Yu Shifang, Huang Huicong, Deng Gang, Xie Zuoting, Ye Yincai, Guo Ruide, Cai Xuejiao, Hong Junying, Qian Dingliang, Zhou Xiangjing, Tao Zhihua, Chen Bile, Li Qiang
The Department of Transfusion Medicine, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China; The Department of Transfusion Medicine, The First Affiliated Hospital of the Wenzhou Medical University, Wenzhou, China.
School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China.
PLoS One. 2015 Apr 13;10(4):e0122784. doi: 10.1371/journal.pone.0122784. eCollection 2015.
Platelets play crucial roles in hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and tumor metastases. Because they are anucleated blood cells, platelets lack nuclear DNA, but they do contain mitochondrial DNA, which plays a key role in regulating apoptosis. Recent evidence has suggested that miRNAs are also involved in regulating gene expression and apoptosis in platelets. Our previous study showed that the expression of miR-326 increased visibly when apheresis platelets were stored in vitro. The antiapoptotic Bcl-2 family regulator Bcl-xL has been identified as a putative target of miR-326. In the present study, dual reporter luciferase assays were used to characterize the function of miR-326 in the regulation of the apoptosis of platelet cells. These assays demonstrated that miR-326 bound to the 3'-translated region of Bcl-xL. To directly assess the functional effects of miR-326 expression, levels of Bcl-xL and the apoptotic status of stored apheresis platelets were measured after transfection of miR-326 mimic or inhibitor. Results indicated that miR-326 inhibited Bcl-xL expression and induced apoptosis in stored platelets. Additionally, miR-326 inhibited Bcl-2 protein expression and enhanced Bak expression, possibly through an indirect mechanism, though there was no effect on the expression of Bax. The effect of miR-326 appeared to be limited to apoptosis, with no significant effect on platelet activation. These results provide new insight into the molecular mechanisms affecting differential platelet gene regulation, which may increase understanding of the role of platelet apoptosis in multiple diseases.
血小板在止血、血栓形成、伤口愈合、炎症、血管生成和肿瘤转移中发挥着关键作用。由于血小板是无核血细胞，缺乏核DNA，但它们确实含有线粒体DNA，其在调节细胞凋亡中起关键作用。最近的证据表明，miRNA也参与调节血小板中的基因表达和细胞凋亡。我们之前的研究表明，当单采血小板在体外储存时，miR-326的表达明显增加。抗凋亡的Bcl-2家族调节剂Bcl-xL已被确定为miR-326的一个假定靶点。在本研究中，使用双报告荧光素酶测定法来表征miR-326在调节血小板细胞凋亡中的功能。这些测定表明，miR-326与Bcl-xL的3'-非翻译区结合。为了直接评估miR-326表达的功能效应，在转染miR-326模拟物或抑制剂后，测量了Bcl-xL的水平和储存的单采血小板的凋亡状态。结果表明，miR-326抑制Bcl-xL表达并诱导储存血小板的凋亡。此外，miR-326可能通过间接机制抑制Bcl-2蛋白表达并增强Bak表达，尽管对Bax的表达没有影响。miR-326的作用似乎仅限于细胞凋亡，对血小板活化没有显著影响。这些结果为影响血小板基因差异调节的分子机制提供了新的见解，这可能会增加对血小板凋亡在多种疾病中作用的理解。
