A comparison of two cellular delivery mechanisms for small interfering RNA.
A comparison of two cellular delivery mechanisms for small interfering RNA.
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DOI：
10.14814/phy2.12286
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发表时间：
2015-02-01
影响因子：
2.5
通讯作者：
Brink PR
中科院分区：
文献类型：
--
作者：
Valiunas V;Wang HZ;Li L;Gordon C;Valiuniene L;Cohen IS;Brink PR
小干扰RNA向组织的靶细胞的细胞递送具有通过两种细胞间途径行进的潜力。对于紧密贴壁的细胞，间隙连接允许排除细胞外空间的运输。对于不紧密接触的细胞，囊泡内容物的胞吐释放和随后通过外泌体和其他囊泡内容物的胞吞作用的回收代表了利用细胞外空间的替代细胞间递送系统。先前的研究已经显示siRNA/miRNA通过间隙连接通道从递送细胞转移到靶细胞。我们假设siRNA可以通过间隙连接传递，并下调受体细胞对中报告基因环核苷酸门控阳离子通道基因（mHCN 2）的表达。采用全细胞膜片钳技术测定mHCN 2诱导的电流和连接电导。靶细胞是内源性表达Cx43的HEK 293细胞或HeLaCx 43细胞，两者均用mHCN 2转染。源细胞是用靶向mHCN 2的荧光标记的siRNA转染的HEK 293或HeLaCx 43细胞。我们发现靶向mHCN 2的siRNA导致单细胞和细胞对的受体细胞中mHCN 2电流的显著下调。此外，我们还记录了不与源细胞接触的靶细胞中的下调，表明细胞外介导的递送。为了进一步测试细胞外递送，将HEK 293/HCN 2或HeLaCx 43/HCN 2细胞在从HEK 293或HeLaCx 43细胞收集的培养基中培养，所述HEK 293或HeLaCx 43细胞用设计为靶向HCN 2的荧光标记的siRNA或荧光标记的吗啉代转染。24 h后，单个HEK 293/HCN 2或HeLaCx 43细胞显示siRNA积累。在siRNA摄取的细胞中，mHCN 2电流也下调。应用200 nmol/L巴弗洛霉素A1，这已被证明会影响内体酸化和内吞活性，导致荧光标记的siRNA在单个靶细胞中的积累较少。与siRNA不同，靶向HCN 2的吗啉代表现出大大降低的细胞外介导的转移，而在细胞对中，靶细胞表现出与有效间隙连接介导的递送一致的降低的HCN 2电流。
Cellular delivery of small interfering RNAs 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 siRNA/miRNA transfer from a delivery cell to a target cell via gap junction channels. We hypothesized that siRNA can be delivered via gap junctions and downregulate the expression of a reporter gene, the cyclic nucleotide-gated cation channel gene (mHCN2), in the recipient cells of cell pairs. Whole-cell patch clamp was used to measure the mHCN2-induced current and junctional conductance. The target cells were HEK293 cells that endogenously express Cx43 or HeLaCx43 cells, both transfected with mHCN2. The source cells were HEK293 or HeLaCx43 cells transfected with fluorescent-labeled siRNA targeting mHCN2. We found that siRNA targeting mHCN2 resulted in significant downregulation of mHCN2 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 HEK293/HCN2 or HeLaCx43/HCN2 cells were cultured in medium collected from HEK293 or HeLaCx43 cells transfected with fluorescent-labeled siRNA or fluorescent-labeled morpholino designed to target HCN2. After 24 h single HEK293/HCN2 or HeLaCx43cells showed accumulation of siRNA. The mHCN2 currents were also down regulated in cells with siRNA 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 siRNA in single target cells. In distinction to siRNA, morpholinos targeting HCN2 exhibited greatly reduced extracellularly mediated transfer while in cell pairs, target cells exhibited reduced HCN2 currents consistent with effective gap junction-mediated delivery.