dsRNA和Aza-CdR转染猪肾细胞的全基因组差异甲基化区域分布特征的比较

本研究旨在通过比对PolyI：C和Aza-CdR转染猪肾细胞后全基因组差异甲基化峰的分布特征，进而筛选Gene Ontology （GO）特有的差异甲基化基因，分析差异甲基化区域。首先，基于MeDIP-chip技术，采用猪385 K全基因组启动子和CpG岛甲基化芯片，分析3组试验材料（病毒模拟物Poly I：C转染的猪PK15细胞、甲基化酶抑制剂Aza-CdR转染的PK15细胞、无处理的mock细胞），通过Peak DM Value和Peak Score值获得试验组间显著性富集的差异甲基化峰；其次，对差异甲基化基因进行GO注释，筛选差异甲基化区域和差异甲基化基因。最终结合Bisulfite克隆测序和mRNA荧光定量表达试验验证差异甲基化区域DMR。试验初步揭示猪肾细胞全基因组DNA甲基化主要分布于5'调控区域。试验在组间比较后，特别是在P vs.C和A vs.C比较中发现DNA甲基化在基因组上的分布特征与CpG岛密度与距离TSS的位置有关，而在近启动子区域（0―+200 bp） DNA甲基化显著影响基因的表达。Poly I：C对PK15作用使得TSS附近200 bp （-200―+500 bp）低甲基化启动子增多，说明Poly I：C与Aza-CdR的作用相似，均具有潜在的去甲基化作用，特别是位于猪14号染色体上BNIP3L基因的10459946―10460615 bp区段共有669 bp Peak Length CG位点发生去甲基化。研究揭示，PolyI：C和Aza-CdR并不是对猪所有基因具有去甲基化作用，主要针对特有基因的特有启动子，证明这些特有启动子的CpG岛对Poly I：C和Aza-CdR具有特别的敏感性。

Comparison of Genome-wide Differential Methylation Regions in dsRNA and Aza-CdR Transfected Porcine Kidney Cells

This study aimed to screen differential methylation genes and analyze the differential methylation regions specific to the Gene Ontology (GO) by comparing the distribution characteristics of differential methylation peaks in the whole genome of PK15 cells transfected by Poly I:C and Aza-CdR. Firstly, the pig 385 K whole genome promoter and CpG Island methylation chip were used based on MeDIP-chip technology. Three groups of experimental materials (porcine PK15 cells transfected with Poly I:C virus mimic, PK15 cells transfected with methylase inhibitor Aza-CdR, and mock cells without treatment) were analyzed to obtain significantly enriched differential methylation peaks between two experimental groups based on Peak DM Value and Peak Score. Secondly, Gene Ontology (GO) annotation of differential methylation genes was performed to screen for differential methylation regions and differential methylation genes. Finally, the differential methylated region DMR was verified by Bisulfite clone sequencing and mRNA fluorescence quantitative expression test. DNA methylation in the whole genome of porcine kidney cells was mainly distributed in the 5' regulatory region. After comparison between groups, especially P vs. C and A vs. C comparisons, it was found that the distribution characteristics of DNA methylation on the genome were related to the density of CpG island and the location of distance from TSS. DNA methylation in the near promoter region (0-+200 bp) significantly affected gene expression. The effect of Poly I:C on PK15 increased the number of hypomethylation promotors at 200 bp (-200-+500 bp) near TSS, indicating that Poly I:C and Aza-CdR have similar potential demethylation effects. Especially, 669 bp Peak Length CG sites were demethylated in the 10459946-10460615 bp region of the BNIP3L gene on pig chromosome 14. It was revealed that Poly I:C and Aza-CdR did not demethylate in all pig genes, but mainly targeted specific promoters of specific genes, demonstrating that CpG islands of these specific promoters were particularly sensitive to Poly I:C and Aza-CdR.