Integrated Proteotranscriptomics of Human Myometrium in Labor Landscape Reveals the Increased Molecular Associated With Inflammation Under Hypoxia Stress

Abstract

During labor, a variety of coordinated physiological and biochemical events cause the myometrium to transition from a quiescent to contractile state; the molecular mechanisms responsible for this transition, however, remain unclear. To better understand this transition at a molecular level, the global transcriptome and proteome of human myometrial samples in labor and those not in labor were investigated through RNA sequencing (RNA-seq) and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) via data-independent acquisition (DIA) and parallel reaction monitoring (PRM) methods. Furthermore, an integrated proteotranscriptomic analysis was performed to explore biological processes and pathway alterations during labor; this analysis identified 1,626 differentially expressed mRNAs (1,101 upregulated, 525 downregulated) and 135 differentially expressed proteins (97 upregulated, 38 downregulated) in myometrium between nonlabor and in labor groups. The comprehensive results of these analyses showed that the upregulated mRNAs and proteins increased inflammation under hypoxia stress in the myometrium under labor, and related proteins and cytokines were validated by PRM and Luminex assays. Our study confirmed the biological process of inflammation and hypoxia in laboring myometrium at the transcriptome and proteome levels and provided recourse to discover new molecular and biological changes during labor.

Keywords: inflammation; labor; myometrium; proteome; transcriptome.

Figure 1

Visualization of the transcriptomics results of mRNAs between nonlabor and in labor myometrium. (A) Overall study design and workflow. (B) Volcano plot of all mRNA alteration between NL and IL groups. The horizontal dashed line corresponds to p value < 0.05, and the vertical dashed line corresponds to a 1.5-fold change decrease or increase in expression levels. Red, green, and black dots represent upregulated, downregulated, and nondifferentially expressed transcripts in the IL group (as compared to the NL group), respectively. Data associated with this figure can be found in Supplemental Table 1 . (C) Bubble diagram of partial significantly enriched pathways for DE mRNAs. (D) Bar diagram of partial biology progress GO term clusters of DE mRNAs. (E) GSEA plots of mRNA sets of hypoxia and inflammatory response.

Figure 2

Visualization of the proteomic results between nonlabor and in labor myometrium. (A) PCA plot based on DE proteins. IL samples (blue dots) clustered distinctly from NL (yellow dots). (B) Volcano plot of all protein alteration between NL and IL groups. The horizontal dashed line corresponds to p value < 0.05, and the vertical dashed line corresponds to a 1.5-fold change decrease or increase. Red, green, and black dots represent upregulated, downregulated, and nondifferentially expressed transcripts in the IL group (as compared to the NL group), respectively. Data associated with this figure can be found in Supplemental Table 4 . (C) Pie graph for classification of DE proteins showing the overall percentages of types to the DE proteins. (D) Bar diagram of partial biology progress GO term clusters of DE proteins. (E) Bubble diagram of partial significantly enriched pathways for DE proteins. (F) GSEA plots of interest sets of myometrial relaxation and contraction pathways and ADORA2B mediated anti-inflammatory cytokine production. (G) Protein–protein interactions between DE proteins. Pink circles represent upregulated proteins, green circles represent downregulated proteins, and the circles with red edges and blue edges represent complement and coagulation cascades and HIF-1 signaling pathway key proteins, respectively.

Figure 3

Visualization of the proteotranscriptomic results between nonlabor and in labor myometrium. (A) Venn diagram of the gene set identified both in transcriptome and proteome. (B) Venn diagram of gene sets of mRNA–protein pairs, DE mRNAs, and DE proteins. (C, D) Heatmaps of expression of the 24 overlapping mRNAs (C) and proteins (D). The color gradient represents gene expression levels as z-scores. (E, F) Correlation matrix of the expression levels of the 24 overlapping mRNAs (E) and proteins (F). The darker the dot, the stronger the correlation. Red indicates positive correlation; blue indicates negative correlation. (G) Quantification validation of proteins by PRM. (H, I) Quantification validation of TNF-α and IL-6 by Luminex assays. *p<0.05, **p<0.01.