High expression of Circ-PALLD in heart failure is transcriptionally regulated by the transcription factor GATA4

Abstract

To determine the changes in the expression of circular RNA Circ-PALLD in heart failure and explore the biogenesis of Circ-PALLD. We analyzed second-generation sequencing results of human and murine heart failure samples to identify the candidate CircRNAs. Sanger generation sequencing was performed after PCR amplification, and the sequencing results were compared to determine the reverse splicing pattern of the corresponding CircRNAs. We further examined the expressions of CircRNAs and linear RNAs in 8 patients with heart failure admitted in our hospital, and RT-qPCR was performed to detect the expression levels of Circ-PALLD and PALLD in the failing myocardium. Bioinformatic analysis was performed to predict the transcription factors that may regulate PALLD. Small interfering RNAs (siRNAs) against GATA4 were used to determine the regulatory effect of the transcription factor GATA4 on PALLD. Sanger sequencing and sequence alignment verified the reverse splicing of Circ-VWA8, Circ-VMP1, Circ-PRDM5, Circ-PLCL2, Circ-PALLD, Circ-NFATC3, Circ-MLIP, Circ-FAM208A, Circ-ANKIB1, and Circ-AGTPBP1, demonstrated their loop-forming nature and determined the exon arrangement of reverse splicing. Semi-quantitative PCR results showed that the expression levels of CircPALLD, Circ-NFATC3 and Circ-AGTPBP1 were significantly increased while the expression level of linear PALLD was significantly decreased in the myocardial tissues of heart failure patients. Bioinformatic analysis suggested that the transcription of PALLD was regulated possibly by the transcription factor GATA4. RT-qPCR showed that the expression level of Circ-PALLD was significantly increased, while PALLD expression was significantly decreased in the failing myocardium, which was consistent with the results of semi-quantitative PCR. In primary mammary rat cardiomyocytes, GATA4 knockdown resulted in lowered expressions of both Circ-PALLD and PALLD. Circ-PALLD is highly expressed in heart failure and can be used as a novel molecular marker for chronic heart failure, and GATA4 may play important role in regulating its transcription. Circ-PALLD points a new direction for investigating the molecular mechanism of heart failure and may also serve as a potential therapeutic target for heart failure.