基于RNA-seq分析TBL2基因在心肌细胞缺氧/复氧损伤中的作用机制

Role and mechanism of TBL2 gene in hypoxia/reoxygenation injury of cardiomyocytes based on RNA⁃seq analysis

目的 采用转录组测序技术探讨转导蛋白β样蛋白2(TBL2)基因在大鼠心肌H9c2细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤中的影响及其作用机制。方法 通过H/R诱导H9c2细胞建立心肌细胞损伤模型,体外模拟心肌缺血。采用Western blot检测H/R心肌细胞与正常H9c2细胞中TBL2表达水平。H9c2心肌细胞分为si-TBL2组和si-NC组,分别转染TBL2-siRNA、NC-siRNA后,再进行H/R处理,每组分别取3个样本进行RNA-seq。采用DESeq2软件筛选差异表达基因(differentially expressed genes,DEGs)。采用ClusterProfiler对数据进行差异基因的GO和KEGG功能注释和富集分析。使用STRING数据库联合Cytoscape软件建立DEGs的蛋白-蛋白相互作用(PPI)网络并筛选核心基因。结果 与正常H9c2细胞相比,TBL2在H/R心肌细胞中显著升高(P=0.002 2)。转录组测序分析发现si-TBL2组和si-NC组DEGs共905个,其中566个下调基因,309个上调基因。GO功能富集分析结果显示,DEGs主要富集在细胞黏附、调节全身动脉血压、钾离子运输等生物过程,以及细胞外基质、细胞外间隙、参与细胞黏附的蛋白质复合物等细胞组分,跨膜受体活性、信号受体活性、无机阳离子跨膜转运蛋白活性、G蛋白偶联受体活性等分子功能。KEGG通路富集分析结果显示,DEGs主要分布在40条信号通路中,主要包括:细胞黏附分子、神经活性配体-受体相互作用、ECM受体相互作用、PI3K/Akt信号通路、钙信号通路、蛋白质消化和吸收等。通过构建PPI网络图共筛选出7个核心基因,包括:IL-6、LEP、IL-1α、FGF9、GFAP、CD68、AGT。结论 TBL2在心肌梗死细胞模型中显著高表达,抑制TBL2基因在H/R诱导的H9c2心肌细胞损伤模型中有明显差异表达基因,并且参与多种生物学进程和信号通路调节,影响心肌细胞的增殖、凋亡、炎症反应的过程,可能是心肌梗死潜在的治疗靶点。

Objective To investigate the effects of transducinβ‐like protein 2(TBL2)gene on hypoxia/reoxygenation(H/R)injury in rat myocardial H9c2 cells by transcriptome sequencing technology.Methods The cardiomyocyte injury model was established in H9c2 cells through H/R induction to simulate myocardial ischemia in vitro.The expression level of TBL2 in H/R cardiomyocytes and normal H9c2 cells was detected by Western blot.H9c2 cardiomyocytes were transfected with TBL2‐siRNA(si‐TBL2 group)and NC‐siRNA(si‐NC group),respectively,and then subjected to H/R treatment.Three samples were taken from each group for RNA‐seq.The differentially expressed genes(DEGs)were screened by DESeq2 software.The GO and KEGG functional annotation and enrich‐ment analysis of DEGs were conducted using ClusterProfiler.The protein‐protein interaction(PPI)network of DEGs was established using the STRING database in combination with Cytoscape software,and the core genes were screened.Results Compared with normal H9c2 cells,TBL2 was significantly elevated in H/R cardiomyocytes(P=0.0022).Transcriptome sequencing analysis identified a total of 905 differentially expressed genes(DEGs)between si‐TBL2 group and si‐NC group,among which 566 were downregulated and 309 were upregulated.GO functional enrichment analysis indicated that the DEGs were mainly enriched in biological processes such as cell adhesion,regulation of systemic arterial blood pressure,and potassium ion transport,cellular components such as extra‐cellular matrix,extracellular space,and protein complexes involved in cell adhesion,and molecular functions such as transmembrane receptor activity,signal receptor activity,inorganic cation transmembrane transporter activity,and G protein‐coupled receptor activity.KEGG pathway enrichment analysis demonstrated that the DEGs were mainly distributed in 40 signaling pathways,mainly including cell adhesion molecules,neuroactive ligand‐receptor interactions,ECM receptor interactions,PI3K/Akt signaling pathway,calcium signaling pathway,protein digestion and absorption,etc.Through the construction of the PPI network diagram,a total of 7 core genes were screened out,including IL‐6,LEP,IL‐1α,FGF9,GFAP,CD68,and AGT.Conclusion TBL2 is significantly highly expressed in the myocardial infarction cell model.There are remarkable differentially expressed genes in the H/R‐induced H9c2 cardiomyocyte injury model after inhibiting TBL2 gene.TBL2 participates in the regulation of multiple biological processes and signaling pathways,affects the processes of proliferation,apoptosis,and inflammatory response of cardiomyocytes.TBL2 may potentially be a therapeutic target for myocardial infarction.