Expression Characteristics of Genes Hypermethylated and Downregulated in Rat Liver Specific to Nongenotoxic Hepatocarcinogens
Expression Characteristics of Genes Hypermethylated and Downregulated in Rat Liver Specific to Nongenotoxic Hepatocarcinogens
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DOI：
10.1093/toxsci/kfz027
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发表时间：
2019-05-01
影响因子：
3.8
通讯作者：
Shibutani, Makoto
中科院分区：
文献类型：
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作者：
Ito, Yuko;Nakajima, Kota;Shibutani, Makoto
本研究通过甲基序列分析和表达芯片分析相结合的方法，检测了四氯化碳(CCl4)特异性基因的高甲基化和下调，并排除了DEN大鼠。在52个基因中，28天的甲基化特异性定量PCR分析证实，Ldlrad4、proc、CDH17和NfiA基因表现出启动子区域的超甲基化。实时逆转录-聚合酶链式反应分析显示，与未处理的对照组和致癌基因相比，经90天的非遗传毒性肝癌诱导剂处理的大鼠肝脏中这4个基因的转录水平降低。免疫组织化学显示，LDLRAD4和Proc在胎盘型谷胱甘肽S转移酶(GST-P)(+)灶中的免疫反应性降低，形成阴性灶，而非遗传毒性致癌物处理84天或90天后，GST-P+灶中LDLRAD4(-)和proc-灶的发生率高于遗传毒性致癌物。相比之下，CDH17和NFIA对致癌物的反应与致癌物的遗传毒性潜力没有任何关系。治疗28天后，所有4个基因对肾脏致癌物均无反应。考虑到Ldlrad4是转化生长因子-β信号的负调控因子，proc通过激活参与p21(WAF1/CIP1)上调，CDH17通过基因敲除导致细胞周期停滞，以及Nfia发挥抑癌作用，这些基因可能是治疗早期非遗传性肝癌的潜在表观遗传学标志物。LDLRAD4和proc可能在非遗传毒性肝癌致癌前病变的发生发展中起一定作用。
This study examined hypermethylated and downregulated genes specific to carbon tetrachloride (CCl4) by Methyl-Seq analysis combined with expressionmicroarray analysis in the liver of rats treated with CCl4 or N-nitrosodiethylamine (DEN) for 28 days, by excluding those with DEN. Among 52 genes, Ldlrad4, Proc, Cdh17, and Nfia were confirmed to show promoter-region hypermethylation bymethylation-specific quantitative PCR analysis on day 28. The transcript levels of these 4 genes decreased by real-time reverse transcription-PCR analysis in the livers of rats treated with nongenotoxic hepatocarcinogens for up to 90 days compared with untreated controls and genotoxic hepatocarcinogens. Immunohistochemically, LDLRAD4 and PROC showed decreased immunoreactivity, forming negative foci, in glutathione S-transferase placental form(GST-P)(+) foci, and incidences of LDLRAD4(-) and PROC- foci in GST-P+ foci induced by treatment with nongenotoxic hepatocarcinogens for 84 or 90 days were increased compared with those with genotoxic hepatocarcinogens. In contrast, CDH17 and NFIA responded to hepatocarcinogens without any relation to the genotoxic potential of carcinogens. All 4 genes did not respond to renal carcinogens after treatment for 28 days. Considering that Ldlrad4 is a negative regulator of transforming growth factor-beta signaling, Proc participating in p21(WAF1/CIP1) upregulation by activation, Cdh17 inducing cell cycle arrest by gene knockdown, and Nfia playing a role in a tumor-suppressor, all these genesmay be potential in vivo epigeneticmarkers of nongenotoxic hepatocarcinogens from the early stages of treatment in terms of gene expression changes. LDLRAD4 and PROC may have a role in the development of preneoplastic lesions produced by nongenotoxic hepatocarcinogens.