肾阳虚体质大鼠肝细胞全基因组DNA甲基化的测序与分析

Whole-genome DNA Methylation Sequencing and Analysis of Hepatocytes in Rats with Kidney Yang Deficiency Constitution

目的探讨肾阳虚体质大鼠肝细胞DNA甲基化特征。方法选取本课题组前期研发的自发性肾阳虚体质模型大鼠子一代、子四代雄性各3只分别组成子一代组和子四代组,3只正常SD大鼠设为空白组。取各组大鼠肝脏组织,进行DNA抽取与质检,构建DNA文库,进行甲基化测序,与参考基因组进行唯一比对,定位富集区域。将子一代组、子四代组富集区域分别与空白组比较,取差异富集区域。子一代组、子四代组差异富集区域取交集,将富集区域关联到基因,并根据组间富集区域富集样本个数差异筛选组间差异的甲基化富集区域。针对差异关联基因集进行GO、KEGG功能分析。结果子一代组共获得276091个富集区域,子四代组共获得309430个富集区域,空白组共获得275413个富集区域。与空白组大鼠相比,子一代组肾阳虚体质大鼠差异富集区域共372个,其中甲基化水平增高89个,甲基化水平降低283个;子四代组肾阳虚体质大鼠差异富集区域共51条,其中甲基化水平增高30个,甲基化水平降低21个。取子一代组与子四代组差异富集区域的交集,初步筛出差异富集区域12个。对差异富集区域进行GO、KEGG功能分析,密切关联到基因11个,其中甲基化水平增高有Gdap1、Cdkal1、Cd300lb,甲基化水平降低有Zmiz1、Fam111a、Sh3bp4、As3mt、Col16a1、LOC103690271、Wdpcp、LOC102553861,基因功能涉及线粒体膜生成、代谢过程、细胞增殖、细胞吞噬、信号转导、生物调节、蛋白定位等。结论肾阳虚体质大鼠存在肝细胞DNA甲基化的差异表达,差异甲基化位点其关联基因集中于肿瘤发生和线粒体能量代谢、糖脂代谢等生物学功能紊乱。

Objective To explore the characteristics of DNA methylation of hepatocytes in rats with kidney yang deficiency constitution.Methods Three first-generation and three fourth-generation male offspring of spontaneous kidney yang deficiency model rats developed by our previous research were selected as the first-generation offspring group and fourth-generation offspring group,respectively,and three normal SD rats were set as the blank group.The liver tissues of rats in each group were collected for DNA extraction and quality inspection,and DNA libraries were then constructed.Methylation sequencing was performed,and the results were uniquely compared with the reference genome to identify the location of enrichment regions.The enriched region of the first-generation offspring group and the fourth-generation offspring group were compared to that of the blank group,respectively,to identify the differentially enriched regions.The differentially enriched regions of first-generation offspring group and the fourth-generation offspring group were intersected,and the enrichment region was associated with genes.The methylation-enriched regions that differ between groups were then identified according to the differences in the number of enriched samples in enriched regions between groups.GO and KEGG functional analysis was performed on differentially expressed genes.Results A total of 276091 enrichment regions were obtained in the first-generation offspring group,while 309430 enrichment regions and 275413 enrichment regions were identified in the fourth-generation offspring group and the blank group,respectively.Compared to the rats in the blank group,there were 372 differentially enriched regions in the kidney yang deficiency rats in the first-generation offspring group,among which 89 regions had increased methylation levels and 283 had decreased methylation levels;there were 51 differentially enriched regions in the kidney yang deficiency rats in the fourth-generation offspring group,among which 30 regions had increased methylation levels and 21 had decreased methylation levels.The intersection of the differentially enriched areas of the first-generation offspring group and the fourth-generation offspring group preliminarily identified 12 differentially enriched regions.GO and KEGG functional analysis of differentially enriched regions showed that 11 genes were closely related.Gdap 1,Cdkall,and Cd3001b had an increase in the methylation level,while Zmiz1,Fam111a,Sh3bp4,As3 mt,Coll6al,LOC103690271,Wdpcp,and LOC102553861 had a decrease in the methylation level.Gene function was involved with mitochondrial membrane formation,metabolic process,cell proliferation,cell phagocytosis,signal transduction,biological regulation,protein localization,etc.Cnclusion DNA methylation of hepatocytes is differentially expressed in rats with kidney yang deficiency constitution,and the genes associated with differential methylation sites are mainly involved with tumorigenesis and biological function disorders such as mitochondrial energy metabolism and glucose and lipid metabolism.