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

doi:10.12122/j.issn.1673-4254.2025.03.16

摘要/Abstract

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。

关键词: 心肌肥厚, 反向剪接, 环状RNA, 剪接因子, HNRNPH1

Abstract:

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.

Key words: cardiac hypertrophy, back-splicing, circular RNA, splicing factors, HNRNPH1

蔡蕊, 黄卓, 贺文霞, 艾添红, 宋晓伟, 胡淑婷. 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[J]. 南方医科大学学报, 2025, 45(3): 587-594.

Rui CAI, Zhuo HUANG, Wenxia HE, Tianhong AI, Xiaowei SONG, Shuting HU. The splicing factor HNRNPH1 regulates Circ-MYOCD back-splicing to modulate the course of cardiac hypertrophy[J]. Journal of Southern Medical University, 2025, 45(3): 587-594.

图/表 7

表1 引物序列

Tab.1 Primer sequences for qRT-PCR

Forward:

Reverse:

AACGACCCCTTCATTGACCTC

CCTTGACTGTGCCGTTGAACT

Rno-ANP

Forward:

Reverse:

GCCCTGAGCGAGCAGACCGA

CGGAAGCTGTTGCAGCCTA

Rno-BNP

Forward:

Reverse:

AGTCCTAGCAGTCTCAGA

GTCTCTCCGGATCCGGAAG

Rno-HnRNPH1

Forward:

Reverse:

GTGCAGTTTGCTTCACAGGA

CCATCCCCATATCTGTGGTC

Rno-HnRNPL

Forward:

Reverse:

AAGAGGCAGGCACTGGTAGA

GGCACTTTGCACAGAATCAA

Forward :

Reverse:

GCTCGCTTCGGCAGCACA

AACGCTTCACGAATTTGCGT

Rno-Circ-MYOCD

Forward:

Reverse:

CTTGCAGATGACCTCAACGA

TGGACTTTTCAGGGGTGGTA

Rno-MYOCD

Forward:

Reverse:

CATTGGGAAGGTGAGCGATG

TGTATGCATAGGGGAGAGCG

图1 Circ-MYOCD在H9C2细胞中的成环性、分布和稳定性鉴定

Fig.1 Identification of the circularity, localization, and stability of Circ-MYOCD in H9C2 Cells. A: Circ-MYOCD is composed of exons 2 to 5 of the MYOCD gene. B: Agarose gel electrophoresis of PCR amplification products of Circ-MYOCD. C: Sanger sequencing results of Circ-MYOCD. D: Stability validation of Circ-MYOCD (n=3). E: Localization of Circ-MYOCD within cells (n=3). ***P<0.001 vs control.

图2 预测与Circ-MYOCD结合的RNA结合蛋白

Fig.2 Predicted RNA-binding proteins (RBPs) associated with Circ-MYOCD.

图3 敲低HNRNPH1和HNRNPL后对Circ-MYOCD与MYOCD的影响

Fig.3 Effects of HNRNPH1 and HNRNPL knockdown on Circ-MYOCD and MYOCD expression in H9C2 cells. A: Knockdown efficiency of si-HNRNPH1 in H9C2 cells. B: Knockdown efficiency of si-HNRNPL in H9C2 cells. C: Expression levels of Circ-MYOCD and MYOCD in H9C2 cells after transfection with si-HNRNPH1. D: Expression levels of Circ-MYOCD and MYOCD after transfection with si-HNRNPL in H9C2 cells. n=3; *P<0.01, **P<0.05, ***P<0.001 vs NC.

图4 HNRNPH1可能调控Circ-MYOCD的反向剪接

Fig.4 HNRNPH1 potentially regulates Circ-MYOCD back-splicing. A: Fluorescence images of H9C2 cells infected with Ad-HNRNPH1 recombinant adenovirus. B: Verification of overexpression efficiency of Ad-HNRNPH1 recombinant adenovirus (n=3). C: Expression levels of Circ-MYOCD and MYOCD after overexpression of HNRNPH1 (n=3). *P<0.01, **P<0.05, ***P<0.001 vs GFP.

图5 HNRNPH1可能促进心肌肥厚的发生

Fig.5 HNRNPH1 potentially promotes the development of cardiac hypertrophy. A: Construction of cardiac hypertrophy model in H9C2 cells by treatment with Ang II. B: Expression of HNRNPH1 in the hypertrophic cell model. C: Changes in ANP and BNP expressions after HNRNPH1 knockdown. D: Changes in ANP and BNP expressions after HNRNPH1 overexpression. n=3, **P<0.05, ***P<0.001 vs ANP or Control.

图6 HNRNPH1影响Circ-myocd的反向剪接且调控心肌肥厚的进展

Fig.6 HNRNPH1 influences Circ-MYOCD back-splicing and regulates the progression of cardiac hypertrophy. A: Knockdown efficiency of HNRNPH1 in the rat cardiac hypertrophy cell model. B: Changes in Circ-MYOCD and MYOCD expression after HNRNPH1 knockdown in the cardiac hypertrophy cell model. C: Changes in ANP and BNP expression after HNRNPH1 knockdown in the cardiac hypertrophy model. n=3, *P<0.01, **P<0.05, ***P<0.001.

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