To better understand the molecular mechanisms underlying altered-FGFR3 oncogenic activity in bladder carcinomas, we made use of RT112 cell lines, which were derived from a human bladder tumor and endogenously expressed the FGFR3-TACC3 fusion protein, the growth and transformation of these cell lines being dependent on activated-FGFR3 activity. We conducted a gene expression analysis using Affymetrix DNA arrays in this cell line treated or not with FGFR3 siRNAs.
An FGFR3/MYC positive feedback loop provides new opportunities for targeted therapies in bladder cancers.
Cell lineView Samples
We differentiated mouse bone marrow cells in the presence of recombinant macrophage colony stimulating (rM-CSF) factor for 14 days during the flight of space shuttle Space Transportation System (STS)-126. We tested the hypothesis that the receptor expression for M-CSF, c-Fms was reduced. We used flow cytometry to assess molecules on cells that were preserved during flight to define the differentiation state of the developing bone marrow macrophages; including CD11b, CD31, CD44, Ly6C, Ly6G, F4/80, Mac2, c-Fos as well as c-Fms. In addition, RNA was preserved during the flight and was used to perform a gene microarray. We found that there were significant differences in the number of macrophages that developed in space compared to controls maintained on Earth. We found that there were significant changes in the distribution of cells that expressed CD11b, CD31, F4/80, Mac2, Ly6C and c-Fos. However, there were no changes in c-Fms expression and no consistent pattern of advanced or retarded differentiation during space flight. We also found a pattern of transcript levels that would be consistent with a relatively normal differentiation outcome but increased proliferation by the bone marrow macrophages that were assayed after 14 days of space flight. There also was a surprising pattern of space flight influence on genes of the coagulation pathway. These data confirm that a space flight can have an impact on the in vitro development of macrophages from mouse bone marrow cells.
Evaluation of in vitro macrophage differentiation during space flight.
Specimen partView Samples
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 andnormalizedoncogenic 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.
Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.
Cell lineView Samples
The tumor suppressor genes EAF2 and p53 are frequently dysregulated in prostate cancers. Recently, we reported that concurrent p53 nuclear staining and EAF2 downregulation were associated with high Gleason score. Combined loss of EAF2 and p53 in a murine model induced prostate tumors, and concurrent knockdown of EAF2 and p53 in prostate cancer cells enhanced proliferation and migration, further suggesting that EAF2 and p53 could functionally interact in the suppression of prostate tumorigenesis. Here, RNA-seq analyses identified differentially regulated genes in response to concurrent knockdown of p53 and EAF2. Several of these genes were associated with the STAT3 signaling pathway, and this was verified by significantly increased p-STAT3 immunostaining in the Eaf2-/-p53-/- mouse prostate. STAT3 knockdown abrogated the stimulation of C4-2 cell proliferation by concurrent knockdown of EAF2 and p53. Furthermore, immunostaining of p-STAT3 was increased in human prostate cancer specimens with EAF2 downregulation and/or p53 nuclear staining. Our findings suggest that simultaneous inactivation of EAF2 and p53 can act to activate STAT3 and drive prostate tumorigenesis. Overall design: C4-2 prostate cancer cells treated with siEAF2 and/or sip53 mRNA profiles were generated by deep sequencing, using Illumina HiSeq 2000.
EAF2 and p53 Co-Regulate STAT3 Activation in Prostate Cancer.
Specimen part, Cell line, SubjectView Samples
Alternative splicing is a mechanism for increasing the protein variety of a limited number of genes. Studies have shown that aberrant regulations of the alternative splicing of apoptotic gene transcripts may contribute to the development of cancer. In this study, we isolated 4ß-Hydroxywithanolide E (4bHWE) from the traditional herb Physalis peruviana, and analyzed its biological effects in cancer cells. The results demonstrated that 4bHWE modulates the alternative splicing of apoptotic genes (e.g., HIPK3, SMAC/DIABLO, and SURVIVIN), changes the expression level of splicing factors (e.g., hnRNP C1/C2, ASF/SF2, SRp20, and SRp55), and induces histone tail posttranslational modifications (e.g., H3K27me1, H3K27me2, H3K36me3, and H3K79me1). Pretreatment with okadaic acid to inhibit protein phosphatase-1 could partly relieve the effects of 4bHWE on the alternative splicing of HIPK3 and SMAC/DIABLO transcripts, as well as on the dephosphorylation of ASF/SF2. Genome-wide detection of alternative splicing further indicated that several other apoptosis-related genes are also regulated by 4bHWE, including APAF1, CARP-1, and RIPK1. Moreover, we extended our study to apoptosis-associated molecules, detecting an increasing level of CASPASE-3 activity and cleavage of poly ADP-ribose polymerase in 4bHWE-induced apoptosis. Furthermore, in vivo experiments showed that the treatment of tumor-bearing mice with 4bHWE resulted in a marked decrease of tumor size and weight. Taken together, this study is the first to show that 4bHWE affects alternative splicing through the modulations of splicing factors, providing a novel view of the antitumor mechanism of 4bHWE. Overall design: Examination of the global genes with altered alternative splicing in 4bHWE-treated Huh-7 cells.
4β-Hydroxywithanolide E Modulates Alternative Splicing of Apoptotic Genes in Human Hepatocellular Carcinoma Huh-7 Cells.
Specimen part, Treatment, SubjectView Samples
Mice lacking the beta 2 subunit (Chrnb2) of the neuronal nicotinic acetylcholine receptor display altered retinal waves and disorganized projections of the retinal ganglion cells to the lateral geniculate nucleus (LGN). mRNA populations from retinas and LGN from Chrnb2-/-and wild type (C57BL/6J) mice were compared at 4 days postnatal, when RGC segregation to the LGN begins in WT mice. Retinal mRNAs were also compared at adulthood.
Mouse mutants for the nicotinic acetylcholine receptor ß2 subunit display changes in cell adhesion and neurodegeneration response genes.
Sex, Specimen partView Samples
The study demontrates differences in the transcriptome ( both of protein coding transcripts and long non-coding RNAs) in the unilateral ureteric obstruction model of renal fibrosis. Overall design: Renal tissue was studied from animals undergoing sham operation (as controls) or right ureteric ligation. Animals were sacrificed 2 and 8 days following ligation and the right kidney tissue was examined.
Whole-transcriptome analysis of UUO mouse model of renal fibrosis reveals new molecular players in kidney diseases.
Sex, Age, Specimen part, Cell line, SubjectView Samples
We demonstrate that GLUT4 up-regulation significantly increased cell migration and invasion in lower magligance head and neck cancer cell lines in vitro.
Glucose transporter 4 promotes head and neck squamous cell carcinoma metastasis through the TRIM24-DDX58 axis.
Specimen part, Cell lineView Samples
Rationale. Lung inflammation in premature infants contributes to development of bronchopulmonary dysplasia (BPD), a chronic lung disease with long-term sequelae. Pilot studies administering budesonide suspended in surfactant have found reduced BPD without apparent adverse effects as occur with systemic dexamethasone therapy. Objectives. To determine effects of budesonide on differential genes expression in human fetal lung Overall design: Methods. We prepared RNA from 3 samples of human fetal lung at 23 weeks gestation before (preculture, PC) and after 4 days culture as explants with (Bud) or without (Way) budesonide (30 nM) and performed RNAseq on the 9 samples.
Antiinflammatory Effects of Budesonide in Human Fetal Lung.
Specimen part, SubjectView Samples