Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Hepatocellular Carcinoma Huh-7 Cells

Screening small molecules and drugs for activity to modulate alternative splicing, we found that amiloride, distinct from four other intracellular pH-affecting analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts in human hepatocellular carcinoma Huh-7 cells. To elucidate the underlying mechanisms, our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF and also decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, while increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulated kinases and up-regulated phosphatases in the signal pathways known to affect the splicing factor protein phosphorylation. The amiloride effects of splicing factor protein hypo-phosphorylation and“normalized”oncogenic RNA splicing were both abrogated by pre-treatment with a PP1 inhibitor. We then performed global exon array analysis of Huh-7 cells treated with amiloride for 24 hours. Using gene array chips (Affymetrix GeneChip® Human Exon 1.0 ST Array of >518000 exons of 42974 genes) for exon array analysis (set parameters of correlation coefficient ≥ 0.7, splicing index ≤ -1.585 , and log2 ratio ≤ -1.585), we found that amiloride influenced the splicing patterns of 551 genes involving at least 584 exons, which included 495 known protein-coding genes involving 526 exons, many of which play key roles in functional networks of ion transport, extracellular matrix, cytoskeletons 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. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of abnormal RNA splicing for cancer therapeutics. Target was prepared from 3 biological replicates of 0.5mM amiloride-treatment for 24hr and 3 control untreatment from Huh7 cell line and hybridized to the Affymetrix Human Exon 1.0 ST Array. The values of the hybridized probesets were normalized and analyzed for gene expression using dChip2010 software.

为筛选可调控可变剪接的小分子化合物与药物，我们发现阿米洛利（amiloride）与其他四种影响细胞内pH的类似物不同，能够在人类肝细胞癌Huh-7细胞中“正常化”BCL-X、HIPK3以及RON/MISTR1转录本的剪接模式。为阐明其潜在作用机制，我们对阿米洛利处理后的细胞开展蛋白质组学分析，检测到剪接因子SF2/ASF呈现低磷酸化状态，同时SRp20与两种未鉴定的SR蛋白的表达水平均出现下调。我们进一步观察到AKT、ERK1/2及PP1（蛋白磷酸酶1）的磷酸化水平降低，而p38与JNK的磷酸化水平升高，这提示阿米洛利处理可下调已知影响剪接因子蛋白磷酸化的信号通路中的激酶活性，并上调磷酸酶的活性。阿米洛利所介导的剪接因子蛋白低磷酸化与“正常化”致癌RNA剪接的效应，均可通过预先使用PP1抑制剂处理而被完全消除。随后我们对经阿米洛利处理24小时的Huh-7细胞进行全外显子组芯片分析。采用基因芯片（Affymetrix GeneChip® Human Exon 1.0 ST Array，覆盖42974个基因的超518000个外显子）开展外显子阵列分析，设定分析参数为相关系数≥0.7、剪接指数≤-1.585且log₂比值≤-1.585，结果显示阿米洛利可影响551个基因（涉及至少584个外显子）的剪接模式，其中包含495个已知蛋白编码基因（涉及526个外显子），多数基因在离子转运、细胞外基质、细胞骨架及基因组维持的功能网络中发挥关键作用。细胞功能分析结果显示，经阿米洛利处理的Huh-7细胞出现侵袭与迁移缺陷、细胞周期紊乱、胞质分裂受损以及致死性DNA降解。本研究因此为利用小分子化合物调控异常RNA剪接以开发癌症治疗手段提供了机制层面的理论依据。本研究的靶标样本来自Huh-7细胞系的3份生物学重复：经0.5mM阿米洛利处理24小时的实验组，与3份未处理的对照组样本，随后将样本与Affymetrix Human Exon 1.0 ST Array进行杂交。杂交后的探针组信号值经过标准化处理，并使用dChip2010软件进行基因表达分析。