MicroRNA-345 induces apoptosis in pancreatic cancer cells through potentiation of caspase-dependent and -independent pathways.
MicroRNA-345 induces apoptosis in pancreatic cancer cells through potentiation of caspase-dependent and -independent pathways.
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DOI：
10.1038/bjc.2015.252
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发表时间：
2015-08-11
影响因子：
8.8
通讯作者：
Singh AP
中科院分区：
文献类型：
--
作者：
Srivastava SK;Bhardwaj A;Arora S;Tyagi N;Singh S;Andrews J;McClellan S;Wang B;Singh AP
此前，miR-345被认为是胰腺癌(PC)中下调最显著的microRNAs之一；然而，其功能意义仍未被探索。用WST-1、Hoechst-33342/Annexin-V和JC-1染色检测miR-345对PC细胞生长、细胞凋亡和线粒体膜电位的影响。用定量逆转录-聚合酶链式反应和/或免疫印迹法检测基因表达，制备亚细胞组分，用商业试剂盒检测caspase-3/7活性。通过突变分析和荧光素酶报告基因分析进行miR-345靶标验证。与正常胰腺细胞相比，miR-345在PC组织和细胞系中的表达显著下调，在PC进展模型细胞系中的表达逐渐降低。强制表达miR-345可诱导PC细胞凋亡，导致PC细胞生长减慢，并伴有线粒体膜电位下降、细胞色素-c释放、caspase-3/7活化、PARP-1裂解，以及凋亡诱导因子的线粒体核转位。这些作用可被反义miR-345寡核苷酸处理过表达miR-345的PC细胞所逆转。Bcl2被认为是miR-345的一个新靶点，它的强制表达在PC细胞中消除了miR-345的作用。MIR-345下调对PC细胞具有抗凋亡作用，其恢复可用于治疗。
Previously, miR-345 was identified as one of the most significantly downregulated microRNAs in pancreatic cancer (PC); however, its functional significance remained unexplored. miR-345 was overexpressed in PC cells by stable transfection, and its effect on growth, apoptosis and mitochondrial-membrane potential was examined by WST-1, Hoechst-33342/Annexin-V, and JC-1 staining, respectively. Gene expression was examined by quantitative reverse-transcription-PCR and/or immunoblotting, and subcellular fractions prepared and caspase-3/7 activity determined by commercially available kits. miR-345 target validation was performed by mutational analysis and luciferase-reporter assay. miR-345 is significantly downregulated in PC tissues and cell lines relative to normal pancreatic cells, and its expression decreases gradually in PC progression model cell lines. Forced expression of miR-345 results in reduced growth of PC cells because of the induction of apoptosis, accompanied by a loss in mitochondrial membrane potential, cytochrome-c release, caspases-3/7 activation, and PARP-1 cleavage, as well as mitochondrial-to-nuclear translocation of apoptosis-inducing factor. These effects could be reversed by the treatment of miR-345-overexpressing PC cells with anti-miR-345 oligonucleotides. BCL2 was characterised as a novel target of miR-345 and its forced-expression abrogated the effects of miR-345 in PC cells. miR-345 downregulation confers apoptosis resistance to PC cells, and its restoration could be exploited for therapeutic benefit.