Arsenic trioxide induced apoptosis in retinoblastoma cells by abnormal expression of microRNA-376a.
Arsenic trioxide induced apoptosis in retinoblastoma cells by abnormal expression of microRNA-376a.
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DOI：
10.4149/neo_2013_033
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发表时间：
2013
期刊：
影响因子：
3
通讯作者：
Y. Zhang;J. Wu;F. Han;J. Huang;S. Shi;R. Gu;X. Chen;B. He
中科院分区：
文献类型：
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作者：
Y. Zhang;J. Wu;F. Han;J. Huang;S. Shi;R. Gu;X. Chen;B. He
三氧化二砷（ATO）已被证明可以诱导视网膜母细胞瘤细胞凋亡，然而，负责这一现象的机制并不完全清楚。在本研究中，我们确定ATO是否通过microRNA的异常表达诱导细胞凋亡。在视网膜母细胞瘤细胞经4 μM ATO处理72 h后的凋亡模型中，我们用miRNA微阵列分析发现14个miRNA发生了2倍以上的变化。这些异常表达的miRNAs中的大多数通过定量RT-PCR证实。miR-376 a是一种显著下调的miRNA，被选择用于进一步研究。由miR-376 a模拟物转染产生的miR-376 a的过表达显著抑制ATO诱导的细胞凋亡。相比之下，miR-376 a抑制剂转染导致的miR-376 a缺陷加重ATO诱导的细胞凋亡。使用生物信息学算法，caspase-3，一个关键的凋亡执行者，被预测为miR-376 a的推定靶点。定量RT-PCR显示miR-376 a模拟物或抑制剂对caspase-3 mRNA水平无影响。然而，caspase-3蛋白的量被miR-376 a模拟物减少，而被miR-376 a抑制剂增加。此外，荧光素酶报告基因检测证实caspase-3是miR-376 a的靶点，并且由miR-376 a抑制剂引起的细胞凋亡被caspase-3抑制剂消除。这些结果表明，ATO诱导的视网膜母细胞瘤细胞凋亡部分是通过降低miR-376 a的表达介导的，其随后增加caspase-3的表达。
Arsenic trioxide (ATO) has been demonstrated to induce apoptosis in retinoblastoma cells, however, mechanisms responsible for this phenomenon are not fully understood. In the present study, we determined whether ATO induced apoptosis by abnormal expression of microRNA. In an apoptosis model of retinoblastoma cells subjected to 4 μM ATO for 72 hours, we found 14 miRNAs changed more than 2-fold by using miRNA microarray analysis. Most of these aberrantly expressed miRNAs were confirmed by quantitative RT-PCR. MiR-376a, a significantly down-regulated miRNA, was selected for further study. The overexpression of miR-376a resulting from miR-376a mimic transfection significantly inhibited ATO-induced apoptosis. By contrast, miR-376a deficiency resulting from miR-376a inhibitor transfection aggravated ATO-induced apoptosis. Using bioinformatic algorithms, caspase-3, a key apoptosis executioner, was predicted as a putative target of miR-376a. The quantitative RT-PCR showed no effects of miR-376a mimic or inhibitor on caspase-3 mRNA level. However, the amount of caspase-3 proteins was reduced by miR-376a mimic, whereas increased by miR-376a inhibitor. Furthermore, the luciferase reporter assay confirmed caspase-3 to be a target of miR-376a, and the apoptosis caused by miR-376a inhibitor were abolished by a caspase-3 inhibitor. These results suggest that ATO -induced apoptosis in retinoblastoma cells is part mediated by decreasing expression of miR-376a, which subsequently increased caspase-3 expression.