Expression Profiles and Bioinformatic Analysis of Circular RNAs in Db/Db Mice with Cardiac Fibrosis

Figures & data

Figure 1 Flow chart of animal experiment.

Abbreviations: BW, body weight; CeRNA, competitive endogenous RNA; DCM, diabetic cardiomyopathy; FBG, fasting blood glucose; GO, Gene Ontology; HE, hematoxylin–eosin; KEGG, Kyoto Encyclopedia of Genes and Genomes; RT-qPCR, Real‐time polymerase chain reaction; T2DM, Type 2 diabetes mellitus; TEM, transmission electron microscopy.

Table 1 The Sequences of Primers of Verified circRNAs

Target	Primer Sequences (5’-3’)
circRNA_28667	Forward, 5’ GTCGCTATTACCCTGCCAAGA3’, reverse, 5’ GTAGTCCAGTGCCTCCAAACG3’;
circRNA_32303	Forward, 5’ AGTGAAGATAGGGCTGGGGA3’, reverse, 5’ AGGCCTGGCATGAAGAACTC 3’;
circRNA_20674	Forward, 5’ AACCTGGCTGCTATCAAGATGC3’, reverse, 5’ TTCACAGAACCAGGGATTCGTC 3’;
circRNA_25425	Forward, 5’ CTTTAGAGTCAGAGTATGAGGGCTG3’, reverse, 5’CATTTACCGAGAGGATCTTATCACCC3’;
circRNA_014302	Forward, 5’ TGGAGAGCTCGAATTCCAGAAC3’, reverse, 5’ AAAACAAGAGAGCCACCAGAGC3’;
circRNA_001535	Forward, 5’ AGCTACGGTGTCCTGAAACCTG3’, reverse, 5’ TTTCACAACGCTGACCACTGAC3’;
circRNA_005522	Forward, 5’ GCACCAAAGGCGATGGAGAAG3’, reverse, 5’GTTGCTCTCTGAAGACCATCTGG3’;
circRNA_013422	Forward, 5’ ATGTTGGTGGATCCTGTTCGC3’, reverse, 5’ GGGTAGACCAAGACTTGTGAGG3’;
ACTB	Forward, 5’ GTACCACCATGTACCCAGGC3’, reverse, 5’ AACGCAGCTCAGTAACAGTCC3’.

Figure 2 Establishment of db/db DCM mouse model. (A) Heart weight/tibia length ratio, body weight, and fasting blood glucose and insulin levels of the db/db and control mice (n = 8). (B) Cardiac tissue was stained with H & E (bar = 20 μm). (C) Masson’s trichrome stain in cardiac tissue and collagen fraction (blue, arrow) (bar = 50 μm). (D) Myocardial tissue and mitochondria under electron microscopy (bar = 5.0 μm). Z fibrils (arrow in NC group), collagen deposition (arrow in DCM group). (B–D) n = 3, ***P < 0.001.

Abbreviations: DCM, diabetic cardiomyopathy; H/T, heart weight/tibia length; NC, normal control.

Figure 3 Changes of cardiac structure and function in DCM mice. (A) Representative pictures of echocardiography. (B) Echocardiogram measurements (LVIDd, LVIDs, EF, FS, E/A, E/e′) of the DCM and NC groups. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

Abbreviations: LVIDd, left ventricular internal diastolic dimension; LVIDs, left ventricular internal diameter in systole; EF, ejection fraction; FS, fractional shortening; E/A: ratio of E wave to A wave; E/e’: ratio of E wave to e’ wave; DCM, diabetic cardiomyopathy; NC, normal control.

Table 2 Differentially Expressed circRNAs in Myocardial Tissues Based on Fold Change

circRNA ID	P-value	FC (abs)	Regulation	GeneSymbol
mmu_circRNA_28667	0.049978294	4.0263555	Up	Phf20l1
mmu_circRNA_32303	0.022305223	3.3373264	Up	Gda
mmu_circRNA_20674	0.018721621	3.070012	Up	Clasp1
mmu_circRNA_25425	0.021272701	2.6734024	Up	Synj2bp
mmu_circRNA_39272	0.009375087	2.632007	Up	Naa25
mmu_circRNA_37101	0.039606401	2.4998485	Up	Mllt3
mmu_circRNA_39303	0.04601282	2.2993219	Up	Rad9b
mmu_circRNA_39417	0.020412384	2.2846202	Up	Aacs
mmu_circRNA_40979	0.013479433	2.2559389	Up	Atf7ip
mmu_circRNA_38420	0.027116693	2.2321756	Up	Depdc5
mmu_circRNA_014302	0.047061334	0.2275387	Down	Slc8a1
mmu_circRNA_001535	0.026882785	0.3689883	Down	Pms1
mmu_circRNA_005522	0.043059227	0.4062490	Down	Mkln1
mmu_circRNA_013422	0.046962145	0.4373786	Down	Hipk3
mmu_circRNA_012617	0.010386744	0.4398317	Down	Mlip

Notes: P-value calculated from unpaired t-test. FC (abs), the absolute ratio (no log scale) of normalized intensities between two conditions. Regulation, it depicts which group has greater or lower intensity values than the other group.

Figure 4 Overview of the detected circRNAs. (A) Distribution of differentially expressed circRNAs in chromosomes between the two groups. Upregulated (red) and downregulated circRNAs (green) in the DCM group vs NC group. (B) Hierarchical cluster analysis (heatmap) of differentially expressed circRNAs between the NC and DCM groups. (C) CircRNA volcano plots. The vertical lines correspond to 1.5-fold up and down, respectively, and the horizontal line represents a P-value of 0.05. The red point in the plot represents the differentially expressed circRNAs with statistical significance. (D) Some representative differentially expressed circRNAs were verified by RT-qPCR. n = 3; circRNA, circular RNA. circPHF20L1, mmu_circRNA_28667; circGDA, mmu_circRNA_32303; circCLASP1, mmu_circRNA_20674; circSYNJ2BP, mmu_circRNA_25425; circSLC8A1, mmu_circRNA_014302; circPMS1, mmu_circRNA_001535; circMKLN1, mmu_circRNA_005522; circHIPK3, mmu_circRNA_013422.

Abbreviations: DCM, diabetic cardiomyopathy; NC, normal control.

Figure 5 CeRNA network and functional analysis. (A) The ceRNA network was predicted by an online bioinformatics program. The top six upregulated circRNAs, and the top four downregulated circRNAs that were indexed by the circBase database, are shown. Nodes with red color are microRNAs, nodes with light-blue color are protein coding RNAs, nodes with brown color are up-circular RNAs, nodes with yellow color are down-circular RNAs. Edges with T-shape arrow represent directed relationships, edges without arrow represent undirected relationships (ceRNA relationship). (B–E) Differentially expressed circRNAs in the DCM group as indicated by GO and KEGG analyses.

Abbreviations: circRNA, circular RNA; DCM, diabetic cardiomyopathy; NC, normal control.

Data Sharing Statement

CircRNA microarray data reported in this paper have been uploaded to NCBI and are publicly accessible at GSE239970.