Secretion of small/microRNAs including miR-638 into extracellular spaces by sphingomyelin phosphodiesterase 3.
Secretion of small/microRNAs including miR-638 into extracellular spaces by sphingomyelin phosphodiesterase 3.
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DOI：
10.3892/or.2014.3605
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发表时间：
2015-01
期刊：
影响因子：
4.2
通讯作者：
Watanabe N
中科院分区：
文献类型：
--
作者：
Kubota S;Chiba M;Watanabe M;Sakamoto M;Watanabe N
最近的一项研究表明，细胞内的小/微RNA从细胞中释放，并且这些细胞外RNA中的一些嵌入脂质双层膜上的囊泡中，例如富含神经酰胺的外泌体。在本研究中，我们研究了鞘磷脂磷酸二酯酶3（SMPD 3），从鞘磷脂产生神经酰胺，对小/microRNA从细胞内释放到细胞外空间的影响。在这些实验中，将SW 480人结肠直肠癌细胞和HuH-7人肝细胞癌细胞在无血清培养基中培养48小时。然后收集培养物上清液，通过离心和0.22 μm过滤器过滤去除漂浮细胞和碎片。纯化的培养物上清液中的细胞外小RNA在室温下、20次冻融循环和暴露于pH 2.0后稳定4周，并且对核糖核酸酶A降解具有抗性。氨基酸序列分析显示SMPD 3在哺乳动物之间具有很高的同源性，表明其具有进化保守性。因此，为了研究细胞小/微小RNA输出的机制，在无血清培养基中用SMPD 3抑制剂GW 4869处理SW 480和HuH-7细胞。收集培养物上清液用于微阵列和/或逆转录定量聚合酶链反应（RT-qPCR）实验。用GW 4869处理后，培养上清液中的microRNA的数量减少。其中，细胞外和细胞内miR-638分别呈剂量依赖性减少和增加。这些数据表明SMPD 3在microRNA释放到细胞外空间中起重要作用。
A recent study demonstrated that intracellular small/microRNAs are released from cells, and some of these extracellular RNAs are embedded in vesicles, such as ceramide-rich exosomes, on lipid-bilayer membranes. In the present study, we examined the effects of sphingomyelin phosphodiesterase 3 (SMPD3), which generates ceramide from sphingomyelin, on the release of small/microRNAs from intracellular to extracellular spaces. In these experiments, SW480 human colorectal and HuH-7 human hepatocellular cancer cells were cultured for 48 h in serum-free media. Culture supernatants were then collected, and floating cells and debris were removed by centrifugation and filtration through a 0.22-μm filter. Extracellular small RNAs in purified culture supernatants were stable for 4 weeks at room temperature, after 20 freeze-thaw cycles and exposure to pH 2.0, and were resistant to ribonuclease A degradation. Amino acid sequence analyses of SMPD3 showed high homology between mammals, indicating evolutionary conservation. Therefore, to investigate the mechanisms of cellular small/microRNA export, SW480 and HuH-7 cells were treated with the SMPD3 inhibitor GW4869 in serum-free media. Culture supernatants were collected for microarray and/or reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments. The number of microRNAs in culture supernatants was decreased following treatment with GW4869. Among these, extracellular and intracellular miR-638 were dose-dependently decreased and increased, respectively. These data suggest that SMPD3 plays an important role in the release of microRNAs into extracellular spaces.
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DOI：
10.1186/1758-907x-1-7
发表时间：
2010-03-01
期刊：
Silence
影响因子：
--
作者：
Kosaka N;Izumi H;Sekine K;Ochiya T
通讯作者：
Ochiya T
影响因子：
64.8
作者：
SOLLNER, T;WHITEHART, SW;ROTHMAN, JE
通讯作者：
ROTHMAN, JE
影响因子：
3.8
作者：
King HW;Michael MZ;Gleadle JM
通讯作者：
Gleadle JM
影响因子：
6.4
作者：
Huang, Zhaohui;Huang, Dan;Du, Xiang
通讯作者：
Du, Xiang
影响因子：
6.7
作者：
Lee, Dong Hun;Kim, Sung Hyun;Kim, Dae Kyong
通讯作者：
Kim, Dae Kyong