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

CAI Rui; HUANG Zhuo; HE Wenxia; AI Tianhong; SONG Xiaowei; HU Shuting

doi:10.12122/j.issn.1673-4254.2025.03.16

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.