a-5p mRNA levels increased 1.5-fold (p= 0.04) in the basal plate of the placenta during spontaneous term Table 2. Human placental miRNAs and their experimentally validated targets. MiRNAs that were either up- (red) or down- (green) regulated at the onset of labor and have validated targets are shown. Listed miRNA targets were obtained with miRTarBase software. All miRNA–target interactions have been validated by reporter assay, western blot, and/or qPCR. miRNAs are in the same order as listed in Table 1. MicroRNA Validated targets hsa-miR-373-3p BTG1, CD44, CSDC2, DKK1, IRF9, JAK1, LATS2, LEFTY1, LEFTY2, MBD2, MRE11, MTOR, MYC, NFIB, PIK3CA, RABEP1, RAD23B, RAD52, RASSF1, RECK, SIRT1, TGFBR2, TNFAIP1, TXNIP, VEGFA, XPA hsa-miR-371a5p CDH1, HSP90AA1, PRPF4B, PTENP1, SOX2 hsa-miR-372-3p ATAD2, BTG1, CCNA1, CDK2, CDKN1A, DKK1, ERBB4, KLF13, LATS2, LEFTY1, MBNL2, NFIB, NR4A2, PHLPP2, RHOC, TGFBR2, TNFAIP1, TRPS1, TXNIP, WEE1, VEGFA hsa-miR-760 CSNK2A1, HIST1H2AD, HIST1H3D hsa-miR-1254 CCAR1 hsa-miR-551a CKB, PTP4A3 hsa-miR-184 AGO2, AKT1, AKT2, BCL2, BIN3, EZR, GAS1, INPPL1, MYC, NFATC2, PDGFB, PKM, PLPP3, PPP1R13L, PRKCB, SND1, SOX7, TNFAIP2, ZFPM2 hsa-miR-509-3p CFTR, MAP3K8, NTRK3, XIAP, YAP1 hsa-miR-504-5p BAX, BBC3, CDK6, DRD1, FAS, GADD45A, MDM2, TCEAL1, TFF1, TP53, TP53I3, VEGFA hsa-miR-99b-3p GSK3B hsa-miR-133a3p CDC42, HCN4, UCP2, KRT7, CACNA1C, HCN2, CASP9, KCNQ1, FSCN1, KCNH2, TAGLN2, LASP1, PNP, MSN, EGFR, VKORC1, PRDM16, ARPC5, FTL, EGFL7, VEGFA, PIK3R2, RGS3, COL1A1, SP1, BCL2L1, MCL1, RFFL, IGF1R, UBA2, ANXA2, MMP14, SNX30, SGMS2, PDLIM5, IGF1, ZEB1, CTGF, LDLRAP1, NR2C2, MEG3, AFTPH, ERBB2, FOXl2, SOX4, RBPJ, NGFR, GSTP1 hsa-miR-205-5p LRP1, ZEB1, DDX5, INPPL1, MED1, E2F5, E2F1, ERBB3, ZEB2, PRKCE, VEGFA, SIGMAR1, IL24, IL32, EGLN2, TP73, CYR61, CTGF, ERBB2, LAMC1, LRRK2, YES1, SRC, BCL2, SMAD1, SMAD4, YY1, PTPRM, AR, BCL6, ACSL4, ITGA5, ACSL1, HMGB3, PTEN, ESRRG, PHLPP2, KCNJ10, EZR, LMNA, RUNX2, HMGB1, PRDX2, SMAD2, UVRAG, CENPF, SATB2, CCNJ hsa-miR-135a5p JAK2, NR3C2, APC, HOXA10, MYC, SMAD5, BMPR2, STAT6, MTSS1, ROCK1, VLDLR, TXNIP, IRS2, APC, HTR1A, SLC6A4, SIAH1, BCL2, KLF8, ESRRA, RUNX2, ROCK2, FOXO1, PHLPP2, E2F1, DAPK2, KLF4, PTPRD, PTK2, CEBPD, PPM1E, MMP11, RBAK, EGFR hsa-miR-501-5p AKT2, DKK1, DNAJB14, INPPL1, LAMTOR5, NFATC2, NKD1 hsa-let-7g-3p CCL2, CCL5, MYBPC3 hsa-miR-12925p SOX4 hsa-miR-4304 AKT2, INPPL1, NFATC2 hsa-miR-451a ABCB1, ADAM10, AKT1, BCL2, CAB39, CDKN2D, CPNE3, DCBLD2, FRZB, IKBKB, IL6, IL6R, MAP3K1, MAPK1, MIF, MMP2, MMP9, MYC, OSR1, OXTR, PKD1, RAB14, RAB5A, ROR2, TMED7, TSC1 https://doi.org/10.1371/journal.pone.0234403.t002 PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 8 / 21 Table 3. Functional annotation of miRNA targets associated with spontaneous term birth. Clustering was performed by DAVID analysis [43] according to KEGG pathways and used all of the identified miRNA targets listed in Table 2. KEGG terms with false discovery rate (FDR)–adjusted pvalues of <0.05 are shown. In functional annotation clustering, KEGG terms highlighted in orange comprised the most significant cluster, with an enrichment score of 16.05. KEGG term Number of genes involved in the KEGG term pValue Benjamini-Hochberg–corrected pvalue FDR-adjusted p value hsa05200:Pathways in cancer 50 6.30 ×10 −24 1.27 ×10 −21 7.90 ×10 −21 hsa05215:Prostate cancer 24 1.07 ×10 −18 1.08 ×10 −16 1.34 ×10 −15 hsa05161:Hepatitis B 28 1.05 ×10 −17 7.05 ×10 −16 1.32 ×10 −14 hsa05205:Proteoglycans in cancer 31 