Identification of circular RNAs regulating cardiomyocyte proliferation in neonatal pig hearts

Little is known about the expression patterns and functions of circular RNAs (circRNAs) in the heart of large mammals. In this study, we examined the expression profiles of circRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in neonatal pig hearts. Pig heart samples collected on postnatal days 1 (P1), 3 (P3), 7 (P7), and 28 (P28) were sent for total RNA sequencing. Our data revealed a total of 7,000 circRNAs in the 24 pig hearts. Pathway enrichment analysis of hallmark gene sets demonstrated that differentially expressed circRNAs were engaged in different pathways. The most significant difference was observed between P1 and the other 3 groups (P3, P7, and P28) in pathways related to cell cycle and muscle development. Out of the 10 circRNAs that were validated through real-time quantitative PCR to verify their expression, 6 exhibited significant effects on cell cycle activity in human induced pluripotent stem cell–derived cardiomyocytes following small interfering RNA–mediated knockdown. circRNA-miRNA-mRNA networks were constructed to understand the potential mechanisms of circRNAs in the heart. In conclusion, our study provided a data set for exploring the roles of circRNAs in pig hearts. In addition, we identified several circRNAs that regulate cardiomyocyte cell cycle.

from 36 circRNA samples obtained from 24 pig hearts sequenced by CD Genomics Inc.
circRNAs were categorized as significantly upregulated if they had a p value < 0.05 and log2FC > 0.5 (Up_significant in red), significantly downregulated if they had a p value < 0.05 and log2FC < -0.5 (Down_significant in blue), significant but neither upregulated nor downregulated if the p value < 0.05 but log2FC < 0.05 (in dark), otherwise nonsignificant (in grey).P7 and P28.miRNAs were categorized as significantly upregulated if they had a p value < 0.05 and log2FC > 0.5 (Up_signficant distinguished in red), significantly downregulated if they had a p value < 0.05 and log2FC < -0.5 (Down_significant distinguished in blue), significant but neither upregulated nor downregulated if the p value < 0.05 but log2FC < 0.05 (distinguished in green), otherwise nonsignificant ( distinguished in grey).

Supplemental Figure 5. Differentially expressed mRNAs in neonatal pig hearts. (A)
PCA visualization of 12 mRNA samples obtained from 12 pig hearts at different postnatal days (P1, P3, P7, and P28).(B) Barplot showing the counts of significantly differentially expressed mRNAs.mRNAs with a log2FC > 0.5 and p value < 0.05 were classified as significantly upregulated (in orange) while mRNAs with a log2FC < -0.5 and p value < 0.05 were classified as significantly downregulated (in teal).(C-F) Volcano plot visualizing differential expression analysis of mRNA from various comparisons including (C) P1 versus P3, (D) P1 versus P7, (E) P1 versus P28, and (F) P1 versus P3, P7 and P28.mRNAs were categorized as significantly upregulated if they had a p value < 0.05 and log2FC > 0.5 (Up_signficant in red), significantly downregulated if they had a p value < 0.05 and log2FC < -0.5 (Down_significant in blue), significant but neither upregulated nor downregulated if p value < 0.05 but log2FC < 0.05 (in dark), otherwise nonsignificant (in grey).

Figure 3 .
Time-varying effect modeling (TVEM) of cell cycle regulating circRNAs.(A-G) Visualization of the changes in the β1 coefficient across different postnatal periods.Significant variation in the β1 coefficients over time in core cell cycle related pathways such as G2M checkpoint (A), mitotic spindle (B), Myc-targets (C-D), myogenesis (E), PI3K-Akt-mTOR signaling (F), and Wnt-beta catenin signaling (G).

