👤 Ryoko Ichikawa

Alternative RNA splicing adds diverse variations to gene function, and its abnormalities are occasionally associated with the etiology of disease. We examined this possibility in pre-eclampsia. We performed transcriptome analysis of placentas from pre-eclamptic and normotensive pregnancies and screened for disease-specific aberrant splicing. We identified aberrant splicing at exon 14 in the ZC3H4 gene. This in-frame exon is generally skipped in placentas from normal pregnancies but often observed in those from pre-eclampsia patients. The level of exon inclusion did not correlate with disease severity, such as blood pressure or fetal weight, but showed an association with the decrease in placental weight. Significantly, placental blood flow resistance measured by Doppler ultrasound correlated with the level of ZC3H4 exon 14 inclusion, suggesting that this retention leads to the onset and/or symptoms of pre-eclampsia. ZC3H4 is known to act on transcriptional regulation via suppression of lncRNA expression. Moreover, the SOD1 gene, encoding superoxide dismutase that eliminates toxic free superoxide radicals, was identified in the downstream gene group for ZC3H4. Indeed, the expression of SOD1 was found in this current study to be decreased in the pre-eclamptic placenta in correlation with the levels of ZC3H4 exon 14 retention. Aberrant splicing of ZC3H4 gene may induce excessive oxidative stress in the placenta via the downregulation of downstream SOD1 expression thereby leading to the onset and development of pre-eclampsia. Show less

Primary haematological neoplasms of the larynx are uncommon; therefore, information regarding their epidemiology is limited and the diagnosis of histological types requires careful consideration. The current study describes the case of a 72-year-old male patient with primary laryngeal lymphoplasmacytic lymphoma (LPL) that was difficult to distinguish from plasmacytoma. Imaging examinations of the neck revealed a mass in the right laryngeal folds, 25×12×25 mm in size, which was surgically resected by direct laryngoscopy. Histopathologically, the mass showed diffuse proliferation of plasma cells with CD138 (+) and IgG (+) in the submucosal stroma. Flow cytometry revealed the tumour was positive for CD19 and negative for CD56. Based on these findings, the final diagnosis was confirmed as LPL, albeit similar to plasmacytoma regarding phenotypic features. There was no evidence of local or systemic recurrence following surgery, and the patient has been followed up without additional treatment. This case highlights the unique presentation of laryngeal lymphoma mimicking solitary plasmacytoma. The key factor in the diagnosis was the expression pattern of surface antigen markers. Show less

There is a lack of information available on the anorexic action of fusarenon-x (FX), which is a sesquiterpenoid mycotoxin. In this study, we investigated the changes in the hypothalamus and small intestine related to appetite after oral FX exposure. The time-course change of food intake after oral FX exposure (0.5, 1.0, and 2.5 mg/kg bw) in B6C3F1 mice showed that 2.5 mg/kg bw of FX significantly suppressed food intake during 3-6 h compared to the control. Furthermore, the total food intake for 24 h was lower in the group exposed to FX than in the control. The FX exposure (2.5 mg/kg bw for 3 h) significantly increased mRNA levels of anorexic hormones (pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcription (CART)) without changing the mRNA levels of orexigenic hormones. In addition, FX exposure indicated significantly higher mRNA levels of possible downstream targets of anorexic POMC neurons, such as the melanocortin 4 receptor (MC4R), brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB), in the hypothalamus compared to the control. FX exposure also significantly increased the mRNA level of inflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)) and activated nuclear factor-kappa B (NF-κB), which is a regulatory factor for POMC in the hypothalamus. In the intestine, FX exposure did not affect the mRNA level of anorexic peptide YY but significantly elevated that of anorexic cholecystokinin (CCK) and regulatory factors for CCK (calcium-sensing receptor (CaSR), the transient receptor potential ankyrin-1 channel (TRPA1), and transient receptor potential cation channel subfamily M member 5 (TRPM5)). These results suggest that FX sequentially induces inflammatory cytokine expression, NF-κB activation, and POMC expression in the hypothalamus. FX also induces CCK expression in the intestine possibly via induction of CaSR, TRPM5, and TRPA1 expression. These changes will eventually lead to the anorexic action of FX. Show less