7.33 ×10 −17 5.55 ×10 −15 1.44 ×10 −13 hsa05212:Pancreatic cancer 19 2.61 ×10 −15 1.03 ×10 −13 3.21 ×10 −12 hsa05206:MicroRNAs in cancer 33 3.87 ×10 −14 1.29 ×10 −12 4.85 ×10 −11 hsa04151:PI3K-Akt signaling pathway 36 4.07 ×10 −14 1.17 ×10 −12 5.10 ×10 −11 hsa05219:Bladder cancer 15 1.44 ×10 −13 3.61 ×10 −12 1.80 ×10 −10 hsa04068:FoxO signaling pathway 23 2.33 ×10 −13 5.21 ×10 −12 2.93 ×10 −10 hsa05214:Glioma 17 7.59 ×10 −13 1.52 ×10 −11 9.52 ×10 −10 hsa05218:Melanoma 16 4.43 ×10 −11 8.09 ×10 −10 5.55 ×10 −08 hsa05222:Small cell lung cancer 17 6.19 ×10 −11 1.04 ×10 −09 7.76 ×10 −08 hsa05210:Colorectal cancer 15 7.80 ×10 −11 1.21 ×10 −09 9.79 ×10 −08 hsa05213:Endometrial cancer 14 9.58 ×10 −11 1.37 ×10 −09 1.20 ×10 −07 hsa04510:Focal adhesion 24 2.35 ×10 −10 3.15 ×10 −09 2.95 ×10 −07 hsa04115:p53 signaling pathway 15 2.38 ×10 −10 2.98 ×10 −09 2.98 ×10 −07 hsa04066:HIF-1 signaling pathway 17 4.19 ×10 −10 4.95 ×10 −09 5.25 ×10 −07 hsa05220:Chronic myeloid leukemia 15 6.57 ×10 −10 7.33 ×10 −09 8.24 ×10 −07 hsa04012:ErbB signaling pathway 16 9.26 ×10 −10 9.80 ×10 −09 1.16 ×10 −06 hsa04550:Signaling pathways regulating pluripotency of stem cells 19 2.55 ×10 −09 2.56 ×10 −08 3.20 ×10 −06 hsa05223:Non-small cell lung cancer 13 3.56 ×10 −09 3.41 ×10 −08 4.46 ×10 −06 hsa05202:Transcriptional misregulation in cancer 20 7.18 ×10 −09 6.56 ×10 −08 9.01 ×10 −06 hsa05230:Central carbon metabolism in cancer 13 1.78 ×10 −08 1.56 ×10 −07 2.24 ×10 −05 hsa05162:Measles 17 5.41 ×10 −08 4.53 ×10 −07 6.79 ×10 −05 hsa04520:Adherens junction 13 6.05 ×10 −08 4.86 ×10 −07 7.59 ×10 −05 hsa04150:mTOR signaling pathway 12 6.35 ×10 −08 4.91 ×10 −07 7.96 ×10 −05 hsa04370:VEGF signaling pathway 12 1.10 ×10 −07 8.17 ×10 −07 1.38 ×10 −04 hsa04210:Apoptosis 12 1.31 ×10 −07 9.39 ×10 −07 1.64 ×10 −04 hsa05166:HTLV-I infection 22 3.12 ×10 −07 2.16 ×10 −06 3.92 ×10 −04 hsa04919:Thyroid hormone signaling pathway 15 3.31 ×10 −07 2.22 ×10 −06 4.16 ×10 −04 hsa04350:TGF-beta signaling pathway 13 4.12 ×10 −07 2.67 ×10 −06 5.17 ×10 −04 hsa04722:Neurotrophin signaling pathway 15 5.64 ×10 −07 3.54 ×10 −06 7.08 ×10 −04 hsa05169:Epstein-Barr virus infection 15 6.92 ×10 −07 4.22 ×10 −06 8.68 ×10 −04 hsa04152:AMPK signaling pathway 15 7.66 ×10 −07 4.53 ×10 −06 9.61 ×10 −04 hsa04110:Cell cycle 15 8.46 ×10 −07 4.86 ×10 −06 1.06 ×10 −03 hsa04390:Hippo signaling pathway 16 1.77 ×10 −06 9.87 ×10 −06 2.22 ×10 −03 hsa04071:Sphingolipid signaling pathway 14 3.40 ×10 −06 1.85 ×10 −05 4.26 ×10 −03 hsa04668:TNF signaling pathway 13 5.71 ×10 −06 3.02 ×10 −05 7.16 ×10 −03 hsa05164:Influenza A 16 1.03 ×10 −05 5.32 ×10 −05 1.30 ×10 −03 hsa04930:Type II diabetes mellitus 9 1.17 ×10 −05 5.90 ×10 −05 1.47 ×10 −02 hsa05203:Viral carcinogenesis 17 1.83 ×10 −05 8.98 ×10 −05 2.30 ×10 −02 hsa05231:Choline metabolism in cancer 12 1.88 ×10 −05 9.01 ×10 −05 2.36 ×10 −02 hsa04666:Fc gamma R-mediated phagocytosis 11 2.08 ×10 −05 9.73 ×10 −05 2.61 ×10 −02 hsa05142:Chagas disease (American trypanosomiasis) 12 2.49 ×10 −05 1.14 ×10 −05 3.12 ×10 −02 https://doi.org/10.1371/journal.pone.0234403.t003 PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 9 / 21 as a hydatidiform mole (HM), which may develop into malignant gestational trophoblastic neoplasia (GTN). MiR-371a-5p levels are upregulated in GTN, and elevated levels are associated with enhanced proliferation, differentiation, and invasion in choriocarcinoma cells [54]. Recurrent pregnancy loss is a condition in which apoptosis dominates