Objective: To determine the changes in the expression of circular RNA Circ-PALLD in heart failure and explore the biogenesis of Circ-PALLD.
Methods: 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.
Results: 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.
Conclusion: 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.
目的: 确定环状RNA-Circ-PALLD在心力衰竭中的表达变化；初步探究Circ-PALLD的生物学发生。
方法: 通过现有分析人和鼠心衰样本中的二代测序结果，确定候选CircRNAs；PCR扩增后进行Sanger一代测序，比对测序结果，确定对应CircRNA的反向剪接方式；纳入我院心衰患者8例，提取RNA后进行PCR扩增，检测CircRNAs与线性RNA的表达；RT-qPCR检测在衰竭心肌中Circ-PALLD与PALLD的表达水平；生物信息学预测可能调控基因PALLD的转录因子；设计并合成针对GATA4的siRNA，确定转录因子GATA4对PALLD的调控作用。
结果: Sanger测序和序列比对验证了Circ-VWA8，Circ-VMP1，Circ-PRDM5，Circ-PLCL2，Circ-PALLD，Circ-NFATC3，Circ-MLIP，Circ-FAM208A，Circ-ANKIB1，Circ-AGTPBP1的反向剪接，证明了其成环性，确定了反向剪接的外显子排列；半定量PCR结果表明Circ-PALLD，Circ-NFATC3和Circ-AGTPBP1在心衰患者的心肌组织中表达水平明显增加，而其中线性PALLD的表达水平明显降低，提示Circ-PALLD在慢性心衰中的潜在作用；通过生物信息学分析后发现PALLD的转录可能受转录因子GATA4所调控；RT-qPCR结果表明，Circ-PALLD在衰竭心肌中表达水平明显升高，而PALLD表达水平明显降低，其结果与半定量PCR结果一致；在原代乳鼠心肌细胞中敲降GATA4后Circ-PALLD与PALLD表达水平均降低。
结论: Circ-PALLD在心衰中高表达，可作为慢性心衰的新型分子标志物，且转录因子GATA4在PALLD的转录中或有重要作用，因此Circ-PALLD可能为心力衰竭发生的分子机制提供新的研究方向并且为心衰治疗提供了潜在的药物靶点。
Keywords: Circ-PALLD; GATA4; biomarker; heart failure.