Circular RNA Expression Profiling and Selection of Key Circular RNAs in the Hypothalamus of Heat-Acclimated Rats

FIGURE 1. Data are expressed as mean ± SD. HA, heat acclimation (n = 17); C, control (n = 11). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with the C group. ##P < 0.01, ###P < 0.001 compared with the same group before heat exposure. P < 0.05 was considered statistically significant. (A) Rats weight in the HA group was significantly lower than C group (P = 0.003 for 10 days, P = 0.011 for 20 days, and P = 0.019 for 30 days). (B) Tre in the HA group on days 1 and 10 differed significantly from that in the C group after heat exposure (P = 0.000 for 1 days and P = 0.025 for 10 days); there was a significant difference of Tre in the HA group after heat exposure on days 1 and 10 compared with the same group before heat exposure (P = 0.000 for 1 day and P = 0.004 for 10 days). (C) During 2-h continuous monitoring, Tre showed an initial rising trend, followed by a decrease and gradual stabilization. There was no difference in Tre between the HA and C groups on day 30.

FIGURE 2. Differential expression of circRNAs in rat hypothalamus. (A) Box plot showing the distribution of circRNA expression values in the HA and C groups. (B) Hierarchical clustering of circRNAs differentially expressed between the hypothalamus of rats in the HA and C groups; each group contained three individuals (>1.5-fold difference in expression; P < 0.05). Expression levels above and below the median expression level were indicated with different colors in all samples. (C) Volcano plots constructed using the fold changes (FCs) and P-values are shown. Vertical lines correspond to 1.5-FC in the up- and downregulation between the HA and C groups, and horizontal line indicates the P-value. The red dot in the figure indicates circRNAs with significant difference in expression. (D) Visual assessment of differences in circRNA expression between HA and C using scatter plots. The values corresponding to the x- and y-axes in the scatter plot are the normalized signals of the samples (Log2 scaled). Green lines indicate FCs. The circRNAs above the top green line and below the bottom green line show a >1.5-FCs between the HA and C groups. (E) Chromosomal distributions of differentially…

FIGURE 3. (A) Fifteen circRNAs for GO enrichment analysis in terms of BP, CC, and MF. The top significantly enriched target genes and their scores (negative logarithm of P-value) are listed as x-axis and y-axis, respectively. The significant level of GOs are represented in horizontal axis. (B) KEGG pathway analysis for 8 up-regulated circRNAs and the top 10 significantly enriched pathways. (C) KEGG pathway analysis for 7 down-regulated circRNAs and the top 10 significantly enriched pathways. The significant level of pathways represented in horizontal axis.

FIGURE 4. Based on sequence-pairing prediction predicted 15 circRNAs targeted circRNA–miRNA–mRNA/gene network. The results of miRNA-binding sites predicted by mirSVR, and targeted TargetScan and miRanda predicted miRNAs and mRNAs, were considered. The red circles represent mRNAs and green nodes represent miRNAs.

FIGURE 5. Seed sequence matching predicted the direct interaction of rno_circRNA_014301, rno_circRNA_010393, and rno_circRNA_011190 with the three miRNAs: rno-miR-3575, rno-miR-20b-3p, and rno-miR-667-5p.

FIGURE 6. (A) GAPDH was measured as a reference gene, and three independent times for each sample of RNA were detected for these circRNAs and mRNAs. ∗P < 0.05, ∗∗P < 0.01. (B) U6 was measured as a reference gene, and three independent times for each sample of RNA were detected for microRNAs. ∗P < 0.05, ∗∗P < 0.01.

FIGURE 7. Validation of HIF-1α and MFAP4 as HA-related proteins by ELISA analysis. Data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01. (A) The expression level of HIF-1α of HA group was higher than that of control group (P = 0.14, P < 0.05). (B) A significant lower expression level of MFAP4 was found the hypothalamus from HA group (P = 0.002, P < 0.05).