Table 6_The combination of decitabine with multi-omics confirms the regulatory pattern of the correlation between DNA methylation of the CACNA1C gene and atrial fibrillation.xlsx

BackgroundStudies have shown that DNA methylation of the CACNA1C gene is involved in the pathogenesis of various diseases and the mechanism of drug action. However, its relationship with atrial fibrillation (AF) remains largely unexplored.
ObjectiveTo investigate the association between DNA methylation of the CACNA1C gene and AF by combining decitabine (5-Aza-2′-deoxycytidine, AZA) treatment with multi-omics analysis.
MethodsHepG2 cells were treated with AZA to observe the expression of the CACNA1C gene, which was further validated using gene expression microarrays. Pyrosequencing was employed to validate differentially methylated sites of the CACNA1C gene observed in DNA methylation microarrays. A custom DNA methylation dataset based on the MSigDB database was combined with ChIP-sequencing and RNA-sequencing data to explore the regulatory patterns of DNA methylation of the CACNA1C gene.
ResultsTreatment of HepG2 cells with three different concentrations of AZA (2.5 µM, 5.0 µM, and 10.0 µM) resulted in 1.6, 2.5, and 2.9-fold increases in the mRNA expression of the CACNA1C gene, respectively, compared to the DMSO group, with statistical significance at the highest concentration group (p < 0.05). Similarly, AZA treatment of T47D cells showed upregulated mRNA expression of the CACNA1C gene in the gene expression microarray results (adj P < 0.05). DNA methylation microarray analysis revealed that methylation of a CpG site in intron 30 of the CACNA1C gene may be associated with AF (adj P < 0.05). Pyrosequencing of this site and its adjacent two CpG sites demonstrated significant differences in DNA methylation levels between AF and sinus rhythm groups (p < 0.05). Subsequent multivariate logistic regression models confirmed that the DNA methylation degree of these three sites and their average was associated with AF (p < 0.05). Additionally, the UCSC browser combined with ChIP-sequencing revealed that the aforementioned region was enriched in enhancer markers H3K27ac and H3K4me1. Differential expression and pathway analysis of RNA-sequencing data ultimately identified ATF7IP and KAT2B genes as potential regulators of the CACNA1C gene.
ConclusionThe DNA methylation levels at three CpG sites in intron 30 of the CACNA1C gene are associated with AF status, and potentially regulated by ATF7IP and KAT2B.

【背景】已有研究证实，CACNA1C基因的DNA甲基化（DNA methylation）参与多种疾病的发病过程与药物作用机制，但该基因甲基化与心房颤动（atrial fibrillation, AF）的关联仍未得到充分探索。
【目的】本研究通过联合地西他滨（decitabine, 5-Aza-2′-脱氧胞苷, AZA）处理与多组学分析，探究CACNA1C基因DNA甲基化与AF的关联。
【方法】本研究采用AZA处理HepG2细胞以观测CACNA1C基因的表达水平，并通过基因表达微阵列（gene expression microarrays）进一步验证该结果；利用焦磷酸测序（Pyrosequencing）对DNA甲基化微阵列中筛选出的CACNA1C基因差异甲基化位点进行验证。此外，本研究整合基于MSigDB数据库构建的定制化DNA甲基化数据集与染色质免疫共沉淀测序（ChIP-sequencing）、RNA测序（RNA-sequencing）数据，以探究CACNA1C基因DNA甲基化的调控模式。
【结果】与二甲基亚砜（DMSO）对照组相比，分别以2.5 μM、5.0 μM及10.0 μM三种浓度的AZA处理HepG2细胞后，CACNA1C基因的mRNA表达水平分别升高1.6倍、2.5倍及2.9倍，其中最高浓度组的差异具有统计学意义（p < 0.05）。同样，基因表达微阵列结果显示，AZA处理T47D细胞后可上调CACNA1C基因的mRNA表达（校正后P < 0.05）。DNA甲基化微阵列分析显示，CACNA1C基因第30内含子区域的一个CpG位点甲基化可能与AF相关（校正后P < 0.05）。对该位点及其邻近的两个CpG位点进行焦磷酸测序验证，结果显示AF组与窦性心律（sinus rhythm）组的DNA甲基化水平存在显著差异（p < 0.05）。后续多因素logistic回归模型证实，这三个位点的甲基化水平及其平均值均与AF显著相关（p < 0.05）。此外，通过UCSC基因组浏览器（UCSC browser）结合染色质免疫共沉淀测序数据分析发现，上述区域富集增强子标记H3K27ac与H3K4me1。对RNA测序数据进行差异表达分析及通路富集分析后，最终筛选出ATF7IP与KAT2B基因作为CACNA1C基因的潜在调控因子。
【结论】CACNA1C基因第30内含子区域的三个CpG位点的DNA甲基化水平与AF患病状态显著相关，且其可能受ATF7IP与KAT2B基因的调控。