目的 探讨组蛋白去甲基化酶含Jumonji结构域的蛋白3（Jumonji domain-containing protein D3，JMJD3）在新生儿坏死性小肠结肠炎（necrotizing enterocolitis，NEC）肠道组织中的表达情况。 方法 收集对照（先天性小肠闭锁）患儿及NEC患儿手术中切除的回肠组织进行RNA测序，筛选出差异基因。对差异基因进行组蛋白甲基化修饰酶相关基因聚类分析，筛选出目的基因赖氨酸特异性去甲基化酶6B[lysine（K）-specific demethylase 6B，KDM6B]，并采用实时荧光定量聚合酶链反应扩大样本量进行验证。蛋白免疫印迹和免疫组织化学检测JMJD3以及其底物组蛋白H3第27位赖氨酸三甲基化（trimethylation of histone H3 at lysine 27，H3K27me3）在肠道组织中的蛋白表达情况。组间比较根据数据分布特点采用t检验或Mann-Whitney U检验。 结果 RNA测序筛查出1 481条差异基因，其中643条基因上调，838条基因下调。筛查NEC肠道组织中30种组蛋白甲基化修饰酶的表达情况，其中KDM6B在NEC样本中差异表达最显著[Log2（fold change）=0.769 104，P=0.009 009]。实时荧光定量聚合酶链反应结果显示，与对照组相比，KDM6B在NEC肠道组织中高表达（P<0.05）；蛋白免疫印迹和免疫组织化学结果显示JMJD3蛋白水平在NEC肠道组织中明显增高，H3K27me3蛋白水平在NEC肠道组织中降低。 结论 在NEC肠道组织中组蛋白去甲基化酶JMJD3高表达，H3K27me3低表达。JMJD3可能通过降低H3K27me3的表达水平调控NEC的发生发展。 Objective To explore the expression of histone demethylase Jumonji domain-containing protein D3 (JMJD3) in intestinal tissue of necrotizing enterocolitis (NEC). Methods RNA-sequencing was conducted for screening out the differential genes in intestinal tissues from controls (congenital intestinal atresia) and NEC children. And cluster analysis of histone methylation modification-related genes was utilized for screening target gene lysine (K)-specific demethylase 6B (KDM6B). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for verifying the expression of KDM6B in intestinal tissues. Western blot and immunohistochemistry were utilized for detecting the protein expressions of JMJD3 and trimethylation of histone H3 at lysine 27 (H3K27me3) in intestinal tissues. Group comparisons were made by t or Mann-Whitney U test, depending on data distribution. Results RNA sequencing revealed 1481 differentially expressed genes, including 643 up-regulated and 838 down-regulated genes. Among 30 histone methylation modifying enzymes, KDM6B was the most differentially expressed in NEC intestinal tissues [Log2(fold change)=0.769104, P=0.009009]. qRT-PCR indicated a higher expression of KDM6B in NEC intestinal tissues compared to controls (P<0.05). Western blot and immunohistochemistry indicated a higher level of JMJD3 protein and a lower level of H3K27me3 protein in NEC intestinal tissues. Conclusions A high expression of histone demethylase JMJD3 and a low expression of H3K27me3 are detected in NEC intestinal tissues. JMJD3 may participate in the process of NEC through down-regulating the expression of H3K27me3.
Expression of histone demethylase JMJD3 in necrotizing enterocolitis
Objective To explore the expression of histone demethylase Jumonji domain-containing protein D3 (JMJD3) in intestinal tissue of necrotizing enterocolitis (NEC). Methods RNA-sequencing was conducted for screening out the differential genes in intestinal tissues from controls (congenital intestinal atresia) and NEC children. And cluster analysis of histone methylation modification-related genes was utilized for screening target gene lysine (K)-specific demethylase 6B (KDM6B). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for verifying the expression of KDM6B in intestinal tissues. Western blot and immunohistochemistry were utilized for detecting the protein expressions of JMJD3 and trimethylation of histone H3 at lysine 27 (H3K27me3) in intestinal tissues. Group comparisons were made by t or Mann-Whitney U test, depending on data distribution. Results RNA sequencing revealed 1481 differentially expressed genes, including 643 up-regulated and 838 down-regulated genes. Among 30 histone methylation modifying enzymes, KDM6B was the most differentially expressed in NEC intestinal tissues [Log2(fold change)=0.769104, P=0.009009]. qRT-PCR indicated a higher expression of KDM6B in NEC intestinal tissues compared to controls (P<0.05). Western blot and immunohistochemistry indicated a higher level of JMJD3 protein and a lower level of H3K27me3 protein in NEC intestinal tissues. Conclusions A high expression of histone demethylase JMJD3 and a low expression of H3K27me3 are detected in NEC intestinal tissues. JMJD3 may participate in the process of NEC through down-regulating the expression of H3K27me3.
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