Integrated microRNA and mRNA network analysis of the human myometrial transcriptome in the transition from quiescence to labor†,‡

Abstract

We conducted integrated transcriptomics network analyses of miRNA and mRNA interactions in human myometrium to identify novel molecular candidates potentially involved in human parturition. Myometrial biopsies were collected from women undergoing primary Cesarean deliveries in well-characterized clinical scenarios: (1) spontaneous term labor (TL, n = 5); (2) term nonlabor (TNL, n = 5); (3) spontaneous preterm birth (PTB) with histologic chorioamnionitis (PTB-HCA, n = 5); and (4) indicated PTB nonlabor (PTB-NL, n = 5). RNAs were profiled using RNA sequencing, and miRNA-target interaction networks were mined for key discriminatory subnetworks. Forty miRNAs differed between TL and TNL myometrium, while seven miRNAs differed between PTB-HCA vs. PTB-NL specimens; six of these were cross-validated using quantitative PCR. Based on the combined sequencing data, unsupervised clustering revealed two nonoverlapping cohorts that differed primarily by absence or presence of uterine quiescence, rather than gestational age or original clinical cohort. The intersection of differentially expressed miRNAs and their targets predicted 22 subnetworks with enriched representation of miR-146b-5p, miR-223-3p, and miR-150-5p among miRNAs, and of myocyte enhancer factor-2C (MEF2C) among mRNAs. Of four known MEF2 transcription factors, decreased MEF2A and MEF2C expression in women with uterine nonquiescence was observed in the sequencing data, and validated in a second cohort by quantitative PCR. Immunohistochemistry localized MEF2A and MEF2C to myometrial smooth muscle cells and confirmed decreased abundance with labor. Collectively, these results suggest altered MEF2 expression may represent a previously unrecognized process through which miRNAs contribute to the phenotypic switch from quiescence to labor in human myometrium.

Transcriptomic analysis of fetal membranes reveals pathways involved in preterm birth

“…In contrast, PTB chorioamniotic expression analysis revealed a more diverse group, further stressing the heterogeneity of this condition. This may be a consequence of the complexity of the syndrome characterization as it has been previously reported in the expression of other tissues related to PTB like the myometrium [62]. In concordance, Ngo et al [38] developed a gene expression model based on GA that predicted time until delivery for full-term but that failed for preterm deliveries.…”

Dynamic Transcriptome, Accessible Genome and PGR Cistrome Profiles in the Human Myometrium

“…Motif enrichment analysis on the DEG-associated dynamic OCRs further reveals candidate transcription regulators that may control transcription of the associated DEGs and whose actions are possibly affected by change of chromatin structure over the course of pregnancy (Table S8). Among the DNA-binding proteins that may occupy these enriched motifs, estrogen receptor, glucocorticoid receptor, progesterone receptor, MEF2C, NFAT, NFkB, AP-1 and STAT transcription factors have been studied for their roles in the myometrial biology [6, 51-54]; and CTCF and YY1 are known for their functions in regulation of chromatin conformation [55, 56]. The presence of these motifs in the dynamic OCRs suggest that changes in chromatin structure may also have an impact on the actions of these transcription and chromatin conformation regulators in modulation of associated DEGs between the two states of pregnancy.…”

“…For example, binding motifs of the estrogen receptor and the cell cycle regulator E2F1 are over-represented in the open chromatin regions found only in the NP myometrium (Table 4), which reflects the higher expression levels of genes for transcription and gene control (Table 2) and implicates the preparation of smooth muscle proliferation for the early gestation myometrial expansion under the control of estrogen receptor. Another example is the parturition associated gene MEF2A [51]. Enrichment of the MEF2A binding motif in the class III open chromatin regions (Table 4), suggest that accessibility of MEF2A to its target sites could be another layer of regulation of its action in addition to changes in expression levels [51].…”

“…Another example is the parturition associated gene MEF2A [51]. Enrichment of the MEF2A binding motif in the class III open chromatin regions (Table 4), suggest that accessibility of MEF2A to its target sites could be another layer of regulation of its action in addition to changes in expression levels [51]. Our genome-wide integrative analysis of genome accessibility and transcriptome have identified potential regulatory elements and associated molecular pathways that may be affected the chromatin accessibility (Table S8 and table S9).…”

Toll‐like receptor 9, maternal cell‐free DNA and myometrial cell response to CpG oligodeoxynucleotide stimulation

“…TLR9 mRNA levels were quantified by real‐time PCR using TaqMan probes from Applied Biosystems (Hs00152973_m1) relative to the housekeeping genes β2‐microglobulin and RPL30, as previously described . In addition, to compare TLR9 levels with other TLRs, we referred to our previously published RNA‐seq datasets of human myometrium and placenta …”