The Neuropeptide-Related HERC5/TAC1 Interactions May Be Associated with the Dysregulation of lncRNA GAS5 Expression in Gestational Diabetes Mellitus Exosomes

Abstract

Objective: The goal of this work was to look at the expression and probable role of exosomal long noncoding RNA (lncRNA) GAS5 in gestational diabetes mellitus (GDM), as well as forecast the importance of its interaction with neuropeptides in the progression of the disease.

Methods: We divided 44 pregnant women visiting the obstetric outpatient clinics at the Affiliated Hospital of Guilin Medical College from January 2021 to December 2021 into healthy and GDM groups. We measured the expression levels of the lncRNA GAS5 in peripheral blood using PCR and compared the expression levels between the 2 groups. The Gene Expression Omnibus (GEO) database and the R software were used to analyse the differences in the genes expressed in the amniotic fluid cells in the GDM and normal groups. catRAPID was used to identify potential target proteins for GAS5. Key neuropeptide-related proteins and potential target proteins of GAS5 were extracted, and protein interaction networks were mapped. AlphaFold 2 was used to predict the structure of the target protein. The ClusPro tool was used to predict protein-protein interactions. ZDOCK was used to further confirm the protein-nucleic acid docking.

Results: The lncRNA GAS5 was downregulated in the peripheral blood of pregnant women with GDM compared with normal pregnant women. The subcellular localization sites of GAS5 were the nucleus, cytoplasm, and ribosome; in addition, GAS5 was present in exosomes. Intercellular interactions, including neuropeptide receptors, were increased in the amniotic fluid cells of patients with GDM. Venn diagram analysis yielded seven neuropeptide-related proteins and three GAS5 target proteins. Among them, HERC5/TAC1 interacted and GAS5 docked well with HERC5.

Conclusion: The lncRNA GAS5 in the peripheral blood exosomes in patients with GDM may be a new target for the detection of GDM, and the interaction between GAS5 and HERC5/TAC1 may be involved in the pathogenesis of GDM.

Figure 1

Expression and functional characterisation of GAS5 in diabetes mellitus. (a) Expression characteristics of long noncoding RNA (lncRNA) GAS5 in the serum of patients with diabetes. (b) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment profiles of genes that were downregulated in women with GDM. (c) GO and KEGG enrichment profiles of genes that were upregulated in patients with diabetes.

Figure 2

Common DEGs between neuropeptide-related genes and GAS5 target protein genes in the amniotic fluid cells of patients with gestational diabetes mellitus (GDM). (a) Venn diagram showing 91 intersecting genes among the differentially expressed genes and neuropeptide-related genes in the amniotic fluid cells of women with GDM. (b) Heat map and expression characterisation of the 91 intersecting genes. (c) Seven proteins with neuropeptide-related scores > 5 (prepronociceptin (PNOC), secretin (SCT), chromogranin A (CHGA), secretogranin II (SCG2), membrane metalloendopeptidase (MME), tachykinin 1 (TAC1), and neuromedin U receptor 2 (NMUR2)). (d) Intersection analysis of GAS5 target proteins with 91 intersecting genes was performed to identify three target proteins (DEAD box polypeptide 60-like (DDX60L), HECT and RLD domain-containing E3 ubiquitin protein ligase 5 (HERC5), and interferon induced with helicase C domain 1 (IFIH1)). (e) mRNA expression corresponding to the seven neuropeptide-related proteins and three GAS5 target proteins was plotted on a volcano map.

Figure 3

Correlation analysis, screening, and structure prediction of key proteins. (a) Key neuropeptide-related proteins and potential GAS5 target proteins were extracted. (b) Chord diagrams demonstrated the relevance of these key genes in the amniotic cells. (c) Multiple iterative support vector machines (SVM) suggested the best prediction performance was achieved when single feature factors were taken for two feature factors (HECT and RLD domain-containing E3 ubiquitin protein ligase 5 (HERC5) and tachykinin 1 (TAC1)). (d) Protein structure of HERC5 predicted using AlphaFold 2 and (e) heat map analysis of reaction prediction accuracy. (f) Protein structure of TAC1 predicted using AlphaFold 2 and (g) heat map analysis of reaction prediction accuracy.

Figure 4

Prediction of GAS5/HERC5/TAC1 interaction. (a) Predicted HERC5 and TAC1 interaction. (b) Predicted pattern diagram of HECT and RLD domain-containing E3 ubiquitin protein ligase 5 (HERC5) and tachykinin 1 (TAC1) interaction. (c) Predicted pattern diagram of HERC5/lncRNA GAS5 interaction.