Chang J.-G.Yang D.-M.Chang W.-H.LU-PING CHOWChan W.-L.Lin H.-H.Huang H.-D.Chang Y.-S.Hung C.-H.Yang W.-K.2020-01-222020-01-22201119326203https://www.scopus.com/inward/record.uri?eid=2-s2.0-79958294217&doi=10.1371%2fjournal.pone.0018643&partnerID=40&md5=ef32cf353837699d446e8daba329581ahttps://scholars.lib.ntu.edu.tw/handle/123456789/452421Alternative 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. ? 2011 Chang et al.[SDGs]SDG3amiloride; cell DNA; cell protein; mitogen activated protein kinase 1; mitogen activated protein kinase 3; mitogen activated protein kinase p38; protein kinase B; protein SF2; ribonucleoprotein; ribonucleoprotein 20; stress activated protein kinase; unclassified drug; isoprotein; messenger RNA; nuclear protein; phosphoprotein phosphatase 1; protein kinase B; RNA; RNA binding protein; serine arginine rich splicing proteins; serine-arginine-rich splicing proteins; tumor protein; alternative RNA splicing; article; cancer cell; cancer invasion; cell cycle; cell damage; cell migration; controlled study; cytokinesis; cytoskeleton; DNA degradation; down regulation; drug mechanism; enzyme induction; enzyme phosphorylation; enzyme regulation; exon; extracellular matrix; genetic transcription; human; human cell; human genome; ion transport; oncogene; proteomics; apoptosis; cell nucleus; chemistry; DNA fragmentation; DNA repair; drug effect; enzyme activation; gene expression regulation; genetics; metabolism; mitosis; neoplasm; pathology; phosphorylation; tumor cell line; tumor gene; Alternative Splicing; Amiloride; Apoptosis; Cell Line, Tumor; Cell Nucleus; Cytokinesis; Cytoskeleton; DNA Fragmentation; DNA Repair; Enzyme Activation; Exons; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Genome, Human; Humans; Mitosis; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms; Nuclear Proteins; Phosphorylation; Protein Isoforms; Protein Phosphatase 1; Proteomics; Proto-Oncogene Proteins c-akt; RNA, Messenger; RNA, Neoplasm; RNA-Binding Proteinsjournal article10.1371/journal.pone.00186432-s2.0-79958294217