Peptidome analysis of umbilical cord mesenchymal stem cell (hUC-MSC) conditioned medium from preterm and term infants

Abstract

Background: The therapeutic role of mesenchymal stem cells (MSCs) has been widely confirmed in several animal models of premature infant diseases. Micromolecule peptides have shown promise for the treatment of premature infant diseases. However, the potential role of peptides secreted from MSCs has not been studied. The purpose of this study is to help to broaden the knowledge of the hUC-MSC secretome at the peptide level through peptidomic profile analysis.

Methods: We used tandem mass tag (TMT) labeling technology followed by tandem mass spectrometry to compare the peptidomic profile of preterm and term umbilical cord MSC (hUC-MSC) conditioned medium (CM). Gene Ontology (GO) enrichment analysis and ingenuity pathway analysis (IPA) were conducted to explore the differentially expressed peptides by predicting the functions of their precursor proteins. To evaluate the effect of candidate peptides on human lung epithelial cells stimulated by hydrogen peroxide (H₂O₂), quantitative real-time PCR (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay (ELISA) were, respectively, adopted to detect inflammatory cytokines (TNF-α, IL-1β, and IL-6) expression levels at the mRNA and protein levels.

Results: A total of 131 peptides derived from 106 precursor proteins were differentially expressed in the preterm hUC-MSC CM compared with the term group, comprising 37 upregulated peptides and 94 downregulated peptides. Bioinformatics analysis showed that these differentially expressed peptides may be associated with developmental disorders, inflammatory response, and organismal injury. We also found that peptides ⁷¹¹⁸TGAKIKLVGT⁷¹²⁷ derived from MUC19 and ⁵⁰⁸AAAAGPANVH⁵¹⁷ derived from SIX5 reduced the expression levels of TNF-α, IL-1β, and IL-6 in H₂O₂-treated human lung epithelial cells.

Conclusions: In summary, this study provides further secretomics information on hUC-MSCs and provides a series of peptides that might have antiinflammatory effects on pulmonary epithelial cells and contribute to the prevention and treatment of respiratory diseases in premature infants.

Keywords: Infant diseases; LC-MS/MS; Peptidomics; TMT labeling; hUC-MSCs.

Fig. 1

Differentially expressed peptides in hUC-MSC conditioned medium (CM) from preterm infants compared with term infants. a, b Upregulated and downregulated peptides visualized using heatmaps (n = 3 per group, P1–3 represent preterm infants and T1–3 represent term infants). c, d Top 20 upregulated and top 20 downregulated peptides visualized using heatmaps

Fig. 2

Basic features of the differentially expressed peptides in hUC-MSC CM from preterm and term infants. a, b Molecular weights (MW) and isoelectric points (PI) of the differentially expressed peptides. c Scatter plot of MW versus PI of the differentially expressed peptides. d Distributions of the N- and C-terminals of the differentially expressed peptides

Fig. 3

Gene Ontology (GO) and subcellular location analysis of the differentially expressed peptide precursors. a Molecular functions. b Cellular components. c Biological processes. d Subcellular locations

Fig. 4

Diseases and regulator effects networks associated with the differentially expressed peptide precursors. Functional proteins related to a developmental disorder and b inflammatory response. Regulator effects network related to c cellular development, embryonic development, and organismal development and d organismal injury and abnormalities. Precursor proteins, diseases, and functions are shown as nodes, and the biological relationships between nodes are represented as lines with arrows. All lines are supported by at least one literature reference from the ingenuity pathway analysis (IPA) analysis. The intensity of the node color indicates the degree of upregulation (red) or downregulation (green)

Fig. 5

Function analysis of differentially expressed peptides in vitro. (a) TNF-α, IL-1β, and IL-6 mRNA expression assessed by qRT-PCR in A549 cells stimulated by H₂O₂ (1 mM) with or without peptides ⁷¹¹⁸TGAKIKLVGT⁷¹²⁷ (MUC19) and ⁵⁰⁸AAAAGPANVH⁵¹⁷ (SIX5) at concentrations of 1, 10, and 100 μM. b The protein levels of TNF-α, IL-1β, and IL-6 measured by ELISA analyze in H₂O₂-treated A549 cells with or without peptides. c, d The protein levels of TNF-α, IL-1β, and IL-6 measured by western blot in H₂O₂-treated A549 cells with or without peptides (n = 3 biological independent samples per group in qRT-PCR and WB, technical replication = 3 in ELISA. *P < 0.05, **P < 0.01, ***P < 0.001)