Fig. 1

Identification and characteristics of circ_005077 in H9c2 cells and myocardial tissues. Rats were fed a normal chow diet (NCD) or high-fat diet (HFD) for 20 consecutive weeks and heart tissues were harvested for detection. (A, B) Representative image of hematoxylin and eosin staining representative image (400× magnification) with the cross-sectional area of the cardiomyocyte evaluated; Masson staining (200× magnification) with the myofibrillar density evaluated; Sirius Red staining (200× magnification) with the collagen volume evaluated. (C) Analysis of relative expression of hypertrophy and fibrosis indicators (ANP, BNP, Collagen I, and Collagen III) in the myocardium identified by qRT-PCR. (D, E) Typical images taken in M mode. Measurement of the thickness of the left ventricular posterior wall at the end-diastole (LVPW; d) left ventricular end-diastolic chamber diameters (LVID; d), and cardiac output (CO). (F) Hierarchical clustering heatmap showing differences in the circRNAs of greatest expression between NCD and HFD myocardium with the top 10 upregulated or downregulated genes selected (n = 3, P < 0.05). In a heat map, red indicates upregulation. Blue denotes downregulation. (G) qRT-PCR validation of microarray-based expression of the top five upregulated circRNAs. Scatter plots displayed the results of ten independent experiments conducted for each group. (H) In situ hybridization showed the expression of circ_005077in NCD and HFD myocardial tissue Sect. (400× magnification). (I) Exons 2–4 of the crmp1 gene were used to generate circ_005077 in the schematic diagram (top). Circ_005077 was identified by Sanger sequencing in H9c2 cells. The black arrow marked the back-splicing site conjunction. (J) Circ_005077 and β-actin were detected from the cDNA or genomic DNA (gDNA) of the H9c2 cell using divergent and convergent primers by agarose gel electrophoresis experiments. (K) Circ_005077 and Crmp1 abundance were determined by qRT-PCR in H9c2 cells treated with actinomycin D at the specified time points (n = 4). (L) qRT-PCR for circ_005077 and crmp1 abundance in H9c2 cells treated with RNase R versus mock cells (n = 3). (M) RNA-FISH test demonstrated the localization of circ_005077 in the cytoplasm and nucleus regions of H9c2 cells using a junction-specific antisense probe (green, 400× magnification) and nuclear labeling with DAPI (blue, 400× magnification). Data were presented as mean ± SD. *P < 0.05, ** P < 0.01, *** P < 0.001, NS, no significance