The current study evaluated the outcome of dietary folate modulations on the expression of tumor suppressor genes (TSGs) during developmental stages of hepatocellular carcinoma (HCC) in a Wistar rat model. In addition to dietary folate modulations, male rats were administered diethylnitrosamine (DEN) intraperitoneally once a week upto 18 weeks to induce HCC. Serum folate levels were found to be decreased and increased in folate deficiency (FD) and folate-oversupplemented (FO) groups respectively when compared to folate normal (FN) rats. Apoptosis was observed in FD in fibrosis and HCC stages. mRNA expression analysis by RT-PCR of TSGs (DPT, p16, RUNX3, RASSF1A and SOCS1) and protein expression by western blot (RASSF1A, RUNX3 and p16) depicted differential expression in FD and FO in various stages of HCC development. Bisulfite sequencing for p16 and RASSF1A promoter was performed. The promoter region of p16 gene was hypermethylated at 7th and that of RASSF1A was hypomethylated at 10th CpG in cirrhotic category in FD rats. Hyper and hypomethylation at 10th and 24th CpG respectively in RASSF1A promoter was observed in HCC category in both FD and FO groups. All TSGs showed differential expression at transcript and protein level. Increased expression of DPT, RASSF1A, SOCS1 and decreased expression of RUNX3 could be playing role in HCC development in FD rats. Reduced expression of RUNX3, RASSF1A and SOCS1 in HCC category was demonstrated in FO rats. Thus, the studied TSGs are differentially expressed with dietary folate modulations during the development of HCC in DEN-treated rat model and the promoter methylation might be a contributing mechanism under these conditions.

中文翻译：

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在肝细胞癌大鼠模型中，膳食叶酸调节对肿瘤抑制基因进行差异调节。

目前的研究评估了 Wistar 大鼠模型中肝细胞癌 (HCC) 发育阶段膳食叶酸调节对肿瘤抑制基因 (TSG) 表达的影响。除了饮食叶酸调节外，雄性大鼠每周腹腔注射一次二乙基亚硝胺 (DEN)，长达 18 周，以诱导 HCC。与叶酸正常（FN）大鼠相比，叶酸缺乏（FD）组和叶酸过量（FO）组的血清叶酸水平分别降低和升高。在纤维化和 HCC 阶段的 FD 中观察到细胞凋亡。通过 RT-PCR 对 TSG（DPT、p16、RUNX3、RASSF1A 和 SOCS1）进行 mRNA 表达分析，并通过蛋白质印迹（RASSF1A、RUNX3 和 p16）进行蛋白质表达分析，显示了 HCC 发展各个阶段 FD 和 FO 中的差异表达。对 p16 和 RASSF1A 启动子进行亚硫酸氢盐测序。FD大鼠肝硬化组中p16基因启动子区第7位CpG高甲基化，RASSF1A启动子区第10位CpG低甲基化。在 FD 和 FO 组的 HCC 类别中，分别观察到 RASSF1A 启动子中第 10 个和第 24 个 CpG 的高甲基化和低甲基化。所有 TSG 在转录物和蛋白质水平上均表现出差异表达。DPT、RASSF1A、SOCS1 表达增加和 RUNX3 表达减少可能在 FD 大鼠 HCC 发展中发挥作用。在 FO 大鼠中，HCC 类别中 RUNX3、RASSF1A 和 SOCS1 的表达降低。因此，在 DEN 处理的大鼠模型中，在 HCC 的发展过程中，所研究的 TSG 随饮食叶酸调节而差异表达，并且启动子甲基化可能是这些条件下的一个促成机制。