Micro-RNAs in Adult Rat Liver are Refractory to Dioxin Treatment. Rattus norvegicus

Dioxin-like chemicals are well-known for their ability to upregulate expression of numerous genes via the AH receptor (AHR). However, recent transcriptomic analyses in several laboratories indicate that dioxin-like chemicals or AHR genotype itself also can downregulate levels of mRNAs encoded by numerous genes. The mechanism responsible for such downregulation is unknown. We hypothesized that microRNAs (miRNAs), which have emerged as powerful negative regulators of mRNA levels in several systems, might be responsible for mRNA downregulation in dioxin/AHR pathways. We used the Exiqon miRNA array platform as well as quantitative RT-PCR to measure miRNA levels in dioxin-sensitive Long-Evans (Turku/AB; L-E) rats vs. dioxin-resistant Han/Wistar(Kuopio; H/W) rats. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo caused few changes in miRNA levels in rat livers and those changes that were statistically significant were of modest magnitude. AHR genotype had little effect on hepatic miRNA levels, either in constitutive expression or in response to TCDD – only a few miRNAs differed in expression between between L-E rats (that have wildtype AHR) compared to H/W rats (whose AHR has a large deletion in the transactivation domain). It is unlikely that mRNA downregulation by dioxins is mediated by miRNAs, nor are miRNAs likely to play a significant role in dioxin toxicity in adult rodent liver. We conducted a thorough investigation to address the following questions: (1) does AHR genotype itself affect constitutive expression of microRNAs? (2) does TCDD affect microRNA levels and, if so, is this response dependent on the AHR? (3) does TCDD affect microRNA levels differently in animals that are sensitive to dioxin toxicity versus those that are dioxin-resistant? We assessed the in vivo effect of TCDD on microRNA levels in liver at multiple time points after TCDD treatment using microRNA arrays along with quantitative RT-PCR. The cumulative results of our experiments indicate that downregulation of mRNA levels by dioxins in adult rodent livers is very unlikely to involve microRNAs. Manuscript Submitted: Moffat ID, Boutros PC, Celius T, Pohjanvirta R & Okey AB. Micro-RNAs in rodent liver are refractory to dioxin treatment. Toxicological Sciences May, 2007. Keywords: miRNA expression, Time course, response to xenobiotics, genetic modification, comparative genome hybridization Overall design: We examined strain differences in miRNA expression independent of TCDD: 19-h vehicle-treated dioxin-resistant H/W AHRH/W/H/W rats (HC19) vs. vehicle- control dioxin-sensitive L-E AHRWT/WT rats (LC19). In the dioxin-sensitive rats we compared miRNA expression levels 3 h (LT3) or 19 h (LT19) post-TCDD treatment vs. LC19. In the dioxin-resistant rats we compared miRNA levels 3 h (HT3), 19 h (HT19), or 96 h (HT96) post-TCDD treatment vs. HC19.

类二噁英化合物可通过芳香烃受体（AH receptor, AHR）上调大量基因的表达，这一功能早已为人熟知。然而，近期多个实验室的转录组学分析显示，类二噁英化合物或AHR基因型本身，同样可下调大量基因编码的信使RNA（messenger RNA, mRNA）水平。介导此类下调反应的具体机制目前仍不明确。我们提出假说：微小RNA（microRNAs, miRNAs）作为一类在多个系统中被证实的强效mRNA水平负调控因子，可能介导了二噁英/AHR通路中的mRNA下调事件。本研究采用Exiqon miRNA芯片平台结合定量逆转录聚合酶链反应（quantitative RT-PCR），检测了二噁英敏感型Long-Evans（Turku/AB；L-E）大鼠与二噁英抵抗型Han/Wistar（Kuopio；H/W）大鼠体内的miRNA水平。体内给予2,3,7,8-四氯二苯并对二噁英（2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD）后，大鼠肝脏内的miRNA水平几乎未发生明显变化；仅少数具有统计学意义的改变幅度较小。无论是基础表达还是经TCDD处理后，AHR基因型对肝脏miRNA水平的影响均微乎其微；仅野生型AHR的L-E大鼠与AHR基因反式激活结构域存在大片段缺失的H/W大鼠之间，少数miRNA的表达存在差异。由此可见，二噁英介导的mRNA下调几乎不可能由miRNA介导，且miRNA在成年啮齿动物肝脏的二噁英毒性中也不太可能发挥显著作用。本研究开展了全面的探究以解答以下问题：（1）AHR基因型本身是否会影响miRNA的基础表达？（2）TCDD是否会改变miRNA水平？若存在改变，该反应是否依赖于AHR？（3）对于二噁英毒性敏感与抵抗的动物，TCDD对其miRNA水平的影响是否存在差异？本研究采用miRNA芯片结合定量RT-PCR，检测了TCDD处理后多个时间点下，TCDD对大鼠肝脏miRNA水平的体内影响。本研究所有实验的累积结果显示，成年啮齿动物肝脏中，二噁英介导的mRNA下调几乎不涉及miRNA。投稿信息：Moffat ID、Boutros PC、Celius T、Pohjanvirta R及Okey AB。《啮齿动物肝脏中的微小RNA对二噁英处理无响应》。《毒理学科学》，2007年5月。关键词：miRNA表达、时间进程、异生物质响应、基因修饰、比较基因组杂交。实验整体设计：本研究先分析了不依赖TCDD的品系间miRNA表达差异：以19小时赋形剂处理的二噁英抵抗型H/W AHRH/W/H/W大鼠（HC19）作为对照，与赋形剂处理的二噁英敏感型L-E AHRWT/WT大鼠（LC19）进行对比。对于二噁英敏感型大鼠，分别将TCDD处理3小时（LT3）、19小时（LT19）后的miRNA表达水平与LC19组进行比较；对于二噁英抵抗型大鼠，分别将TCDD处理3小时（HT3）、19小时（HT19）、96小时（HT96）后的miRNA水平与HC19组进行比较。