Examination of artificial MiRNA mimics with centered-site complementarity for gene targeting.
BACKGROUND: MiRNA primarily acts to repress gene expression at the post-transcriptional level through imperfect complementarity of its 5' region to the "seed site" in the 3' untranslated region of target mRNAs, with its "3'-supplementary site" and "center site...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3754971?pdf=render |
| _version_ | 1819289852792799232 |
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| author | Shu-Guang Zhang Chun-Yan Liu Li Li Tong-Wen Sun Yong-Gang Luo Wen-Jing Yun Jin-Ying Zhang |
| author_facet | Shu-Guang Zhang Chun-Yan Liu Li Li Tong-Wen Sun Yong-Gang Luo Wen-Jing Yun Jin-Ying Zhang |
| author_sort | Shu-Guang Zhang |
| collection | DOAJ |
| description | BACKGROUND: MiRNA primarily acts to repress gene expression at the post-transcriptional level through imperfect complementarity of its 5' region to the "seed site" in the 3' untranslated region of target mRNAs, with its "3'-supplementary site" and "center site" also playing important roles under certain circumstances. The aim of this study was to test if artificial miRNA mimics (miR-Mimics) that are designed to target the "centered sites" without "seed sites" complementarity are able to repress gene expression as natural miRNAs. METHODS: We designed miR-Mimics carrying centered-site matches (CS-miR-Mimics) or seed-site matches (SS-miR-Mimics) and siRNA to two antiapoptotic genes BCL2 and AKT1. We tested the gene targeting of these constructs using real-time RT-PCR and Western blot to quantify mRNA and protein levels of BCL2 and AKT1, respectively, luciferase reporter gene assay to investigate the interaction between miR-Mimics and their target sites, and cell survival assay to study the functional outcomes of the miR-Mimics. RESULTS: We found that CS-miR-Mimic, SS-miR-Mimic and siRNA, all down regulated the mRNA and protein levels of their cognate target BCL2 or AKT1 in a concentration-dependent manner. Luciferase reporter gene assay further confirmed the functional interactions of CS-miR-Mimic, SS-miR-Mimic and siRNA with their target sites. We then observed that the miR-Mimics and siRNAs were all able to induce cell death, as indicated by the reduced survival rate of cells. CONCLUSIONS: We have provided evidence for the feasibility of CS-miR-Mimics for post-transcriptional repression of genes, which can be designed to have reduced numbers of seed type off-target sites compared to the number of target sites from an average endogenous seed-site miRNA. CS-miR-Mimics may be a novel approach for miRNA research requiring miRNA gain-of-function. |
| first_indexed | 2024-12-24T03:13:26Z |
| format | Article |
| id | doaj.art-eac14c756f094503b1ce02ecfdca2527 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-24T03:13:26Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-eac14c756f094503b1ce02ecfdca25272022-12-21T17:17:42ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0188e7206210.1371/journal.pone.0072062Examination of artificial MiRNA mimics with centered-site complementarity for gene targeting.Shu-Guang ZhangChun-Yan LiuLi LiTong-Wen SunYong-Gang LuoWen-Jing YunJin-Ying ZhangBACKGROUND: MiRNA primarily acts to repress gene expression at the post-transcriptional level through imperfect complementarity of its 5' region to the "seed site" in the 3' untranslated region of target mRNAs, with its "3'-supplementary site" and "center site" also playing important roles under certain circumstances. The aim of this study was to test if artificial miRNA mimics (miR-Mimics) that are designed to target the "centered sites" without "seed sites" complementarity are able to repress gene expression as natural miRNAs. METHODS: We designed miR-Mimics carrying centered-site matches (CS-miR-Mimics) or seed-site matches (SS-miR-Mimics) and siRNA to two antiapoptotic genes BCL2 and AKT1. We tested the gene targeting of these constructs using real-time RT-PCR and Western blot to quantify mRNA and protein levels of BCL2 and AKT1, respectively, luciferase reporter gene assay to investigate the interaction between miR-Mimics and their target sites, and cell survival assay to study the functional outcomes of the miR-Mimics. RESULTS: We found that CS-miR-Mimic, SS-miR-Mimic and siRNA, all down regulated the mRNA and protein levels of their cognate target BCL2 or AKT1 in a concentration-dependent manner. Luciferase reporter gene assay further confirmed the functional interactions of CS-miR-Mimic, SS-miR-Mimic and siRNA with their target sites. We then observed that the miR-Mimics and siRNAs were all able to induce cell death, as indicated by the reduced survival rate of cells. CONCLUSIONS: We have provided evidence for the feasibility of CS-miR-Mimics for post-transcriptional repression of genes, which can be designed to have reduced numbers of seed type off-target sites compared to the number of target sites from an average endogenous seed-site miRNA. CS-miR-Mimics may be a novel approach for miRNA research requiring miRNA gain-of-function.http://europepmc.org/articles/PMC3754971?pdf=render |
| spellingShingle | Shu-Guang Zhang Chun-Yan Liu Li Li Tong-Wen Sun Yong-Gang Luo Wen-Jing Yun Jin-Ying Zhang Examination of artificial MiRNA mimics with centered-site complementarity for gene targeting. PLoS ONE |
| title | Examination of artificial MiRNA mimics with centered-site complementarity for gene targeting. |
| title_full | Examination of artificial MiRNA mimics with centered-site complementarity for gene targeting. |
| title_fullStr | Examination of artificial MiRNA mimics with centered-site complementarity for gene targeting. |
| title_full_unstemmed | Examination of artificial MiRNA mimics with centered-site complementarity for gene targeting. |
| title_short | Examination of artificial MiRNA mimics with centered-site complementarity for gene targeting. |
| title_sort | examination of artificial mirna mimics with centered site complementarity for gene targeting |
| url | http://europepmc.org/articles/PMC3754971?pdf=render |
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