Jiang Shuxia, Guo Shihui, Li Huifang, Ni Yingdong, Ma Wenqiang, Zhao Ruqian
Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing, China.
Front Physiol. 2019 Jun 27;10:819. doi: 10.3389/fphys.2019.00819. eCollection 2019.
Divalent metal transporter 1 (DMT1) is a key transporter of iron uptake and delivering in human and animals. However, post-transcriptional regulation of DMT1 is poorly understood. In this study, bioinformatic algorithms (TargetScan, PITA, miRanda, and miRDB) were applied to predict, screen, analyze, and obtain microRNA-16 family members (miR-16, miR-195, miR-497, and miR-15b) targeting DMT1, seed sequence and their binding sites within DMT1 3' untranslated region (3' UTR) region. As demonstrated by dual-luciferase reporter assays, luciferase activity of DMT1 3' UTR reporter was impaired/enhanced when microRNA-16 family member over-expression plasmid/its inhibitor was transfected to HCT116 cells. Corroboratively, co-transfection of microRNA-16 family member over-expression plasmid and DMT1 3' UTR mutant reporter repressed the luciferase activity in HCT116 cells. In addition, over-expression microRNA-16 family member augmented its expression and diminished DMT1 protein expression in HCT116 cells. Interestingly, tail vein injection of miR-16 assay revealed reduced plasma iron levels, higher miR-16 expression, and lower DMT1 protein expression in the duodenum of mice. Taken together, we provide evidence that microRNA-16 family (miR-16, miR-195, miR-497, and miR-15b) is confirmed to repress intestinal DMT1 expression and , which will give valuable insight into post-transcriptional regulation of DMT1.
二价金属转运蛋白1（DMT1）是人和动物体内铁摄取与转运的关键转运蛋白。然而，对DMT1的转录后调控了解甚少。在本研究中，应用生物信息学算法（TargetScan、PITA、miRanda和miRDB）预测、筛选、分析并获得靶向DMT1的微小RNA-16家族成员（miR-16、miR-195、miR-497和miR-15b）、种子序列及其在DMT1 3'非翻译区（3'UTR）内的结合位点。双荧光素酶报告基因检测表明，当将微小RNA-16家族成员过表达质粒/其抑制剂转染至HCT116细胞时，DMT1 3'UTR报告基因的荧光素酶活性受损/增强。同样，共转染微小RNA-16家族成员过表达质粒和DMT1 3'UTR突变报告基因可抑制HCT116细胞中的荧光素酶活性。此外，过表达微小RNA-16家族成员可增强其在HCT116细胞中的表达，并降低DMT1蛋白表达。有趣的是，尾静脉注射miR-16检测显示，小鼠十二指肠中的血浆铁水平降低、miR-16表达升高且DMT1蛋白表达降低。综上所述，我们提供的证据表明，微小RNA-16家族（miR-16、miR-195、miR-497和miR-15b）可抑制肠道DMT1表达，这将为DMT1的转录后调控提供有价值的见解。