trophoblastic growth, which results in early pregnancy termination. Expression of miR-371-5p and its target gene Xlinked inhibitor of apoptosis protein (XIAP) are greatly decreased and associated with increased apoptosis leading to pregnancy loss [55]. These findings suggest that in uncomplicated pregnancies, miR-371a-5p levels increase toward the end of pregnancy and aberrant expression of miR-317a-5p, especially in early pregnancy, results in severe adverse outcomes. In addition to miR-371-3 cluster miRNAs, there are putative binding sites for C19MC miRNAs in the 30UTR region of CPPED1. We tested the effect of two miRNAs, miR-520d-5p and miR-524-5p of the C19MC, on the expression of CPPED1 and showed that miR-520d-5p binds to the 30UTR of CPPED1. Earlier studies showed that miR-520d is a placenta-specific miRNA and is secreted into the maternal circulation [9,56]. Targets of miR-520d-5p are Fig 3. Binding of miRNAs in 30UTR of CPPED1.Seed locations of miRNAs analyzed for binding to the 30UTR of CPPED1 by luciferase reporter assay (a). MiR-371a-5p has three seed locations, starting at 1602, 2033, and 4607 nucleotides. MiR-520d-5p and miR-524-5p share the same seed location in the end of 30 UTR of CPPED1, which starts at nucleotide 5076. Seed locations are not to scale with respect to one another. For the luciferase assays, two variants of the 30UTR of CPPED1 were constructed. One construct contained the full-length 30UTR (WT). The other construct (Mut) lacked the 33 terminal nucleotides containing the miRNA binding sites for miR-520d-5p and miR-524-5p. Black dots roughly mark every thousand kilobases (kb). Bottom part of the figure shows luciferase reporter–based assays to detect binding of miR-371a-5p (b), miR-520d-5p (c), and miR-524-5p (d) mimics to the 30UTR of CPPED1 mRNA. WT or Mut constructs were cotransfected with miR371a-5p mimic (WT/miR-371a-5p) (b), miR-520d-5p mimic (WT/miR-520d-5p or Mut/miR-520d-5p) (c), or miR-5245p mimic (WT/miR-524-5p or Mut/miR-524-5p) (d) into HEK-293T cells. For WT miR-neg and Mut miR-neg, the miRNA mimic negative control was cotransfected with WT or Mut constructs, respectively. Luciferase activities were measured by dual luciferase reporter assay, in which Firefly luciferase was the experimental reporter and Renilla luciferase the control reporter. Negative control values were set to 1.00 (WT/miR-neg), to which the rest of the samples were compared. Columns show the mean value of relative luciferase activity of six replicates; maximum and minimum values are also indicated. Statistically significant changes were determined by Mann–Whitney Utest and are indicated by an asterisk (p<0.05) or two asterisks (p<0.01). NS, not significant. https://doi.org/10.1371/journal.pone.0234403.g003 PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 16 / 21 related to cancer progression. In colorectal cancer, reduced levels of miR-520d-5p lead to cancer progression by enhancing proliferation, migration, and invasion of cancer cells [57]. By contrast, higher levels of miR-520d-5p restore E-cadherin expression, which results in reduced cancer cell motility and invasiveness. Consequently, higher miR-520d-5p levels are associated with higher survival rates in cancer patients [58]. Specific cancer types and the placenta share certain properties, such as a high proliferation rate, invasion of tissue, and modulation of the host immune response [59]. Previous findings have indicated that expression of