Identification of circRNAs expression profiles and functional networks in parotid gland of type 2 diabetes mouse

Abstract

Background: Circular RNAs (circRNAs) are a novel kind of non-coding RNAs proved to play crucial roles in the development of multiple diabetic complications. However, their expression and function in diabetes mellitus (DM)-impaired salivary glands are unknown.

Results: By using microarray technology, 663 upregulated and 999 downregulated circRNAs companied with 813 upregulated and 525 downregulated mRNAs were identified in the parotid glands (PGs) of type2 DM mice under a 2-fold change and P < 0.05 cutoff criteria. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis of upregulated mRNAs showed enrichments in immune system process and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Infiltration of inflammatory cells and increased inflammatory cytokines were observed in diabetic PGs. Seven differently expressed circRNAs validated by qRT-PCR were selected for coding-non-coding gene co-expression (CNC) and competing endogenous RNA (ceRNA) networks analysis. PPAR signaling pathway was primarily enriched through analysis of circRNA-mRNA networks. Moreover, the circRNA-miRNA-mRNA networks highlighted an enrichment in the regulation of actin cytoskeleton.

Conclusion: The inflammatory response is elevated in diabetic PGs. The selected seven distinct circRNAs may attribute to the injury of diabetic PG by modulating inflammatory response through PPAR signaling pathway and actin cytoskeleton in diabetic PGs.

Keywords: Actin cytoskeleton; Circular RNAs; Diabetes mellitus; Inflammation response; Parotid gland.

Fig. 1

Confirmation of db/db mice a diabetic mice model. (A) Blood glucose of db/m and db/db mice. (B) Serum insulin levels of db/m and db/db mice. (C) Insulin resistance indicator (HOMA-IR) of db/m and db/db mice. **P < 0.01, versus db/m mice, n = 4

Fig. 2

Differentially expressed circRNAs and mRNAs in PGs of db/db mice comparing to db/m mice. (A) Volcano plots presenting differences in the expression of circRNAs between db/db and db/m mice. (B) Volcano plots presenting differences in the expression of mRNAs between db/db and db/m mice. (C) Heat map of top 30 upregulated and downregulated circRNAs with raw intensity higher than 500. (D) Heat map of top 30 upregulated and downregulated mRNAs with raw intensity higher than 500. Values plotted on the x- and y-axes represent the averaged normalized signal values of each group (log2-scaled). CircRNA, circular RNA; PGs, parotid glands; mRNA, massage RNA

Fig. 3

Validation of DE circRNAs and mRNAs by qRT-PCR. (A) Expression of 5 selected upregulated circRNAs in db/db mice and db/m mice. (B) Expression of 9 selected downregulated circRNAs in db/db mice and db/m mice. (C) Expression of 10 selected upregulated mRNAs in db/db mice and db/m mice. (D) Expression of 10 selected downregulated mRNAs in db/db mice and db/m mice. *P < 0.05 and **P < 0.01, versus db/m mice, n = 4. DE, differently expressed; circRNA, circular RNA; mRNA, massage RNA

Fig. 4

GO and KEGG pathway analysis of DE mRNAs. (A) GO analysis of upregulated mRNAs in the PGs of db/db mice. (B) GO analysis of downregulated mRNAs in the PGs of db/db mice. (C) KEGG pathway analysis of upregulated mRNAs in the PGs of db/db mice. (D) KEGG pathway analysis of downregulated mRNAs in the PGs of db/db mice. GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes; DE, differently expressed; mRNA, massage RNA; PGs, parotid glands

Fig. 5

Inflammatory level in the PGs of db/db mice. (A) H&E staining of the PGs from db/m and db/db mice. (B) Expression of inflammatory cytokines in the PGs from db/m and db/db mice, *P < 0.05 and **P < 0.01, versus db/m mice, n = 4. PGs, parotid glands

Fig. 6

GO and KEGG pathway analysis based on CNC analysis of 7 selected circRNAs. (A) GO analysis of co-expressed mRNAs on cellular component (CC), biological process (BP), and molecular function (MF). (B) KEGG pathway analysis of co-expressed mRNAs of 7 selected circRNAs. GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes; CNC, coding–non-coding gene co-expression; mRNA, massage RNA; circRNA, circular RNA

Fig. 7

Coding and non-coding co-expression network involving in PPAR signaling pathway, NF-kappa B signaling pathway, and cytokine-cytokine receptor interaction. Red nodes represent circRNAs; blue nodes represent mRNAs. Positive correlation is a solid line, negative correlation is a dashed line. PPAR, peroxisome proliferator-activated receptor; NF-kappa B, nuclear factor-kappa B; circRNA, circular RNA; mRNA, massage RNA

Fig. 8

GO and KEGG pathway analysis based on ceRNA analysis of 7 selected circRNAs. (A) GO analysis of ceRNA function-related mRNAs on cellular component (CC), biological process (BP), and molecular function (MF). (B) KEGG pathway analysis of ceRNA function-related mRNAs. GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes; ceRNA, competing endogenous RNA; circRNA, circular RNA; mRNA, massage RNA

Fig. 9

CircRNA-miRNA-mRNA network involving in the regulation of actin cytoskeleton, leukocyte trans-endothelial migration, and PPAR signaling pathways. Green node represents upregulated circRNAs; yellow nodes represent downregulated circRNAs; red nodes represent miRNAs; blue nodes represent mRNAs. Purple lines with T-shape arrow represent directed relationships; orange lines without arrow represent undirected relationships. CircRNA, circular RNA; miRNA, microRNA; mRNA, massage RNA; PPAR, peroxisome proliferator-activated receptor