| Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions. | |
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MedLine Citation:
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PMID: 16037090 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase beta (pol beta) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol beta was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mbeta16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mbeta16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol beta. These two pol beta knockdown cell lines (NRK-kcdc and Mbeta16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mbeta16tsA-wt cells or N2A cells. The levels of pol beta mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mbeta16tsA-kcdc cells with Mbeta16tsA-wt, N2A or NRK-wt cells had no effect on pol beta levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol beta levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol beta in the wt cells. The inability of Mbeta16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions. |
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Authors:
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V Valiunas; Y Y Polosina; H Miller; I A Potapova; L Valiuniene; S Doronin; R T Mathias; R B Robinson; M R Rosen; I S Cohen; P R Brink |
Publication Detail:
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Type: Comparative Study; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. Date: 2005-07-21 |
Journal Detail:
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Title: The Journal of physiology Volume: 568 ISSN: 0022-3751 ISO Abbreviation: J. Physiol. (Lond.) Publication Date: 2005 Oct |
Date Detail:
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Created Date: 2005-10-17 Completed Date: 2005-12-27 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 0266262 Medline TA: J Physiol Country: England |
Other Details:
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Languages: eng Pagination: 459-68 Citation Subset: IM |
Affiliation:
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Department of Physiology and Biophysics, SUNY at Stony Brook, NY 11794, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Cell Communication Coculture Techniques Connexin 43 / chemistry, genetics, metabolism* DNA Polymerase beta / genetics, metabolism* Gap Junctions / chemistry, metabolism* Genetic Vectors Hela Cells Humans Mice Oligonucleotides / chemistry, metabolism RNA, Small Interfering / genetics, metabolism* Rats Transfection |
| Grant Support | |
ID/Acronym/Agency:
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CA17395/CA/NCI NIH HHS; DK060037/DK/NIDDK NIH HHS; GM55263/GM/NIGMS NIH HHS; HL20558/HL/NHLBI NIH HHS; HL28958/HL/NHLBI NIH HHS; HL67101/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Connexin 43; 0/Oligonucleotides; 0/RNA, Small Interfering; EC 2.7.7.-/DNA Polymerase beta |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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