Figure 6 .
Intracellular localizations of circRNAs.hiPSC-CMs at day 28 after the initiation of cardiac differentiation were utilized.The intracellular location of each circRNA was visualized using an Alexa Fluorescent 488 probe targeting the specific circRNA.Cardiomyocytes were identified using an Alexa Fluorescent 568 probe targeting human cTnT RNA.All cell nuclei were stained with DAPI.Representative images displaying the cytoplasmic and nuclear distribution of hsa-ABLIM1_0001 (A), hsa-RNF13_0004 (B), hsa-KIF1B_0001 (C), hsa-MYOM1_0001 (D), hsa-AC096949_0001 (E), and hsa-PDLIM5_0001 (F) in hiPSC-CMs were captured.Supplemental Figure 7. Integrated networking analysis of circRNA-miRNA-mRNA interactions.Network diagram illustrates the interactions between circRNAs and miRNAs, as well as miRNA and mRNAs in the comparisons of P1 versus P3 (A), P1 versus P7 (B), and P1 versus P28 (C).Supplemental Figure 8. Evaluation of the cell cycle regulatory function of miRNAs.hiPSC-CMs at day 28 after initiation of cardiac differentiation were utilized.The cells were treated with either negative control siRNA, or various concentration (1, 4, 10, 20, and 100nM) of specific human miRNA siRNAs for 3 days.Cell number was measured using bioluminescence analysis for hiPSC-CMs treated with siRNAs or mimics for (A) hsa-miR-128-3p, (B) has-miR-197-3p, (C) has-miR-215-3p, (D) has-miR-140-5p, (E) has-miR-15a-3p, and (F) has-miR-128-3p.All data were presented as mean ± SEM.Statistical analysis was performed via the Student's t-test.n=4 in each group.*p<0.05,**p<0.01,***p<0.001,and ****p<0.0001.Supplemental Figure 9. Evaluation of the cell cycle regulatory function of has-miR-128-3p mimics.hiPSC-CMs at day 28 after initiation of cardiac differentiation were used.The efficiency of miRNA mimic-based activation in hiPSC-CMs was determined through qRT-PCR.The expression levels of circRNAs were normalized to GAPDH reference gene.The expression levels of miRNAs were normalized to U6 reference gene.Cell numbers were measured using bioluminescence analysis.Cell cycle activity was determined by immunostaining using antibodies against BrdU and PH3.Cardiomyocytes were identified using anti-human cTnT immunostaining.All cell nuclei were stained with DAPI.The BrdU or PH3 positively stained cardiomyocyte nuclei were normalized to the total number of cardiomyocyte nuclei and the results were presented as a percentage.(A-B) Expression of hsa-miR-128-3p (A) and its target circRNA hsa-AC096949-0001 (B) in the cells treated with has-miR-128-3p mimics was evaluated.(C-E) The hsa-miR-128-3p mimics inhibited cell proliferation as indicated by reduced bioluminescence signal (C) and the decrease in the prevalence of BrdU-and PH3-positively stained cardiomyocyte nuclei (D and E).(F) hsa-miR-128-3p mimics also inhibited the expression of MME.All data were presented as mean ± SEM.Statistical analysis was performed via the Student's t-test.n=3 technical replicates in each group for panels A and F. n=4 technical replicates in each group for panels B and C. n=15 technical replicates in each group for panel D. n=20 technical replicates in each group for panel E. *p<0.05,**p<0.01,***p<0.001,and ****p<0.0001.GAPDH reference gene.Cell numbers were measured using bioluminescence analysis.Cell cycle activity was determined by immunostaining using antibodies against BrdU and PH3.Cardiomyocytes were identified using anti-human cTnT immunostaining.All cell nuclei were stained with DAPI.The BrdU or PH3 positively stained cardiomyocyte nuclei were normalized to the total number of cardiomyocyte nuclei and the results were presented as a percentage.(A-B) Expression of hsa-miR-128-3p (A) and its target circRNA hsa-AC096949-0001 (B) in the cells treated with has-miR-128-3p siRNAs was evaluated.(C-E) The hsa-miR-128-3p siRNAs promoted cell proliferation as shown by the increase in bioluminescence signal (C) and the greater prevalence of BrdU-and PH3positively stained cardiomyocyte nuclei (D and E).(F) hsa-miR-128-3p siRNAs also enhanced the expression of MME.All data were presented as mean ± SEM.Statistical analysis was performed via the Student's t-test.n=3 technical replicates in each group for panels A and F. n=4 technical replicates in each group for panels B and C. n=23 technical replicates in each group for panels D and E. *p<0.05,**p<0.01,***p<0.001,and ****p<0.0001.