miR-371a-5p or miR-520d-5p affects cancer phenotypes, including cell proliferation, cell motility, and, ultimately, cancer progression. We found that miR-371a-5p levels were upregulated in spontaneous term delivery compared to elective caesarean section delivery without signs of labor. Thus, during spontaneous labor, upregulation of miRNA-371a-5p in the human placenta could suppress cell functions related to immune tolerance in a manner similar to what is observed during cancer progression [58]. We propose that this may be mediated in part by decreasing levels of the phosphatase CPPED1. In line with this, siRNA silencing of CPPED1 in cultured trophoblasts affects pathways involved in blood vessel development and cytokine activity [23], which is also seen in tumors and trophoblastic invasion [60,61]. Our pathway analysis of the validated targets of miRNAs that we identified in our investigation of the placental miRNAome was enriched for several cancer-related pathways. This provides further evidence that miRNAs associated with spontaneous labor are also involved in carcinogenesis. Additionally, the PI3K-AKT and FOXO signalling pathways were among the enriched pathways. AKT regulates the activity of FOXO, which affects genes involved in the cell cycle, apoptosis, and the immune system [62]. FOXOs also contribute to preeclampsia, fetal growth restriction, and spontaneous labor [63,64]. In conclusion, our results suggest that high expression levels of miR-371a-5p are associated with decreased levels of CPPED1. In our previous study, high levels of CPPED1 were associated with a long duration of pregnancy whereas low expression and low levels of CPPED1 signaled spontaneous labor, with possible involvement of CPPED1 phosphatase activity [23]. Earlier studies also showed that high levels of miR-371a-5 and low levels of CPPED1 are associated with striking functional effects in cancer tissue [24,54]. The mechanism by which placental miR-371a-5p and CPPED1 promote labor is a topic for future investigations. Acknowledgments We acknowledge the technical assistance of Maarit Haarala and sample collection by Dr. Mari Mahlman, Riitta Vikeva¨inen, Miia Lehto, and Sonja Eeli. HEK-293T cells were kindly provided by Professor Peppi Karppinen, Faculty of Biochemistry and Molecular Medicine and Biocenter, Oulu, Finland. We thank the Medical Bioinformatics Centre of Turku Bioscience for the miRNA-seq data analysis. The Centre is supported by University of Turku, Åbo Akademi University, Biocenter Finland, and Elixir-Finland. Author Contributions Formal analysis: Ravindra Daddali, Antti M. Haapalainen. Funding acquisition: Mikko Hallman, Mika Ra¨met, Antti M. Haapalainen. Investigation: Ravindra Daddali, Marja Ojaniemi, Mikko Hallman, Mika Ra¨met, Antti M. Haapalainen. Methodology: Ravindra Daddali, Antti M. Haapalainen. Project administration: Mikko Hallman, Mika Ra¨met. PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 17 / 21 Supervision: Mikko Hallman, Mika Ra¨met, Antti M. Haapalainen. Validation: Ravindra Daddali, Marja Ojaniemi, Antti M. Haapalainen. Writing – original draft: Ravindra Daddali, Mikko Hallman, Mika Ra¨met, Antti M. Haapalainen. Writing – review & editing: Ravindra Daddali, Marja Ojaniemi, Mikko Hallman, Mika Ra¨met, Antti M. Haapalainen. References 1. Schumacher A, Costa SD, Zenclussen AC. Endocrine factors modulating immune responses in pregnancy. Front Immunol. 