Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
J Biol Chem. 2010 Feb 12;285(7):4415-22. doi: 10.1074/jbc.M109.085431. Epub 2009 Dec 15.
Hepatocyte nuclear factor (HNF) 4alpha is a key transcription factor regulating endo/xenobiotic-metabolizing enzymes and transporters. We investigated whether microRNAs are involved in the regulation of human HNF4alpha. Potential recognition elements for miR-24 (MRE24) were identified in the coding region and the 3'-untranslated region (3'-UTR), and those for miR-34a (MRE34a) were identified in the 3'-UTR in HNF4alpha mRNA. The HNF4alpha protein level in HepG2 cells was markedly decreased by the overexpression of miR-24 and miR-34a. The HNF4alpha mRNA level was significantly decreased by the overexpression of miR-24 but not by miR-34a. In luciferase analyses in HEK293 cells, the reporter activity of plasmid containing the 3'-UTR of HNF4alpha was significantly decreased by miR-34a. The reporter activity of plasmid containing the HNF4alpha coding region downstream of the luciferase gene was significantly decreased by miR-24. These results suggest that the MRE24 in the coding region and MRE34a in the 3'-UTR are functional in the negative regulation by mRNA degradation and translational repression, respectively. The down-regulation of HNF4alpha by these microRNAs resulted in the decrease of various target genes such as cytochrome P450 7A1 and 8B1 as well as morphological changes and the decrease of the S phase population in HepG2 cells. We also clarified that the expressions of miR-24 and miR-34a were regulated by protein kinase C/mitogen-activated protein kinase and reactive oxygen species pathways, respectively. In conclusion, we found that human HNF4alpha was down-regulated by miR-24 and miR-34a, the expression of which are regulated by cellular stress, affecting the metabolism and cellular biology.
肝细胞核因子 (HNF) 4alpha 是调节内源性/外源性代谢酶和转运体的关键转录因子。我们研究了 microRNAs 是否参与人类 HNF4alpha 的调节。在 HNF4alpha mRNA 的编码区和 3'-非翻译区 (3'-UTR) 中鉴定到 miR-24 的潜在识别元件 (MRE24)，在 3'-UTR 中鉴定到 miR-34a 的潜在识别元件 (MRE34a)。miR-24 和 miR-34a 的过表达显著降低了 HepG2 细胞中 HNF4alpha 蛋白水平。miR-24 的过表达显著降低了 HNF4alpha mRNA 水平，但 miR-34a 没有。在 HEK293 细胞中的荧光素酶分析中，miR-34a 显著降低了含有 HNF4alpha 3'-UTR 的报告质粒的活性。miR-24 显著降低了含有 HNF4alpha 编码区下游荧光素酶基因的报告质粒的活性。这些结果表明，编码区中的 MRE24 和 3'-UTR 中的 MRE34a 分别通过 mRNA 降解和翻译抑制负调控。这些 microRNAs 下调 HNF4alpha 导致各种靶基因如细胞色素 P450 7A1 和 8B1 的表达下调，以及 HepG2 细胞形态变化和 S 期细胞群减少。我们还阐明了 miR-24 和 miR-34a 的表达分别受蛋白激酶 C/丝裂原活化蛋白激酶和活性氧途径调节。总之，我们发现人类 HNF4alpha 被 miR-24 和 miR-34a 下调，其表达受细胞应激调节，影响代谢和细胞生物学。
