Downregulation of Gabra4 expression during alcohol withdrawal is mediated by specific microRNAs in cultured mouse cortical neurons.
Downregulation of Gabra4 expression during alcohol withdrawal is mediated by specific microRNAs in cultured mouse cortical neurons.
复制标题
戒酒过程中GABRA4表达的下调是由培养的小鼠皮质神经元中的特定microRNA介导的。
DOI：
10.1002/brb3.355
复制
发表时间：
2015-08
影响因子：
3.1
通讯作者：
Harrison NL
中科院分区：
文献类型：
--
作者：
Bekdash RA;Harrison NL
酒精滥用和依赖是一个严重的公共卫生问题。大量的酒精调节基因（ARGs）被认为是受酒精使用和戒断（AW）的影响，最近的证据表明，酒精的神经适应可能部分是由于ARGs表达的表观遗传变化。Gabra 4编码GABAA受体α4亚基（GABAAR），是对酒精反应显示出显著可塑性的许多ARG之一，急性酒精暴露可迅速上调。本研究探讨了AW对Gabra 4和编码GABAARs其他亚基的相关基因表达变化的影响，以及microRNA对Gabra 4的潜在调控。我们使用RT-PCR和microRNA微阵列研究了用酒精处理的培养皮质神经元中的基因和microRNA表达，然后去除酒精以模拟体外AW。我们还使用了microRNA模拟物或抑制剂，以及携带Gabra 4 3′UTR的启动子-报告基因构建体。在去除酒精后11小时，Gabra 4下调，Gabrg 2的表达适度增加，但Gabra 1，Gabrd或Gabrb 2的表达没有变化。在接受AW的神经元中的microRNA分析显示，在AW 8 h后，miR-155、miR-186、miR-24和miR-375的表达上调。用miR-186、miR-24或miR-375的分子模拟物转染也下调Gabra 4表达，而用这些microRNA的相应抑制剂转染使AW神经元中的Gabra 4表达正常化至对照神经元中测量的水平。启动子-报告基因实验支持miR-155、miR-186、miR-24、miR-27 b或miR-375与Gabra 4的3′UTR结合从而抑制蛋白质产生的观点。我们的数据表明，AW减少Gabra 4的表达，这可能是介导的特定microRNA在皮层神经元在AW诱导的一部分。
Alcohol abuse and dependence are a serious public health problem. A large number of alcohol-regulated genes, (ARGs) are known to be influenced by alcohol use and withdrawal (AW), and recent evidence suggests that neuroadaptation to alcohol may be due in part to epigenetic changes in the expression of ARGs. Gabra4, which encodes the α4 subunit of GABAA receptors (GABAARs), is one of a number of ARGs that show remarkable plasticity in response to alcohol, being rapidly upregulated by acute alcohol exposure. This study addressed the effects of AW on changes in the expression of Gabra4 and related genes that encode other subunits of GABAARs, and the potential regulation of Gabra4 by microRNAs. We studied gene and microRNAs expression, using RT-PCR and microRNA microarray in cultured cortical neurons treated with alcohol, which was then removed in order to simulate AW in vitro. We also used microRNA mimics or inhibitors, and a promoter-reporter construct carrying the 3′UTR of Gabra4. Eleven hours after removal of alcohol, Gabra4 was downregulated, with a modest increase in the expression of Gabrg2, but no change in the expression of Gabra1, Gabrd, or Gabrb2. microRNA profiling in neurons undergoing AW revealed upregulation in the expression of miR-155, miR-186, miR-24, and miR-375 after 8 h of AW. Transfection with molecular mimics of miR-186, miR-24, or miR-375 also downregulated Gabra4 expression, whereas transfection with the corresponding inhibitors of these microRNAs normalized Gabra4 expression in AW neurons to the level measured in control neurons. Promoter-reporter experiments supported the idea that miR-155, miR-186, miR-24, miR-27b, or miR-375 bind to the 3′UTR of Gabra4 and thereby inhibit protein production. Our data suggest that AW decreases Gabra4 expression, and that this may be mediated in part by the induction of specific microRNAs in cortical neurons during AW.