2014; 5: 196. https://doi.org/10.3389/fimmu.2014.00196 PMID: 24847324 2. Nair RR, Verma P, Singh K. Immune-endocrine crosstalk during pregnancy. Gen Comp Endocrinol. 2016. 3. Casey ML, MacDonald PC. The endocrinology of human parturition. Ann N Y Acad Sci. 1997; 828: 273–284. https://doi.org/10.1111/j.1749-6632.1997.tb48548.x PMID: 9329848 4. Smith R. Parturition. N Engl J Med. 2007; 356: 271–283. https://doi.org/10.1056/NEJMra061360 PMID: 17229954 5. Leung TN, Chung TK, Madsen G, Lam PK, Sahota D, Smith R. Rate of rise in maternal plasma corticotrophin-releasing hormone and its relation to gestational length. BJOG. 2001; 108: 527–532. https://doi. org/10.1111/j.1471-0528.2001.00112.x PMID: 11368140 6. Kiss H, Ahner R, Hohlagschwandtner M, Leitich H, Husslein P. Fetal fibronectin as a predictor of term labor: a literature review. Acta Obstet Gynecol Scand. 2000; 79: 3–7. PMID: 10646808 7. Valinezhad Orang A, Safaralizadeh R, Kazemzadeh-Bavili M. Mechanisms of miRNA-Mediated Gene Regulation from Common Downregulation to mRNA-Specific Upregulation. Int J Genomics. 2014; 2014: 970607. https://doi.org/10.1155/2014/970607 PMID: 25180174 8. Ambros V. The functions of animal microRNAs. Nature. 2004; 431: 350–355. https://doi.org/10.1038/ nature02871 PMID: 15372042 9. Morales-Prieto DM, Ospina-Prieto S, Chaiwangyen W, Schoenleben M, Markert UR. Pregnancy-associated miRNA-clusters. J Reprod Immunol. 2013; 97: 51–61. https://doi.org/10.1016/j.jri.2012.11.001 PMID: 23432872 10. Morales-Prieto DM, Chaiwangyen W, Ospina-Prieto S, Schneider U, Herrmann J, Gruhn B, et al. MicroRNA expression profiles of trophoblastic cells. Placenta. 2012; 33: 725–734. https://doi.org/10.1016/j. placenta.2012.05.009 PMID: 22721760 11. Gu Y, Sun J, Groome LJ, Wang Y. Differential miRNA expression profiles between the first and third trimester human placentas. Am J Physiol Endocrinol Metab. 2013; 304: 836. 12. Chang G, Mouillet JF, Mishima T, Chu T, Sadovsky E, Coyne CB, et al. Expression and trafficking of placental microRNAs at the feto-maternal interface. FASEB J. 2017; 31: 2760–2770. https://doi.org/10. 1096/fj.201601146R PMID: 28289056 13. Kotlabova K, Doucha J, Hromadnikova I. Placental-specific microRNA in maternal circulation—identification of appropriate pregnancy-associated microRNAs with diagnostic potential. J Reprod Immunol. 2011; 89: 185–191. https://doi.org/10.1016/j.jri.2011.02.006 PMID: 21513988 14. Winger EE, Reed JL, Ji X. Early first trimester peripheral blood cell microRNA predicts risk of preterm delivery in pregnant women: Proof of concept. PLoS One. 2017; 12: e0180124. https://doi.org/10.1371/ journal.pone.0180124 PMID: 28692679 15. Gray C, McCowan LM, Patel R, Taylor RS, Vickers MH. Maternal plasma miRNAs as biomarkers during mid-pregnancy to predict later spontaneous preterm birth: a pilot study. Sci Rep. 2017; 7: 815–8. https://doi.org/10.1038/s41598-017-00713-8 PMID: 28400603 16. Zhu XM, Han T, Sargent IL, Yin GW, Yao YQ. Differential expression profile of microRNAs in human placentas from preeclamptic pregnancies vs normal pregnancies. Am J Obstet Gynecol. 2009; 200: 661.e1–661.e7. 17. Elovitz MA, Brown AG, Anton L, Gilstrop M, Heiser L, Bastek J. Distinct cervical microRNA profiles are present in women destined to have a preterm birth. Am J Obstet Gynecol. 2014; 210: 221.e1–221.11. 18. Morales Prieto DM, Markert UR. MicroRNAs in pregnancy. J Reprod Immunol. 