Exosomal miR-7 Mediates Bystander Autophagy in Lung after Focal Brain Irradiation in Mice.
Exosomal miR-7 Mediates Bystander Autophagy in Lung after Focal Brain Irradiation in Mice.
复制标题
小鼠局灶性脑照射后，外泌体 miR-7 介导肺中的旁观者自噬
DOI：
10.7150/ijbs.18890
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发表时间：
2017
影响因子：
9.2
通讯作者：
Chen Q
中科院分区：
文献类型：
--
作者：
Cai S;Shi GS;Cheng HY;Zeng YN;Li G;Zhang M;Song M;Zhou PK;Tian Y;Cui FM;Chen Q
本研究调查了小鼠局灶性脑照射后外泌体 microRNA-7 (miR-7) 是否介导肺旁观者自噬。小鼠大脑经10 Gy或假照射后，提取肺组织，采用免疫组化、蛋白印迹、定量实时逆转录PCR（qRT-PCR）检测自噬标志物，同时解离大脑，分离神经元/星形胶质细胞/小胶质细胞/少突胶质细胞，分别检测各群体中miR-7的表达量。开发了双荧光素酶报告基因测定法来鉴定 Bcl-2 是否是 miR-7 的靶基因。对星形胶质细胞进行 10 Gy 或假辐射后，提取外泌体，用 Dil（1,1'-双十八烷基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐）染色，并添加到未辐射的星形胶质细胞中。同时，将 10 Gy 或假照射的星形胶质细胞释放的 Dil 染色的外泌体通过尾静脉注射到 LC3B-GFP 小鼠体内。然后提取肺组织进行蛋白质印迹和qRT-PCR。小鼠大脑的辐射增加了未辐射肺组织中的 LC3B-II/I 比值、Beclin-1 和 miR-7 水平，同时降低了 Bcl-2 水平。有趣的是，脑照射显着增加了星形胶质细胞和少突胶质细胞中 miR-7 的表达。 MiR-7 显着抑制野生型 Bcl-2-3'-非翻译区 (UTR) 报告载体的荧光素酶活性，但不抑制 Bcl-2-3'-UTR 突变载体的荧光素酶活性，表明 Bcl-2 是 miR-7 直接靶向的。在体外研究中，添加受辐射的星形胶质细胞分泌的外泌体增加了未受辐射星形胶质细胞中的 LC3B-II/I 比率、Beclin-1 和 miR-7 水平，同时降低了 Bcl-2 水平。此外，通过尾静脉注射经过辐照的星形胶质细胞分泌的外泌体增加了肺LC3B-II/I比值、Beclin-1和miR-7水平，但降低了体内Bcl-2水平。我们得出的结论是，外泌体 miR-7 靶向 Bcl-2，介导脑照射后肺部的远距离旁观者自噬。
This study investigated whether exosomal microRNA-7 (miR-7) mediates lung bystander autophagy after focal brain irradiation in mice. After 10 Gy or sham irradiation of mice brains, lung tissues were extracted for the detection of autophagy markers by immunohistochemistry, western blotting, and quantitative real-time reverse transcription PCR (qRT-PCR), meanwhile the brains were dissociated, the neuron/astrocyte/microglia/oligodendrocyte were isolated, and the miR-7 expression in each population were detected, respectively. A dual-luciferase reporter assay was developed to identify whether Bcl-2 is a target gene of miR-7. After 10 Gy or sham irradiation of astrocytes, exosomes were extracted, stained with Dil (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate), and added into non-irradiated astrocytes. Meanwhile, Dil-stained exosomes released from 10 Gy or sham irradiated astrocytes were injected into LC3B-GFP mice via the tail vein. Lung tissues were then extracted for western blotting and qRT-PCR. Irradiation of mouse brains increased the LC3B-II/I ratio, Beclin-1 and miR-7 levels, while decreased the Bcl-2 level in non-irradiated lung tissue. Interestingly, brain irradiation remarkably increased the miR-7 expression in astrocyte and oligodendrocyte. MiR-7 significantly inhibited the luciferase activity of the wild-type Bcl-2-3′-untranslated regions (UTR) reporter vector, but not that of the Bcl-2-3′-UTR mutant vector, indicating that Bcl-2 is directly targeted by miR-7. In in vitro study, the addition of irradiated astrocyte-secreted exosomes increased the LC3B-II/I ratio, Beclin-1 and miR-7 levels, while decreased the Bcl-2 level in non-irradiated astrocytes. Further, the injection of irradiated astrocyte-secreted exosomes through the tail vein increased the lung LC3B-II/I ratio, Beclin-1 and miR-7 level, but decreased the Bcl-2 level in vivo. We concluded that exosomal miR-7 targets Bcl-2 to mediate distant bystander autophagy in the lungs after brain irradiation.
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