Table_5_The Novel Non-coding Transcriptional Regulator Gm18840 Drives Cardiomyocyte Apoptosis in Myocardial Infarction Post Ischemia/Reperfusion.XLSX

BackgroundIschemia/reperfusion-mediated myocardial infarction (MIRI) is a major pathological factor implicated in the progression of ischemic heart disease, but the key factors dysregulated during MIRI have not been fully elucidated, especially those essential non-coding factors required for cardiovascular development.
MethodsA murine MIRI model and RNA sequencing (RNA-seq) were used to identify key lncRNAs after myocardial infarction. qRT-PCR was used to validate expression in cardiac muscle tissues and myocardial cells. The role of Gm18840 in HL-1 cell growth was determined by flow cytometry experiments in vitro. Full-length Gm18840 was identified by using a rapid amplification of cDNA ends (RACE) assay. The subcellular distribution of Gm18840 was examined by nuclear/cytoplasmic RNA fractionation and qRT-PCR. RNA pulldown and RNA immunoprecipitation (RIP)-qPCR assays were performed to identify Gm18840-interacting proteins. Chromatin isolation by RNA purification (ChIRP)-seq (chromatin isolation by RNA purification) was used to identify the genome-wide binding of Gm18840 to chromatin. The regulatory activity of Gm18840 in transcriptional regulation was examined by a luciferase reporter assay and qRT-PCR.
ResultsGm18840 was upregulated after myocardial infarction in both in vivo and in vitro MIRI models. Gm18840 was 1,471 nt in length and localized in both the cytoplasm and the nucleus of HL-1 cells. Functional studies showed that the knockdown of Gm18840 promoted the apoptosis of HL-1 cells. Gm18840 directly interacts with histones, including H2B, highlighting a potential function in transcriptional regulation. Further ChIRP-seq and RNA-seq analyses showed that Gm18840 is directly bound to the cis-regulatory regions of genes involved in developmental processes, such as Junb, Rras2, and Bcl3.
ConclusionGm18840, a novel transcriptional regulator, promoted the apoptosis of myocardial cells via direct transcriptional regulation of essential genes and might serve as a novel therapeutic target for MIRI.

背景 缺血再灌注介导的心肌梗死（Ischemia/Reperfusion-mediated Myocardial Infarction, MIRI）是缺血性心脏病进展过程中的主要病理诱因，但MIRI发生过程中失调的关键调控因子，尤其是心血管发育所必需的非编码因子，目前尚未得到完全阐明。
方法 本研究通过构建小鼠MIRI模型并结合RNA测序（RNA-seq），筛选心肌梗死发生后的关键长链非编码RNA（lncRNAs）。采用定量实时聚合酶链反应（qRT-PCR）验证该类RNA在心肌组织与心肌细胞中的表达水平。通过体外流式细胞术实验，明确Gm18840对HL-1细胞增殖的调控作用。利用cDNA末端快速扩增（RACE）实验获取Gm18840的全长序列。通过核质RNA分离结合qRT-PCR，检测Gm18840的亚细胞定位。采用RNA下拉实验与RNA免疫沉淀（RIP）-qPCR，筛选与Gm18840相互作用的蛋白质。通过染色质RNA纯化测序（ChIRP-seq，全称Chromatin Isolation by RNA Purification），分析Gm18840在全基因组范围内与染色质的结合情况。通过荧光素酶报告基因实验与qRT-PCR，验证Gm18840的转录调控活性。
结果 无论是在体内还是体外MIRI模型中，心肌梗死后Gm18840的表达均显著上调。Gm18840的全长序列为1471 nt，且同时定位于HL-1细胞的细胞质与细胞核内。功能实验结果显示，敲低Gm18840可促进HL-1细胞的凋亡。Gm18840可直接与包括H2B在内的组蛋白相结合，提示其可能参与转录调控过程。进一步的ChIRP-seq与RNA-seq分析表明，Gm18840可直接结合发育相关基因的顺式调控区域，如Junb、Rras2及Bcl3。
结论 Gm18840作为一种新型转录调控因子，可通过直接调控核心基因的转录进而促进心肌细胞凋亡，有望成为治疗MIRI的新型潜在靶点。