Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.
Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.
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DOI：
10.1371/journal.pone.0018643
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Yang WK
中科院分区：
文献类型：
--
作者：
Chang JG;Yang DM;Chang WH;Chow LP;Chan WL;Lin HH;Huang HD;Chang YS;Hung CH;Yang WK
选择性剪接涉及基因转录物的差异外显子选择以产生具有结构和功能多样性的mRNA和蛋白质同种型。异常选择性剪接已被证明与癌细胞的恶性表型相关，例如化学抗性和侵袭活性。筛选调节人肝癌细胞系Huh-7中RNA剪接的小分子和药物，我们发现阿米洛利，不同于四个pH影响阿米洛利类似物，可以“正常化”BCL-X，HIPK 3和罗恩/MISTR 1转录本的剪接。我们对阿米洛利处理的细胞进行的蛋白质组学分析检测到剪接因子SF 2/ASF的低磷酸化，以及SRp 20和两种未鉴定的SR蛋白的水平降低。我们进一步观察到AKT、ERK 1/2和PP 1的磷酸化减少，p38和JNK的磷酸化增加，表明阿米洛利治疗下调了已知影响剪接因子蛋白磷酸化的信号通路中的激酶，上调了磷酸酶。这些阿米洛利效应的“正常化”致癌RNA剪接和剪接因子低磷酸化都废除了预处理与PP 1抑制剂。阿米洛利处理的Huh-7细胞的全球外显子阵列检测到剪接模式的变化，涉及551个基因转录本中的584个外显子，其中许多编码在离子转运、细胞基质形成、细胞骨架重塑和基因组维持中发挥关键作用的蛋白质。细胞功能分析显示，随后的入侵和迁移缺陷，细胞周期中断，胞质分裂障碍，并在阿米洛利处理的Huh-7细胞的致命DNA降解。其他人实体瘤和白血病细胞，但不是少数正常细胞，显示出类似的阿米洛利改变RNA剪接与失活的后果。因此，这项研究为利用RNA剪接的小分子调节用于癌症治疗提供了机制基础。
Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.
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