2011; 88: 106–111. https://doi.org/10.1016/j.jri.2011.01.004 PMID: 21353310 PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 18 / 21 19. Fu G, Brkic J, Hayder H, Peng C. MicroRNAs in Human Placental Development and Pregnancy Complications. Int J Mol Sci. 2013; 14: 5519–5544. https://doi.org/10.3390/ijms14035519 PMID: 23528856 20. Hromadnikova I, Kotlabova K, Ondrackova M, Kestlerova A, Novotna V, Hympanova L, et al. Circulating C19MC microRNAs in preeclampsia, gestational hypertension, and fetal growth restriction. Mediators Inflamm. 2013; 2013: 186041. https://doi.org/10.1155/2013/186041 PMID: 24347821 21. Hromadnikova I, Kotlabova K, Ondrackova M, Pirkova P, Kestlerova A, Novotna V, et al. Expression profile of C19MC microRNAs in placental tissue in pregnancy-related complications. DNA Cell Biol. 2015; 34: 437–457. https://doi.org/10.1089/dna.2014.2687 PMID: 25825993 22. Elovitz MA, Anton L, Bastek J, Brown AG. Can microRNA profiling in maternal blood identify women at risk for preterm birth? Am J Obstet Gynecol. 2015; 212: 782.e1–782.e5. 23. Haapalainen AM, Karjalainen MK, Daddali R, Ohlmeier S, Anttonen J, Maatta TA, et al. Expression of CPPED1 in human trophoblasts is associated with timing of term birth. J Cell Mol Med. 2018; 22: 968– 981. https://doi.org/10.1111/jcmm.13402 PMID: 29193784 24. Zhuo DX, Zhang XW, Jin B, Zhang Z, Xie BS, Wu CL, et al. CSTP1, a novel protein phosphatase, blocks cell cycle, promotes cell apoptosis, and suppresses tumor growth of bladder cancer by directly dephosphorylating Akt at Ser473 site. PLoS One. 2013; 8: e65679. https://doi.org/10.1371/journal. pone.0065679 PMID: 23799035 25. Vaittinen M, Kaminska D, Kakela P, Eskelinen M, Kolehmainen M, Pihlajamaki J, et al. Downregulation of CPPED1 expression improves glucose metabolism in vitro in adipocytes. Diabetes. 2013; 62: 3747– 3750. https://doi.org/10.2337/db13-0830 PMID: 23939394 26. Johnson ME, Zhao J, Schug J, Deliard S, Xia Q, Guy VC, et al. Two novel type 2 diabetes loci revealed through integration of TCF7L2 DNA occupancy and SNP association data. BMJ Open Diabetes Res Care. 2014; 2: e000052. https://doi.org/10.1136/bmjdrc-2014-000052 PMID: 25469308 27. Zhuo D, Wu Y, Luo J, Deng L, Niu X. CSTP1 inhibits IL-6 expression through targeting Akt/FoxO3a signaling pathway in bladder cancer cells. Exp Cell Res. 2019; 380: 80–89. https://doi.org/10.1016/j.yexcr. 2019.04.019 PMID: 31002815 28. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007; 129: 1261–1274. https://doi.org/10.1016/j.cell.2007.06.009 PMID: 17604717 29. Yang ZZ, Tschopp O, Hemmings-Mieszczak M, Feng J, Brodbeck D, Perentes E, et al. Protein kinase B alpha/Akt1 regulates placental development and fetal growth. J Biol Chem. 2003; 278: 32124–32131. https://doi.org/10.1074/jbc.M302847200 PMID: 12783884 30. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer. 2002; 2: 489–501. https://doi.org/10.1038/nrc839 PMID: 12094235 31. Gao T, Furnari F, Newton AC. PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell. 2005; 18: 13–24. https://doi.org/10.1016/j.molcel. 2005.03.008 PMID: 15808505 32. Hemmings BA, Restuccia DF. PI3K-PKB/Akt pathway. Cold Spring Harb Perspect Biol. 2012; 4: a011189. https://doi.org/10.1101/cshperspect.a011189 PMID: 22952397 33. Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, et al. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell. 2006; 127: 125–137. https://doi.org/10.1016/j.cell.2006.08.033 PMID: 16962653 34. Rena G, Guo S, Cichy SC, Unterman TG, Cohen P. Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B. J Biol Chem. 1999; 274: 17179–17183. https://doi.org/10. 