剪接因子HNRNPH1通过调控Circ-MYOCD的反向剪接影响心肌肥厚的发生  

The splicing factor HNRNPH1 regulates Circ-MYOCD back-splicing to modulate the course of cardiac hypertrophy

作　　者：蔡蕊 黄卓 贺文霞 艾添红 宋晓伟 胡淑婷[1] 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)

机构地区：[1]宁夏医科大学基础医学院,宁夏银川750004 [2]海军军医大学第一附属医院心内科,上海200433

出　　处：《南方医科大学学报》2025年第3期587-594,共8页Journal of Southern Medical University

基　　金：国家自然科学基金(81760076)。

摘　　要：目的探索Circ-MYOCD的反向剪接机制及该分子的反向剪接是如何调控心肌肥厚的发生的。方法Sanger测序和RNase R实验验证Circ-MYOCD的呈环性和稳定性,核质分提确定Circ-MYOCD的分布情况。生物信息学分析及pull-down的质谱结果分析预测与Circ-MYOCD结合的RNA结合蛋白(RBPs)。敲低心肌细胞中HNRNPH1和HNRNPL筛选可能影响到Circ-MYOCD反向剪接的RBPs,过表达HNRNPH1确定影响Circ-MYOCD反向剪接的RBPs。血管紧张素Ⅱ(AngⅡ)诱导建立大鼠心肌肥大模型,检测HNRNPH1在肥大心肌细胞中的表达情况。敲低和过表达心肌细胞中HNRNPH1检测其对心肌肥厚进程的影响。在大鼠心肌肥大细胞模型中敲低HNRNPH1的表达,检测HNRNPH1对Circ-MYOCD反向剪接的影响及对心肌肥厚进程的影响。结果Sanger测序结果显示,接头引物可以扩增出正确的Circ-MYOCD的序列;RNase R实验和核质分提实验结果显示Circ-MYOCD具有稳定性且主要分布在细胞质中(P<0.001)。生物信息学分析及Circ-MYOCD的pull-down的质谱结果分析显示HNRNPH1和HNRNPL是与Circ-MYOCD结合的RBPs。敲低和过表达心肌细胞中RBPs,检测Circ-MYOCD与MYOCD的表达情况,结果显示HNRNPH1影响且抑制Circ-MYOCD的反向剪接(P<0.01,P<0.05)。AngⅡ诱导建立小鼠心肌肥大模型中,检测到HNRNPH1的表达升高(P<0.001)。过表达HNRNPH1可以增加心肌肥厚标志物分子ANP、BNP的表达(P<0.05),敲低的结果与之相反(P<0.05,P<0.001)。在肥大的心肌细胞中敲低HNRNPH1后,Circ-MYOCD表达升高(P<0.001),MYOCD的表达降低(P<0.01);检测心肌肥大相关分子ANP、BNP的表达均降低(P<0.05,P<0.001)。讨论Circ-MYOCD的反向剪接受剪接因子HNRNPH1的调控进而影响了心肌肥厚的进程。Objective To explore the mechanism of Circ-MYOCD back-splicing and its regulatory role in myocardial hypertrophy.Methods 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.Results 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.Conclusion HNRNPH1 regulates Circ-MYOCD back-splicing to influence the progression of myocardial hypertrophy.

关 键 词：心肌肥厚 反向剪接 环状RNA 剪接因子 HNRNPH1 

分 类 号：R542.2[医药卫生—心血管疾病]