Identification and Functional Verification of MicroRNA-16 Family Targeting Intestinal Divalent Metal Transporter 1 (DMT1) in vitro and in vivo
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,...
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Published in | Frontiers in physiology Vol. 10; p. 819 |
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Language | English |
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Abstract | 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. |
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AbstractList | 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 in vitro and in vivo, which will give valuable insight into post-transcriptional regulation of DMT1. 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 in vitro and in vivo , which will give valuable insight into post-transcriptional regulation of DMT1. 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. |
Author | Zhao, Ruqian Li, Huifang Ma, Wenqiang Ni, Yingdong Jiang, Shuxia Guo, Shihui |
AuthorAffiliation | 1 Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing , China 2 MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University , Nanjing , China |
AuthorAffiliation_xml | – name: 1 Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing , China – name: 2 MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University , Nanjing , China |
Author_xml | – sequence: 1 givenname: Shuxia surname: Jiang fullname: Jiang, Shuxia organization: MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing, China – sequence: 2 givenname: Shihui surname: Guo fullname: Guo, Shihui organization: MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing, China – sequence: 3 givenname: Huifang surname: Li fullname: Li, Huifang organization: MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing, China – sequence: 4 givenname: Yingdong surname: Ni fullname: Ni, Yingdong organization: MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing, China – sequence: 5 givenname: Wenqiang surname: Ma fullname: Ma, Wenqiang organization: MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing, China – sequence: 6 givenname: Ruqian surname: Zhao fullname: Zhao, Ruqian organization: MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing, China |
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Copyright | Copyright © 2019 Jiang, Guo, Li, Ni, Ma and Zhao. 2019 Jiang, Guo, Li, Ni, Ma and Zhao |
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Keywords | functional verification intestine regulation DMT1 microRNA-16 family |
Language | English |
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Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Reviewed by: Jinlian Hua, Northwest A&F University, China; Shikha Prasad, Northwestern University, United States This article was submitted to Gastrointestinal Sciences, a section of the journal Frontiers in Physiology Edited by: Stephen J. Pandol, Cedars-Sinai Medical Center, United States |
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Title | Identification and Functional Verification of MicroRNA-16 Family Targeting Intestinal Divalent Metal Transporter 1 (DMT1) in vitro and in vivo |
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