1074/jbc.274.24.17179 PMID: 10358075 35. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 1999; 96: 857–868. https://doi.org/10.1016/ s0092-8674(00)80595-4 PMID: 10102273 36. Lam EW, Shah K, Brosens JJ. The diversity of sex steroid action: the role of micro-RNAs and FOXO transcription factors in cycling endometrium and cancer. J Endocrinol. 2012; 212: 13–25. https://doi.org/ 10.1530/JOE-10-0480 PMID: 21382987 37. Kim JJ, Buzzio OL, Li S, Lu Z. Role of FOXO1A in the regulation of insulin-like growth factor-binding protein-1 in human endometrial cells: interaction with progesterone receptor. Biol Reprod. 2005; 73: 833– 839. https://doi.org/10.1095/biolreprod.105.043182 PMID: 15987820 38. Vasquez YM, Mazur EC, Li X, Kommagani R, Jiang L, Chen R, et al. FOXO1 is required for binding of PR on IRF4, novel transcriptional regulator of endometrial stromal decidualization. Mol Endocrinol. 2015; 29: 421–433. https://doi.org/10.1210/me.2014-1292 PMID: 25584414 39. Karjalainen MK, Ojaniemi M, Haapalainen AM, Mahlman M, Salminen A, Huusko JM, et al. CXCR3 Polymorphism and Expression Associate with Spontaneous Preterm Birth. J Immunol. 2015; 195: 2187–2198. https://doi.org/10.4049/jimmunol.1501174 PMID: 26209629 PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 19 / 21 40. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002; 30: 207–210. https://doi.org/10.1093/nar/30.1.207 PMID: 11752295 41. Janas MM, Novina CD. Not lost in translation: stepwise regulation of microRNA targets. EMBO J. 2012; 31: 2446–2447. https://doi.org/10.1038/emboj.2012.119 PMID: 22522704 42. Chou CH, Shrestha S, Yang CD, Chang NW, Lin YL, Liao KW, et al. miRTarBase update 2018: a resource for experimentally validated microRNA-target interactions. Nucleic Acids Res. 2018; 46: D296–D302. https://doi.org/10.1093/nar/gkx1067 PMID: 29126174 43. Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009; 37: 1–13. https://doi.org/10. 1093/nar/gkn923 PMID: 19033363 44. Wong N, Wang X. miRDB: an online resource for microRNA target prediction and functional annotations. Nucleic Acids Res. 2015; 43: 146. 45. Wang X. Improving microRNA target prediction by modeling with unambiguously identified microRNAtarget pairs from CLIP-ligation studies. Bioinformatics. 2016; 32: 1316–1322. https://doi.org/10.1093/ bioinformatics/btw002 PMID: 26743510 46. Gracie S, Pennell C, Ekman-Ordeberg G, Lye S, McManaman J, Williams S, et al. An integrated systems biology approach to the study of preterm birth using "-omic" technology—a guideline for research. BMC Pregnancy Childbirth. 2011; 11: 71–71. https://doi.org/10.1186/1471-2393-11-71 PMID: 21992798 47. Kacerovsky M, Lenco J, Musilova I, Tambor V, Lamont R, Torloni MR, et al. Proteomic biomarkers for spontaneous preterm birth: a systematic review of the literature. Reprod Sci. 2014; 21: 283–295. https://doi.org/10.1177/1933719113503415 PMID: 24060632 48. Ngo TTM, Moufarrej MN, Rasmussen MH, Camunas-Soler J, Pan W, Okamoto J, et al. Noninvasive blood tests for fetal development predict gestational age and preterm delivery. Science. 2018; 360: 1133–1136. https://doi.org/10.1126/science.aar3819 PMID: 29880692 49. Paikari A D Belair C, Saw D, Blelloch R. The eutheria-specific miR-290 cluster modulates placental growth and maternal-fetal transport. Development. 2017; 144: 3731–3743. https://doi.org/10.1242/dev. 151654 PMID: 28935707 50. Pfaff N, Moritz T, Thum T, Cantz T. miRNAs involved in the generation, maintenance, and differentiation of pluripotent cells. J Mol Med (Berl). 