To identify key oncogenes and proteins that are controlled by the microRNA miR-29 family (miR-29a, miR-29b and miR-29c) in renal cell carcinoma pathogenesis. Genome-wide gene expression and in silico database analyses were carried out. The Cancer Genome Atlas database was used to investigate the clinical significance of gene expression data in renal cell carcinoma patients. Loss-of-function assays were applied to investigate the function of target genes. We identified 47 possible target genes that might be regulated by the miR-29 family in renal cell carcinoma cells. Among the targets of the miR-29 family, high expression of 10 genes (ADAMTS14, TRIB13, SERPINH1, FCGR1B, COL1A1, LAIR2, WISP2, TREM1, TNKS1BP1 and GBP2) significantly predicted poor patient prognosis (P < 0.001). SERPINH1 was directly regulated by the miR-29 family, and its overexpression was detected in renal cell carcinoma surgical specimens and tyrosine kinase inhibitor failure autopsy specimens. High expression of SERPINH1 was significantly associated with tumor stage, pathological grade and poor prognosis (P < 0.0001). Knockdown assays showed that its expression enhanced cancer cell migration and invasive abilities. Genes regulated by the anti-tumor miR-29 family are closely involved in the molecular pathogenesis of renal cell carcinoma. Our approach based on anti-tumor microRNAs might contribute to the development of new diagnostic markers and therapeutic strategies. Show less

The aim of this study was to identify pathways that have a significant impact during renal carcinogenesis. Sixty-seven paired samples of both noncancerous renal cortex tissue and cancerous tissue from patients with clear cell renal cell carcinomas (RCCs) were subjected to whole-exome, methylome and transcriptome analyses using Agilent SureSelect All Exon capture followed by sequencing on an Illumina HiSeq 2000 platform, Illumina Infinium HumanMethylation27 BeadArray and Agilent SurePrint Human Gene Expression microarray, respectively. Sanger sequencing and quantitative reverse transcription-PCR were performed for technical verification. MetaCore software was used for pathway analysis. Somatic nonsynonymous single-nucleotide mutations, insertions/deletions and intragenic breaks of 2,153, 359 and 8 genes were detected, respectively. Mutations of GCN1L1, MED12 and CCNC, which are members of CDK8 mediator complex directly regulating β-catenin-driven transcription, were identified in 16% of the RCCs. Mutations of MACF1, which functions in the Wnt/β-catenin signaling pathway, were identified in 4% of the RCCs. A combination of methylome and transcriptome analyses further highlighted the significant role of the Wnt/β-catenin signaling pathway in renal carcinogenesis. Genetic aberrations and reduced expression of ERC2 and ABCA13 were frequent in RCCs, and MTOR mutations were identified as one of the major disrupters of cell signaling during renal carcinogenesis. Our results confirm that multilayer-omics analysis can be a powerful tool for revealing pathways that play a significant role in carcinogenesis. Show less

We examined the expression profiles of doxorubicin-resistant K562 cells by serial analysis of gene expression (SAGE) to identify novel and/or partially characterized genes that might be related to drug resistance in human leukemia. SAGE complementary DNA (cDNA) libraries were constructed from K562 and doxorubicin-resistant K562 (K562/ADM) cells, and concatamer sequences were analyzed with SAGE 2000 software. We used 9792 tags in the identification of 1076 different transcripts, 296 of which were similarly expressed in K562 and K562/ADM cells. There were 343 genes more actively expressed in K562/ADM than in parental K562 cells and 437 genes expressed less often in K562/ADM cells. K562/ADM cells showed increased expression of well-known genes, including the genes for spectrin beta, eukaryotic translation initiation factor 1A (EIF1A), RAD23 homolog B, laminin receptor 1, and polyA-, RAN-, and PAI-1 messenger RNA-binding proteins. K562/ADM cells showed decreased expression of the genes for fatty acid desaturase 1 (FADS1), hemoglobin epsilon 1, N-myristoyltransferase 1, hemoglobin alpha 2, NADH dehydrogenase Fe-S protein 6, heat shock 90-kDa protein, and karyopherin beta 1. Quantitative reverse transcription-polymerase chain reaction analysis confirmed the increased expression of EIF1A and the decreased expression of FADS1 in K562/ADM cells. Prior to this investigation, such differences in the expression of these genes in doxorubicin-resistant leukemia cells were unknown. Although we do not provide any evidence in the present report for the potential roles of these genes in drug resistance, SAGE may provide a perspective into our understanding of drug resistance in human leukemia that is different from that provided by cDNA microarray analysis. Show less

