Cai Rui, Huang Zhuo, He Wenxia, Ai Tianhong, Song Xiaowei, Hu Shuting
School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, China.
Department of Cardiology,First Affiliated Hospital,Naval Medical University, Shanghai 200433, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2025 Mar 20;45(3):587-594. doi: 10.12122/j.issn.1673-4254.2025.03.16.
To explore the mechanism of Circ-MYOCD back-splicing and its regulatory role in myocardial hypertrophy.
Sanger sequencing and RNase R assays were performed to verify the circularity and stability of Circ-MYOCD, whose subcellular distribution was determined by nuclear-cytoplasmic fractionation. Bioinformatics analysis and mass spectrometry from pull-down assays were conducted to predict the RNA-binding proteins (RBPs) interacting with Circ-MYOCD. In rat cardiomyocytes H9C2 cells, the effects of HNRNPH1 and HNRNPL knockdown and overexpression on Circ-MYOCD back-splicing were evaluated. In a H9C2 cell model of angiotensin II (Ang II)-induced myocardial hypertrophy, the expression of HNRNPH1 was detected, the effects of HNRNPH1 knockdown and overexpression on progression of myocardial hypertrophy were assessed, and the regulatory effect of HNRNPH1 on Circ-MYOCD back-splicing was analyzed.
Sanger sequencing confirmed that the junction primers could amplify the correct Circ-MYOCD sequence. RNase R and nuclear-cytoplasmic fractionation assays showed that Circ-MYOCD was stable and predominantly localized in the cytoplasm. Bioinformatics analysis and mass spectrometry from the Circ-MYOCD pull-down assay identified HNRNPH1 and HNRNPL as the RBPs interacting with Circ-MYOCD. In H9C2 cells, HNRNPH1 knockdown significantly enhanced while its overexpression inhibited Circ-MYOCD back-splicing; HNRNPH1 overexpression obviously increased the expressions of myocardial hypertrophy markers ANP and BNP, while its knockdown produced the opposite effect. In Ang II-induced H9C2 cells, which exhibited a significant increase of HNRNPH1 expression and increased expressions of ANP and BNP, HNRNPH1 knockdown obviously increased Circ-MYOCD expression, decreased MYOCD expression and lowered both ANP and BNP expressions.
HNRNPH1 regulates Circ-MYOCD back-splicing to influence the progression of myocardial hypertrophy.
探讨Circ-MYOCD反向剪接的机制及其在心肌肥大中的调控作用。
进行桑格测序和RNase R分析以验证Circ-MYOCD的环化和稳定性，通过细胞核-细胞质分级分离确定其亚细胞分布。进行生物信息学分析和下拉试验的质谱分析以预测与Circ-MYOCD相互作用的RNA结合蛋白（RBP）。在大鼠心肌细胞H9C2细胞中，评估HNRNPH1和HNRNPL敲低及过表达对Circ-MYOCD反向剪接的影响。在血管紧张素II（Ang II）诱导的心肌肥大H9C2细胞模型中，检测HNRNPH1的表达，评估HNRNPH1敲低及过表达对心肌肥大进展的影响，并分析HNRNPH1对Circ-MYOCD反向剪接的调控作用。
桑格测序证实连接引物可扩增出正确的Circ-MYOCD序列。RNase R和细胞核-细胞质分级分离试验表明Circ-MYOCD稳定且主要定位于细胞质中。Circ-MYOCD下拉试验的生物信息学分析和质谱分析确定HNRNPH1和HNRNPL为与Circ-MYOCD相互作用的RBP。在H9C2细胞中，HNRNPH1敲低显著增强而其过表达抑制Circ-MYOCD反向剪接；HNRNPH1过表达明显增加心肌肥大标志物ANP和BNP的表达，而其敲低则产生相反作用。在Ang II诱导的H9C2细胞中，HNRNPH1表达显著增加，ANP和BNP表达也增加，HNRNPH1敲低明显增加Circ-MYOCD表达，降低MYOCD表达，并降低ANP和BNP表达。
HNRNPH1调节Circ-MYOCD反向剪接以影响心肌肥大的进展。