2012; 90: 747–752. https://doi.org/10.1007/s00109-012-0922-z PMID: 22684238 51. Wu S, Aksoy M, Shi J, Houbaviy HB. Evolution of the miR-290-295/miR-371-373 cluster family seed repertoire. PLoS One. 2014; 9: e108519. https://doi.org/10.1371/journal.pone.0108519 PMID: 25268927 52. Medeiros LA, Dennis LM, Gill ME, Houbaviy H, Markoulaki S, Fu D, et al. Mir-290-295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects. Proc Natl Acad Sci U S A. 2011; 108: 14163–14168. https://doi.org/10.1073/pnas.1111241108 PMID: 21844366 53. Wei Q, Sun Z, He X, Tan T, Lu B, Guo X, et al. Derivation of rhesus monkey parthenogenetic embryonic stem cells and its microRNA signature. PLoS One. 2011; 6: e25052. https://doi.org/10.1371/journal. pone.0025052 PMID: 21966410 54. Zhao JR, Cheng WW, Wang YX, Cai M, Wu WB, Zhang HJ. Identification of microRNA signature in the progression of gestational trophoblastic disease. Cell Death Dis. 2018; 9: 94–2. https://doi.org/10.1038/ s41419-017-0108-2 PMID: 29367697 55. Du E, Cao Y, Feng C, Lu J, Yang H, Zhang Y. The Possible Involvement of miR-371a-5p Regulating XIAP in the Pathogenesis of Recurrent Pregnancy Loss. Reprod Sci. 2019: 1933719119828051. 56. Gilad S, Meiri E, Yogev Y, Benjamin S, Lebanony D, Yerushalmi N, et al. Serum microRNAs are promising novel biomarkers. PLoS One. 2008; 3: e3148. https://doi.org/10.1371/journal.pone.0003148 PMID: 18773077 57. Yan L, Yu J, Tan F, Ye GT, Shen ZY, Liu H, et al. SP1-mediated microRNA-520d-5p suppresses tumor growth and metastasis in colorectal cancer by targeting CTHRC1. Am J Cancer Res. 2015; 5: 1447– 1459. PMID: 26101709 58. Tsukerman P, Yamin R, Seidel E, Khawaled S, Schmiedel D, Bar-Mag T, et al. MiR-520d-5p directly targets TWIST1 and downregulates the metastamiR miR-10b. Oncotarget. 2014; 5: 12141–12150. https:// doi.org/10.18632/oncotarget.2559 PMID: 25426550 59. Holtan SG, Creedon DJ, Haluska P, Markovic SN. Cancer and pregnancy: parallels in growth, invasion, and immune modulation and implications for cancer therapeutic agents. Mayo Clin Proc. 2009; 84: 985–1000. https://doi.org/10.1016/S0025-6196(11)60669-1 PMID: 19880689 PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 20 / 21 60. Bischof P, Campana A. A putative role for oncogenes in trophoblast invasion? Hum Reprod. 2000; 15 Suppl 6: 51–58. 61. Hess AP, Hamilton AE, Talbi S, Dosiou C, Nyegaard M, Nayak N, et al. Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators. Biol Reprod. 2007; 76: 102–117. https://doi.org/10.1095/biolreprod.106.054791 PMID: 17021345 62. Zhang X, Tang N, Hadden TJ, Rishi AK. Akt, FoxO and regulation of apoptosis. Biochim Biophys Acta. 2011; 1813: 1978–1986. https://doi.org/10.1016/j.bbamcr.2011.03.010 PMID: 21440011 63. Lim R, Barker G, Lappas M. A novel role for FOXO3 in human labor: increased expression in laboring myometrium, and regulation of proinflammatory and prolabor mediators in pregnant human myometrial cells. Biol Reprod. 2013; 88: 156. https://doi.org/10.1095/biolreprod.113.108126 PMID: 23636809 64. Sheridan R, Belludi C, Khoury J, Stanek J, Handwerger S. FOXO1 expression in villous trophoblast of preeclampsia and fetal growth restriction placentas. Histol Histopathol. 2015; 30: 213–222. https://doi. org/10.14670/HH-30.213 PMID: 25202916 PLOS ONE MicroRNAs in spontaneous term delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0234403 June 10, 2020 21 / 21