Signature of circular RNAs in human induced pluripotent stem cells and derived cardiomyocytes

Abstract

Background: Circular RNAs (circRNAs) are regarded as a novel class of noncoding RNA regulators. Although a number of circRNAs have been identified by bioinformatics analysis of RNA-seq data, tissue and disease-specific circRNAs are still to be uncovered to promote their application in basic research and clinical practice. The purpose of this study was to explore the circRNA profiles in human induced pluripotent stem cells (hiPSCs) and hiPSC-derived cardiomyocytes (hiPSC-CMs), and to identify cardiac or disease-specific circRNAs.

Methods: hiPSCs were generated from fibroblasts, and then further differentiated to hiPSC-CMs by modulating WNT signaling in RPMI+B27 medium. Following high-throughput RNA sequencing, circRNAs were extracted and quantified by a combined strategy known as CIRCexplorer. Integrative analysis was performed to illuminate the correlation between circRNAs and their parental linear isoforms. Cardiac and disease-specific expression of circRNAs was confirmed by quantitative reverse-transcription PCR.

Results: In this study, a total of 5602 circRNAs were identified in hiPSCs and hiPSC-CMs. Our data indicated, for the first time, more enriched expression of circRNAs in differentiated cardiomyocytes than in undifferentiated hiPSCs. In addition to the host gene-dependent expression, our integrative analysis also identified a number of circRNAs showing host gene-independent expression in hiPSCs and hiPSC-CMs. CircRNAs including circSLC8A1, circCACNA1D, circSPHKAP and circALPK2 showed cardiac-selective expression during cardiac differentiation and human heart-specific enrichment in fetal tissues. Furthermore, circSLC8A1 abnormally increased in heart tissues from patients suffering from dilated cardiomyopathy.

Conclusions: CircRNAs are highly enriched in hiPSC-differentiated CMs, and cardiac-specific circRNAs such as circSLC8A1, circCACNA1D, circSPHKAP and circALPK2 may serve as biomarkers of CMs. Detection of the excessive expression of circSLC8A1 provides a potential approach for pathological status indication of heart disease.

Keywords: Cardiomyocyte; Circular RNA; Induced pluripotent stem cell.

Fig. 1

Identification of hiPSCs and hiPSC-derived cardiomyocytes. a Representative photographs of fibroblasts on day 6 and human fibroblast-derived iPSC colonies on day 30 during hiPSC generation, as well as passaged hiPSCs under bright field at 100× magnification. b Immunofluorescence staining of hiPSCs for pluripotency markers including NANOG, SOX2 and TRA-1-60, and nucleus marker DAPI. c Immunofluorescence staining of hiPSC-derived cell for markers of endoderm (SOX17), mesoderm (SMC), ectoderm (TUJ1) and nucleus (DAPI). d Hematoxylin and eosin staining of hiPSC-derived teratoma comprising of epithelium, cartilage and neuro (asterisks). e Immunofluorescence staining of hiPSC-CMs for cardiac markers including α-SA and TNNT2, and nucleus marker DAPI. f Line-scan images and corresponding spontaneous Ca²⁺ transients in hiPSC-CMs. Ca²⁺ level calculated as ΔF/F0 = (F – F0) / F0, where F0 is diastolic fluorescence level. iPSC induced pluripotent stem cell

Fig. 2

Genomic features of circRNAs in hiPSCs and hiPSC-CMs. a Venn diagram illustrating abundant novel circRNAs in hiPSC (iPSC) and hiPSC-CM (CM) samples. b Most genes in both hiPSCs and hiPSC-CMs generated only one circular isoform. c No significant difference in length of circRNAs observed between hiPSCs and hiPSC-CMs. Most circRNAs spanned less than 45 kb of genomic DNA (d), and were composed of one to eight exons (e). f Average length of exons was larger in single-exon circRNAs than in multiple-exon circRNAs. circRNA circular RNA, CM cardiomyocyte, iPSC induced pluripotent stem cell

Fig. 3

Cell-specific expression of circRNAs in hiPSCs and hiPSC-CMs. a Proportion of circRNA reads among total mapped reads higher in the hiPSC-CM group (CM) than in the hiPSC group (iPSC). b RNA-seq data showed increased expression of Quaking (QKI) and RNA binding Motif protein 20 (RBM20), two alternative factors associated with circRNA formation. c qRT-PCR detection of QKI and RBM20 mRNA in hiPSCs and hiPSC-CMs; data shown as mean ± SD, Student’s t test, *p < 0.05. d Hierarchical clustering (heat map) showed differential expression of circRNAs among hiPSCs and hiPSC-CMs. e Confirmation of differentially expressed circRNAs by RT-PCR. f Representative electrophoretograms of RT-PCR showed differential expression of circRNAs from days 0 to 30 during cardiac differentiation from hiPSCs. g Schematic depiction of circCACNA1D generation by back-splicing of exon 3 to exon 2, and verification of back-splicing site of circCACNA1D by Sanger sequencing. h qRT-PCR detection of QKI mRNA in negative control (NC) and siQKI transfected hiPSC-CMs. i qRT-PCR showed decreased expression of circRNAs in siQKI transfected hiPSC-CMs, compared with that in NC. circRNA circular RNA, CM cardiomyocyte, ESC embryonic stem cell, FB fibroblast, iPSC induced pluripotent stem cell. Data in h and i shown as mean ± SD, one-way ANOVA followed by Turkey’s test, *p < 0.05

Fig. 4

Integrative analysis of circRNAs and their parental linear transcripts. a, b Relationship between top 100 circRNAs and their parental linear transcripts in hiPSCs (iPSC) and hiPSC-CMs (CM). Ratio of circular and linear isoforms of each gene expressed in hiPSCs (c) and hiPSC-CMs (d) were converted from the relative value of circRNA (RPB)/mRNA (RPKM). Based on cutoff value for proportion of circRNAs at 75%, many more circRNAs showed a host gene-independent expression in hiPSC-CMs, compared with that in hiPSCs. Gene Ontology analyses of host genes of upregulated circRNAs in hiPSCs (e) and hiPSC-CMs (f) indicated most enriched 20 signal pathways with p < 10⁻⁵. circRNA circular RNA, CM cardiomyocyte, iPSC induced pluripotent stem cell

Fig. 5

Differential expression of circRNAs in human tissues. a Comparison of our data with circRNAs identified in human tissues by Tan et al. [18] revealed a total of 897 overlapped circRNAs. Quantitative real-time PCR analysis showed significant upregulation of circSLC8A1 (b), circCACNA1D (c), circSPHKAP (d) and circALPK2 (e) transcripts in fetal heart, compared to other human tissues. f No significant change of circFNDC3B expression detected among these fetal tissues. Data shown as mean ± SD, n = 3; one-way ANOVA followed by Turkey’s test, *p < 0.05. CM cardiomyocyte, iPSC induced pluripotent stem cell

Fig. 6

Expression of circRNAs in healthy and diseased human hearts. Quantitative real-time PCR analysis showed significant increased expression of circSLC8A1 (a) in heart samples from patients with dilated cardiomyopathy (DCM), while no change of circCACNA1D (b) and circSPHKAP (c) expression was observed between control (CTR, n = 10) and DCM (n = 14) groups. Expression of SLC8A1 (d), CACNA1D (e) and SPHKAP (f) mRNA also determined by qRT-PCR. Data shown as mean ± SD, Student’s t test, *p < 0.05