组蛋白H3K27乙酰化与H3S28磷酸化改变引起心脏发育相关基因的表达变化

Histone H3K27 acetylation and H3S28 phosphorylation alter gene expressions in heart development

目的通过改变组蛋白H3K27和S28位点整体修饰水平并检测心脏发育相关基因表达,初步探索位点修饰对基因表达的影响,阐明位点修饰在先天性心脏病(CHD)发病中的可能机制。方法 1使用丁酸钠、姜黄素、佛波酯处理培养的C2C12细胞;Western Blot检测组蛋白H3K27乙酰化(H3K27ac)和H3S28磷酸化(H3S28ph)水平;实时荧光定量PCR检测心脏相关基因表达情况。2对培养细胞行丁酸钠和姜黄素浓度梯度处理,检测位点修饰及表达差异相关心脏基因表达。采用Pearson相关检验位点修饰与基因表达的关系。结果 1丁酸钠处理后的C2C12细胞H3K27ac水平较对照组明显上调[(1.90±0.04)vs(1.09±0.01),P<0.05],伴H3S28ph水平明显下调[(0.04±0.01)vs(0.73±0.01),P<0.05)];姜黄素组H3K27ac水平(0.04±0.00)较对照组明显下调(P<0.05),伴H3S28ph水平上调[(0.97±0.06)vs(0.73±0.01),P<0.05)];佛波酯组H3S28ph(1.67±0.00)和H3K27ac水平(1.58±0.03)较对照组上调(P<0.05)。2丁酸钠与姜黄素浓度梯度处理后,H3K27ac、H3S28ph水平与给药浓度的指数有显著的相关性(R2分别为0.993和0.966)。在检测的8个心脏相关基因中,药物处理后均产生了表达改变,差异有统计学意义(ANOVA P<0.05);丁酸钠梯度处理的细胞c Tn T、Cx43和Six1基因表达与H3K27ac水平呈负相关(R2分别为0.709、0.713和0.651),与H3S28ph水平呈正相关(R2分别为0.866、0.822和0.766)。姜黄素梯度处理未有显著的相关性(R2<0.5)。结论组蛋白H3K27和H3S28位点修饰状态的变化影响心脏发育相关基因的表达,可能是CHD的潜在发病机制。

Objective To study the possible relationship between histone H3K27 acetylation and S28 phosphorylation and its role in the pathogenesis in congenital heart disease（ CHD） by changing the global modification stage of histone H3K27 and H3S28 residual in culture cells and detecting heart relating genes expressions. Methods C2C12 cell was treated with sodium butyrate（ Na B）,curcumin and TPA. Western Blot was performed to determine the global H3K27 and H3S28 modification stage. RealtimePCR was performed to examine heart relating genes expressions. Na B and curcumin concentration ladders were performed in culture cells and same test was taken to examine histone modification and relative gene expression. Pearson＇s relative analysis was taken to reveal the relationship between histone modification stage and gene expressions. Results In Na B treated group,H3K27 ac was significant higher than controls[（ 1. 90±0. 04） vs（ 1. 09±0. 01）,P〈0. 05） ]while H3S28 ph was lower[（ 0. 04±0. 01） vs（ 0. 73±0. 01）,P〈0. 05） ]. H3K27 ac decreased in curcumin group[（ 0. 04 ±0. 00） vs（ 1. 09 ± 0. 01）,P〈0. 05） ] while an increase in H3S28 ph [（ 0. 97±0. 06） vs（ 0. 73 ± 0. 01）,P〈0. 05） ]. Both H3K27ac（ 1. 58 ± 0. 03） and H3S28ph（ 1. 67 ± 0. 00） were significantly higher than controls（ P〈0. 05）. The residual modifications were highly related to the concentration of treatments（ R2 was 0. 993 and 0. 966）. Most of the examed genes responded to the treatments and had at least one significant expression change.c Tn T,Cx43 and Six1 gene were found a positive relation to S28ph（ R2 was 0. 707,0. 713 and 0. 651） and negative to K27ac（ R2 was 0. 866,0. 822 and 0. 766） while treated with different concentrations of Na B but no similar results was found in curcumin group（ R20. 5）. Conclusion Histone H3K27 ac and H3S28 ph may interact with each other in some way,the global histone modification stage change could affect heart related gene expression,which could be a potential pathogenesis of CHD.