Open AccessA comparison of two cellular delivery mechanisms for small interfering RNA
Author(s) -
Valiunas Virginijus,
Wang HongZhang,
Li Ling,
Gordon Chris,
Valiuniene Laima,
Cohen Ira S.,
Brink Peter R.
Publication year - 2015
Publication title -
physiological reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.918
H-Index - 39
ISSN - 2051-817X
DOI - 10.14814/phy2.12286
Subject(s) - hek 293 cells , transfection , microbiology and biotechnology , extracellular , intracellular , small interfering rna , endocytosis , microvesicles , gap junction , cell , cell culture , downregulation and upregulation , biology , chemistry , microrna , biochemistry , gene , genetics
Cellular delivery of small interfering RNA s to target cells of a tissue has the potential to travel by two intercellular pathways. For intimately apposed cells gap junctions allow transport exclusive of the extracellular space. For cells not in intimate contact, exocytotic release of vesicular contents and subsequent retrieval via endocytosis of exosomes and other vesicular contents represent an alternative intercellular delivery system that utilizes the extracellular space. Previous studies have shown si RNA /mi RNA transfer from a delivery cell to a target cell via gap junction channels. We hypothesized that si RNA can be delivered via gap junctions and downregulate the expression of a reporter gene, the cyclic nucleotide‐gated cation channel gene ( mHCN 2), in the recipient cells of cell pairs. Whole‐cell patch clamp was used to measure the mHCN 2‐induced current and junctional conductance. The target cells were HEK 293 cells that endogenously express Cx43 or HeLaCx43 cells, both transfected with mHCN 2. The source cells were HEK 293 or HeLaCx43 cells transfected with fluorescent‐labeled si RNA targeting mHCN 2. We found that si RNA targeting mHCN 2 resulted in significant downregulation of mHCN 2 currents both in single cells and the recipient cell of a cell pair. In addition we also documented downregulation in target cells that were not in contact with source cells suggesting an extracellular‐mediated delivery. To test further for extracellular delivery HEK 293/ HCN 2 or HeLaCx43/ HCN 2 cells were cultured in medium collected from HEK 293 or HeLaCx43 cells transfected with fluorescent‐labeled si RNA or fluorescent‐labeled morpholino designed to target HCN 2. After 24 h single HEK 293/ HCN 2 or HeLaCx43cells showed accumulation of si RNA . The mHCN 2 currents were also down regulated in cells with si RNA uptake. Application of 200 nmol/L Bafilomycin A1, which has been shown to affect endosome acidification and endocytotic activity, resulted in a smaller accumulation of fluorescent‐labeled si RNA in single target cells. In distinction to si RNA , morpholinos targeting HCN 2 exhibited greatly reduced extracellularly mediated transfer while in cell pairs, target cells exhibited reduced HCN 2 currents consistent with effective gap junction‐mediated delivery.