紫绀型先心病患儿心肌SOCS3启动子甲基化分析及可能机制研究

发布时间：2018-04-21 00:20

  本文选题：紫绀型 + 心病　； 参考：《第三军医大学》2014年硕士论文

【摘要】：背景： 紫绀型先天性心脏病威胁人类生命与健康，了解其病理生理机制，有助于促进临床诊疗与预后。紫绀型先天性心脏病患者经历着慢性缺氧的病理生理过程，而心肌如何在慢性缺氧应激条件下，完成凋亡、自噬、分泌、代谢、重构等结构功能方面的适应性变化，其具体机制尚未完全阐明。 细胞信号抑制子-3(Suppressor of cell signaling-3，SOCS3)作为Janus激酶-信号转导与转录激活子-3(Janus kinase-Signal transducer and activator of transcription-3，JAK-STAT3)信号通路下游分子，负反馈调控该通路活性，维持细胞信号稳态。我们前期研究发现，相对于非紫绀型先心病患者，紫绀型先心病患者心肌IL-6-JAK-STAT3激活，SOCS3转录和翻译水平上调，且SOCS3蛋白水平的升高依赖于转录水平升高；而缺氧心肌细胞中过表达SOCS3使IL-6-JAK-STAT3信号通路活性抑制，细胞凋亡增加。提示适度表达的SOCS3，对于细胞内IL-6-JAK-STAT3信号稳态至关重要，在心肌慢性缺氧适应过程起到重要调控作用，但并不清楚缺氧心肌中何种因素参与了SOCS3的转录调控。之前肿瘤细胞中的研究显示，SOCS3基因启动子甲基化参与其转录调控，影响蛋白表达，从而调控JAK-STAT3通路活性，维持肿瘤细胞增殖与抗凋亡。因此，我们推测在慢性缺氧心肌中，SOCS3启动子低甲基化可能参与了其转录表达调控，调控JAK-STAT3信号通路活性，参与心肌慢性缺氧适应信号通路平衡。本研究从表观遗传学角度探讨心肌慢性缺氧适应的分子机制，为围手术期心肌保护分子靶点寻找提供理论依据。 目的： 探讨紫绀型先心病患儿心肌组织SOCS3基因启动子甲基化水平及其可能的机制。 方法： 收集紫绀型先天性心脏病(紫绀组，n=18)和非紫绀型先天性心脏病(非紫绀组，n=16)患者术中切除的肥厚的右心室心肌组织。提取心肌组织全基因组DNA，重亚硫酸盐修饰后，，甲基化特异性聚合酶链反应(Methylation specific PCR，MSP)、重亚硫酸盐修饰测序(Bisulfite Sequencing PCR，BSP)检测心肌组织中SOCS3基因启动子CpG岛甲基化差异。 Western blotting检测心肌组织中DNA甲基化转移酶1(DNA methyltransferase1，DNMT1)、DNA甲基化转移酶3A(DNA methyltransferase3A，DNMT3A)、DNA甲基化转移酶3B(DNA methyltransferase3B，DNMT3B)蛋白表达，qRT-PCR检测心肌组织中DNMT1、DNMT3A、DNMT3B mRNA表达。 结果： 1.紫绀组心肌组织SOCS3基因启动子CpG岛呈部分甲基化（甲基化频率为54.287±16.744%），非紫绀组无明显甲基化(甲基化频率为0.000±0.000%)。 2.紫绀组中DNMT3A蛋白表达较非紫绀组增高(0.407±0.469v.s0.160±0.034，P＜0.05)， DNMT1(0.084±0.115v.s0.081±0.085, P0.05)、 DNMT3B(0.054±0.012v.s0.052±0.093，P＞0.05)蛋白表达在紫绀组及非紫绀组间无显著性差异。 3.紫绀组中DNMT1(0.548±0.553v.s0.920±0.456, P0.05)、DNMT3A(0.555±0.395v.s0.756±0.240，P0.05)、DNMT3B (0.473±0.509v.s1.018±0.749，P0.05) mRNA水平较非紫绀组明显降低。 4.紫绀组中DNMT3A蛋白水平与SOCS3基因启动子甲基化程度显著正相关(r=0.359，P=0.047)。 结论： 紫绀型先心病患者心肌组织中，SOCS3基因启动子呈部分甲基化，DNMT3A蛋白水平增高，且DNMT3A蛋白水平与SOCS3基因启动子甲基化程度显著正相关。DNMT3A蛋白可能通过酶促反应，以及招募、锚定含有甲基化CpGs的核小体，参与建立和维持了SOCS3启动子CpG岛甲基化模式。
[Abstract]:Background:
Cyanotic congenital heart disease, which threatens human life and health, and understands its pathophysiological mechanism, helps to promote clinical diagnosis and prognosis. Patients with cyanotic congenital heart disease experience the pathophysiological process of chronic anoxia, and how the myocardium can complete apoptosis, autophagy, secretion, metabolism, remodeling and other structural functions under the condition of chronic hypoxia stress. The specific mechanism of adaptive changes has not yet been fully elucidated.
The cell signal suppressor -3 (Suppressor of cell signaling-3, SOCS3) is the downstream molecule of the Janus kinase signal transduction and transcriptional activator -3 (Janus kinase-Signal transducer and) signaling pathway. Negative feedback regulates the activity of the pathway and maintains the cell signal homeostasis. In patients with non cyanotic congenital heart disease, IL-6-JAK-STAT3 activation, SOCS3 transcriptional and translation levels are up-regulated in patients with cyanotic congenital heart disease, and the increase of SOCS3 protein level depends on the increase of transcriptional level, while the overexpression of SOCS3 in anoxic cardiomyocytes inhibits the activity of IL-6-JAK-STAT3 signaling pathway and increases the apoptosis, suggesting a moderately expressed SOCS3. It is important for the homeostasis of intracellular IL-6-JAK-STAT3 signal and plays an important role in the regulation of chronic hypoxia adaptation, but it is not clear what factors in the hypoxia myocardium are involved in the transcription regulation of SOCS3. Previous studies in tumor cells showed that the promoter methylation of SOCS3 gene involved in its transcriptional regulation and influence protein expression. Therefore, we speculate that hypomethylation of SOCS3 promoter in chronic hypoxia myocardium may participate in the regulation of transcriptional expression, regulate the activity of JAK-STAT3 signaling pathway and participate in the pathway balance of chronic hypoxia adaptation signal in the myocardium of chronic anoxic myocardium. This study explored the heart from the epigenetic point of view. The molecular mechanism of chronic hypoxia adaptation provides a theoretical basis for finding molecular targets of myocardial protection during perioperative period.
Objective:
Objective to investigate the methylation level of SOCS3 gene promoter and its possible mechanism in children with cyanotic congenital heart disease.
Method锛

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