糖尿病性心肌病小鼠心肌组织差异表达环状RNA的探讨

Investigation on the differentially expressed circular RNAs in myocardium of mice with diabetic cardiomyopathy

目的鉴定糖尿病性心肌病(DCM)小鼠心肌组织差异表达的环状RNA(circRNA),并分析其可能的生物学功能及调控网络。方法C57BL/6小鼠,雄性,8周龄,体重21~27 g。选取8只小鼠作为正常组,15只进行建模。通过腹腔注射链脲佐菌素建立糖尿病小鼠模型。注射1周后,测定小鼠空腹血糖水平,经检测12只小鼠建模成功。在相同的实验室条件下饲养小鼠12周,通过超声心动图检测小鼠的心脏结构和功能,筛选出左心室射血分数≤60%,且二尖瓣舒张早期最大血流速度与心房收缩期最大血流速度比值(E/A)≤1.6的糖尿病小鼠作为DCM组(n=3)。于DCM组确立当天麻醉后处死正常组和DCM组小鼠,取出心脏,从心肌组织中提取RNA备用。采用高通量测序对DCM组和正常组小鼠心肌组织中的RNA进行测序和鉴定分析,并利用DeSeq2进行差异性分析,通过实时荧光定量PCR(RT-qPCR)在组织水平上对分析结果进行验证,并对鉴定的差异circRNA进行GO分析和KEGG通路分析。通过微小RNA(miRNA)靶基因预测软件进行差异表达circRNA-miRNA相互作用预测。结果DCM小鼠心肌组织中共鉴定出63种差异表达circRNA。RT-qPCR验证结果示同源性分析筛选出的16种差异表达circRNA中8种组织水平的表达与测序结果一致,其中7种表达上调、1种表达下调。KEGG通路分析结果示,上调的circRNA主要与腺苷酸活化蛋白激酶(AMPK)信号通路以及细胞间的黏着连接通路有关,下调的circRNA主要与心肌病有关。GO功能分析显示,上调表达的circRNA在分子功能方面主要与离子、蛋白质、激酶等因子的结合过程有关,并且在细胞组分方面参与调节细胞内结构尤其是细胞器的构成。分子功能与细胞组分方面的功能分析显示,上调的circRNA与细胞组分起源、募集、组织的生物学过程相关,并由此参与细胞生物学过程的调控;下调的circRNA在分子功能方面与催化活性相关,也与蛋白激酶的结合过程、转移酶及钙调蛋白的活性有关,在细胞组分方面与收缩性纤维的组分、肌原纤维的构成密切相关,而在富集的生物学过程GO分析术语中,包括赖氨酸肽基修饰、肌节的构成、肌原纤维的装配、心肌组织的形态学发育、心肌肥厚等生物学过程。本研究鉴定的差异表达circRNA均存在miRNA的结合位点,且部分miRNA与保护心肌细胞正常生理功能、抗氧化应激损伤、抑制心肌细胞凋亡以及抗心肌肥厚有关。结论DCM小鼠心肌组织中circRNA存在差异表达,且其与DCM的发生发展密切相关。

Objective To identify the differentially expressed circular RNA(circRNA)in the myocardium of diabetic cardiomyopathy(DCM)mice,and analyze their possible biological functions and related regulatory network.Methods C57BL/6 mice,aged 8 weeks,and weighing were 21-27 g.Eight mice were selected as the control group and 15 mice were selected as the experimental group.The diabetic mice model was established by intraperitoneal injection of streptozotocin in the experimental group.One week after injection,the fasting blood glucose level of mice was measured,and 12 diabetic mice were included in the final experimental group.All mice were fed for 12 weeks under the same laboratory conditions.The cardiac structure and function were detected by echocardiography.Diabetic mice with the left ventricular ejection fraction less than 60%and the E/A less than 1.6 were selected as DCM group(n=3).Mice in DCM group and control group were then sacrificed under deep anesthesia.RNA was extracted from myocardial tissue.High-throughput RNA sequencing technology was used to sequence and identify the RNA in the myocardial tissue of DCM group and normal control group,and the difference was analyzed by DeSeq2.The analysis results were verified at the tissue level by RT-qPCR,and the differential circRNA were analyzed by GO and KEGG pathway analysis.The differentially expressed circRNA-microRNA(miRNA)interaction was predicted by the miRNA target gene prediction software.Results A total of 63 differentially expressed circRNAs were found in the myocardium of DCM mice.The results of RT-qPCR showed that the tissue level expression of 8 differentially expressed circRNAs was consistent with the sequencing results,of which 7 were up-regulated and 1 was down-regulated.KEGG pathway analysis showed that the up-regulated circRNAs was mainly related to AMPK signal pathway and intercellular adhesion junction pathway,and the down-regulated circRNA was mainly related to cardiomyopathy.Go analysis showed that the up-regulated circRNA was mainly related to the binding process of ions,proteins,kinases and other factors in terms of molecular function,and was involved in regulating the intracellular structure,especially the composition of organelles in terms of cell components.The functional analysis of molecular function and cell components showed that the up-regulated circRNA were related to the cell component origin,recruitment and tissue,and thus participated in the regulation of cell biological process.The down regulated circRNA was related to catalytic activity in terms of molecular function,protein kinase binding process,transferase and calmodulin activity,and was closely related to the components of contractile fibers and the composition of myofibrils.These differentially expressed circRNAs were also related to biological processes such as lysine peptide modification,sarcomere composition,myofibril assembly,morphological development of myocardial tissue,myocardial hypertrophy and so on.Conclusions In this study,we detected the novel differentially expressed circRNAs in the myocardium of DCM mice,and bioinformatics analysis confirmed that these circRNAs are related to oxidative stress,fibrosis and death of cardiomyocytes,and finally participate in the pathophysiological process of DCM.