The molecular basis by which proteins are transported along cytoskeletal tracts from the trans-Golgi network (TGN) to the cell periphery remains poorly understood. Previously, using human autoimmune sera, we identified and characterized a TGN protein, p230/Golgin-245, an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, that is anchored to TGN membranes and TGN-derived vesicles by its carboxyl-terminal GRIP domain. To identify molecules that interact with the flexible amino-terminal end of p230, we used this domain as bait to screen a human brain cDNA library in a yeast two-hybrid assay. We found that this domain interacts with the carboxyl-terminal domain of MACF1, a protein that cross-links microtubules to the actin cytoskeleton. The interaction was confirmed by co-immunoprecipitation, an in vitro binding assay, double immunofluorescence images demonstrating overlapped localization in HeLa cells, and co-localization of FLAG-tagged constructs containing the interacting domains of these two proteins with their endogenous partners. Expression in HeLa cells of FLAG-tagged constructs containing the interacting domains of p230 and MACF1 disrupted transport of the glycosyl phosphatidyl inositol-anchored marker protein conjugated with yellow fluorescent protein (YFP-SP-GPI), while trafficking of the transmembrane marker protein, vesicular stomatitis virus glycoprotein conjugated with YFP (VSVG3-GL-YFP), was unaffected. Our results suggest that p230, through its interaction with MACF1, provides the molecular link for transport of GPI-anchored proteins along the microtubule and actin cytoskeleton from the TGN to the cell periphery. Show less

Following v-Ha-ras transfection of nonmetastatic dimethylbenz(( a ))anthracene-induced rat mammary cancer (RMC1) cells, occasional transfectants were isolated that acquired high metastatic ability. High metastatic ability is not a simple process regulated by v-Ha-ras p21 levels alone in these v-Ha-ras transfectants but involves the development of cytogenetic changes. If such cytogenetic changes involve only gain in gene expression, then all hybrids formed by fusing highly metastatic v-Ha-ras RMC1 transfectants with the parental nonmetastatic RMC1 should be highly metastatic. If loss of a metastatic suppressor gene(s) is also involved, then such hybrids should be nonmetastatic since chromosomes from the nonmetastatic parental cells should supply the suppressor function. To test this possibility, a highly metastatic cloned v-Ha-ras transfectant was fused with the nonmetastatic parental RMC1 cells. Five hybrid clones were isolated that conserved the chromosomes from their parental cells. When these hybrid clones were injected into animals, primary tumors developed with the same tumor-doubling time as that of the highly metastatic parental v-Ha-ras transfectant (i.e., approximately 2 days). High metastatic ability was, however, suppressed in these hybrid clones. All hybrid clones continued to express v-Ha-ras p21. Thus, suppression of metastatic ability in the hybrids can occur even in the presence of an elevated v-Ha-ras p21 level. This suggests that the acquisition of metastatic ability following v-Ha-ras transfection involves loss of metastasis suppressor gene function in rat mammary cancer cells. Show less

When nonmetastatic dimethylbenzanthracene-induced rat mammary cancer cells (RMC1) were transfected with a control plasmid containing the neomycin resistance gene (i.e., Neo/Only), none of the five clonal Neo/Only transfectants isolated and characterized was metastatic, only one of five had a single structural chromosomal abnormality, and only one of five had a single numerical chromosomal change not present in the untransfected parental RMC1 cells. In contrast, when RMC1 cells were transfected with a plasmid containing both the neo resistance and mutated v-H-ras oncogene (i.e., Neo/Ras), four of nine clonal Neo/Ras transfectants isolated and characterized were highly metastatic, and all nine had multiple structural and/or additional numerical chromosomal abnormalities. The frequency of transfectants which had structural and/or additional numerical chromosomal changes in Neo/Ras transfectants was significantly higher than that in Neo/Only transfectants (P less than 0.05). In addition, seven of nine Neo/Ras transfectants had structural abnormalities in chromosome 1, whereas none of five Neo/Only transfectants had such abnormalities (P less than 0.05). All four Neo/Ras transfectants that were highly metastatic had structural aberrations involving a gain in chromosome 4. In contrast, none of the three Neo/Ras transfectants which were of low metastatic ability had a similar aberration involving chromosome 4. Correlation between a gain in chromosome 4 and a gain of high metastatic ability was significantly (P less than 0.05). These results demonstrate that, when RMC1 cells are transfected with v-H-ras, transfectants expressing the mutated v-H-ras p21 become genetically unstable and undergo chromosomal changes. These studies suggest that, if the appropriate chromosomal changes occur, these v-H-ras transfectants acquire high metastatic ability. Show less