Zheng Yun, Jiang Shibei, Zhang Yihui, Zhang Rui, Gong Daoqing
College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
Int J Mol Sci. 2015 Jun 5;16(6):12737-52. doi: 10.3390/ijms160612737.
miRNAs are single-stranded, small RNA molecules with a length of 18-25 nucleotides. They bind to the 3' untranslated regions of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation. The roles of these molecules differ in biological processes, such as cell differentiation, proliferation, apoptosis and tumor genesis. miRNA-33 is encoded by the gene introns of proteins that bind sterol-regulatory elements. This molecule cooperates with these proteins to control cholesterol homeostasis, fatty acid levels and the genes that are related to the expression of fat metabolism. The examination of miR-33 expression and its target genes can promote the in-depth study of the miRNA regulation mechanism in the formation process of goose fatty liver and can lay a foundation for research into human fatty liver.
METHODOLOGY/PRINCIPAL FINDINGS: (1) Through real-time fluorescent quantitative polymerase chain reaction (TaqMan MicroRNA Assay), we detected the expression of miR-33 during the feeding of Landes geese. The expression level of miR-33 increases significantly in the liver after 19 days in comparison with the control group; (2) By using the bioinformatics software programs TargetScan, miRDB and miRCosm to predict the target genes of miR-33 according to laboratory prophase transcriptome results and references, we screen nine target genes: adenosine triphosphate binding cassette transporters A1, adenosine triphosphate binding cassette transporters G1, Neimann Pick C, carnitine O-octanoyltransferase (CROT), cyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), AMP-activated protein kinase, alpha subunit 1 (AMPKα1), insulin receptor substrate 2, glutamic pyruvate transaminase and adipose differentiation-related protein. The dual luciferase reporter gene system in the CHO cell line verifies that CROT, HADHB and NPC1 are the target genes of miR-33 in geese. The inhibition rate of CROT is highest and reaches 70%; (3) The seed sequence (5' 2-8 bases) is the acting site of miR-33. The two predicted target sites of CROT are the target sites of miR-33. Moreover, the predicted target site of HADHB and NPC1 is the target site of miR-33.
CONCLUSIONS/SIGNIFICANCE: (1) After 19 days of overfeeding, the expression level of miR-33 increases significantly in the livers of geese; (2) CROT, HADHB and NPC1 are the target genes of miR-33 in geese. These genes determine the combined target site.
微小RNA（miRNAs）是长度为18 - 25个核苷酸的单链小RNA分子。它们与mRNA转录本的3'非翻译区结合，以减少这些转录本的翻译或导致其降解。这些分子在细胞分化、增殖、凋亡和肿瘤发生等生物学过程中发挥着不同的作用。miR - 33由结合固醇调节元件的蛋白质基因内含子编码。该分子与这些蛋白质协同作用，以控制胆固醇稳态、脂肪酸水平以及与脂肪代谢表达相关的基因。检测miR - 33的表达及其靶基因，可促进对鹅脂肪肝形成过程中miRNA调控机制的深入研究，并为人类脂肪肝的研究奠定基础。
方法/主要发现：（1）通过实时荧光定量聚合酶链反应（TaqMan MicroRNA检测法），我们检测了朗德鹅饲养过程中miR - 33的表达。与对照组相比，19天后肝脏中miR - 33的表达水平显著升高；（2）根据实验室前期转录组结果和参考文献，使用生物信息学软件TargetScan、miRDB和miRCosm预测miR - 33的靶基因，我们筛选出9个靶基因：三磷酸腺苷结合盒转运体A1、三磷酸腺苷结合盒转运体G1、尼曼-皮克病C型、肉碱O - 辛酰基转移酶（CROT）、线粒体三功能蛋白β亚基（HADHB）、腺苷酸活化蛋白激酶α1亚基（AMPKα1）、胰岛素受体底物2、谷丙转氨酶和脂肪分化相关蛋白。CHO细胞系中的双荧光素酶报告基因系统验证了CROT、HADHB和NPC1是鹅中miR - 33的靶基因。CROT的抑制率最高，达到70%；（3）种子序列（5' 2 - 8个碱基）是miR - 33的作用位点。CROT的两个预测靶位点是miR - 33的靶位点。此外，HADHB和NPC1的预测靶位点也是miR - 33的靶位点。
结论/意义：（1）过度喂养19天后，鹅肝脏中miR - 33的表达水平显著升高；（2）CROT、HADHB和NPC1是鹅中miR - 33的靶基因。这些基因确定了联合靶位点。
