Kenta Hanada, Yusuke Osaki, Ryosuke Miyamoto, Kohei Muto, Shotaro Haji, Keyoumu Nazere, Yuki Kuwano, Hiroyuki Morino, Yoshiteru Azuma, Satoko Miyatake, Naomichi Matsumoto and Yuishin Izumi : Intermediate phenotype between CMT2Z and DIGFAN associated with a novel MORC2 variant: a case report., Human Genome Variation, Vol.11, No.1, 2024.
(Summary)
Charcot-Marie-Tooth disease type 2Z is caused by MORC2 mutations and presents with axonal neuropathy. MORC2 mutations can also manifest as developmental delay, impaired growth, dysmorphic facies, and axonal neuropathy (DIGFAN). We report a patient exhibiting an intermediate phenotype between these diseases associated with a novel MORC2 variant. A literature review revealed that the genotype‒phenotype correlation in MORC2-related disorders is complex and that the same mutation can cause a variety of phenotypes.
Tatsuya Nishikawa, Yuki Kuwano, Mayu Nakata, Kazuhito Rokutan and Kensei Nishida : Multiple G-quadruplexes in the LMNA promoter regulate LMNA variant 6 transcription and promote colon cancer cell growth., Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Vol.1864, No.10, 2021.
(Summary)
Lamin A/C proteins, major components of the nuclear lamina, are encoded by the LMNA gene. These proteins have multiple cellular functions, including DNA transcription and replication, chromatin organization, regulation of the cell cycle, and apoptosis. Mutations in LMNA are associated with a variety of diseases called laminopathies. LMNA has implications in cancer; however, its mechanisms of dysregulation in cancer cells are not yet fully understood. In this study, among the LMNA transcript variants, we focused on a transcriptional variant 6 (termed LMNA-V6), which contains unique 3 exons upstream of exon 1 of LMNA. The promoter region of LMNA-V6 formed multiple G-quadruplexes and increased its transcriptional activity. Moreover, LMNA-V6 negatively regulated other LMNA mRNA variants, lamin A and lamin C, via direct interaction with their promoter. Knockdown of LMNA-V6 decreased the proliferation of colon cancer cells, whereas overexpression of the unique 3 exons of LMNA-V6 increased cell growth. Furthermore, microarray gene expression profiling showed that alteration of LMNA-V6 levels influenced the expression of p53 in colon cancer cells. Taken together, the results suggest that LMNA-V6 may be a novel functional RNA whose expression is regulated through multiple G-quadruplexes in colon cancer cells.
Kensei Nishida, Yuki Kuwano and Kazuhito Rokutan : The MicroRNA-23b/27b/24 Cluster Facilitates Colon Cancer Cell Migration by Targeting FOXP2, Cancers, Vol.12, No.1, 174, 2020.
(Summary)
Acquisition of cell migration capacity is an early and essential process in cancer development. The aim of this study was to identify microRNA gene expression networks that induced high migration capacity. Using colon cancer HCT116 cells subcloned by transwell-based migrated cell selection, microRNA array analysis was performed to examine the microRNA expression profile. Promoter activity and microRNA targets were assessed with luciferase reporters. Cell migration capacity was assessed by either the transwell or scratch assay. In isolated subpopulations with high migration capacity, the expression levels of the cluster increased in accordance with the increased expression of the short transcript, a host gene of the cluster. E2F1-binding sequences were involved in the basic transcription activity of the short expression, and E2F1-small-interfering (si)RNA treatment reduced the expression of both the and clusters. Overexpression experiments showed that and promoted cell migration, but the opposite effect was observed with . Forkhead box P2 (FOXP2) mRNA and protein levels were reduced by both/either and . Furthermore, FOXP2 siRNA treatment significantly promoted cell migration. Our findings demonstrated a novel role of the cluster in cell migration through targeting FOXP2, with potential implications for the development of microRNA-based therapy targeted at inhibiting cancer migration.
Kensei Nishida, Sawada Daisuke, Yuki Kuwano, Hiroki Tanaka and Kazuhito Rokutan : Health Benefits of Lactobacillus gasseri CP2305 Tablets in Young Adults Exposed to Chronic Stress: A Randomized, Double-Blind, Placebo-Controlled Study, Nutrients, Vol.11, No.8, 1859, 2019.
(Summary)
Short-term administration of CP2305 improves stress-associated symptoms and clinical symptoms in healthy young adults and in patients with irritable bowel syndrome, respectively. We evaluated the efficacy and health benefits of the long-term use of a tablet containing heat-inactivated, washed CP2305 (CP2305) in healthy young adults. Sixty Japanese medical students (41 men and 19 women) preparing for the national examination for medical practitioners ingested CP2305-containing or placebo tablets once daily for 24 weeks. Intake of the CP2305 tablet significantly reduced anxiety and sleep disturbance relative to placebo, as quantitated by the Spielberger State-Trait Anxiety Inventory and the Pittsburgh Sleep Quality Index. Single-channel sleep electroencephalograms show that CP2305 significantly shortened sleep latency and wake time after sleep onset and increased the delta power ratio in the first sleep cycle. CP2305 also significantly lowered salivary chromogranin A levels compared with placebo. Furthermore, 16S rRNA gene sequencing of participant feces demonstrated that CP2305 administration attenuated the stress-induced decline of spp. and the stress-induced elevation of spp. We conclude that the long-term use of CP2305-containing tablets may improve the mental state, sleep quality, and gut microbiota of healthy adults under stressful conditions.
Tatsuya Nishikawa, Yuki Kuwano, Yumiko Takahara, Kensei Nishida and Kazuhito Rokutan : HnRNPA1 interacts with G-quadruplex in the TRA2B promoter and stimulates its transcription in human colon cancer cells., Scientific Reports, Vol.9, No.1, 2019.
(Summary)
The human TRA2B gene consists of 10 exons and 9 introns and produces 5 splice isoforms (TRA2β1 to TRA2β5). TRA2B exon 2 encodes multiple premature termination codons. TRA2β1 lacks exon 2 and is translated into a functional transformer 2β (Tra2β) protein, whereas TRA2β4 contains 10 exons and works as a functional RNA. Overexpressed Tra2β and ectopic expression of TRA2β4 may be oncogenic. We found that heterogeneous nuclear ribonucleoprotein (hnRNP)A1 and hnRNPU interacted with TRA2β4 exon 2. Minigene assays revealed that hnRNPA1 facilitated inclusion of exon 2, whereas hnRNPU promoted its skipping. However, knockdown of hnRNPA1 or hnRNPU reduced both TRA2β1 and TRA2β4 levels, and overexpression of these hnRNPs increased levels of both isoforms, suggesting that hnRNPA1 and hnRNPU mainly regulate the transcription of TRA2B. In fact, hnRNPA1 and hnRNPU positively regulated the promoter activity of TRA2B. Circular dichroism analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated the presence of G-quadruplex (G4) formation in the promoter of TRA2B. Formation of G4 suppressed TRA2B transcription, whereas hnRNPA1, but not hnRNPU, interacted with the G4 to facilitate transcription. Our results suggest that hnRNPA1 may modulate TRA2B transcription through its regulation of G4 formation in its promoter in colon cancer cells.
Saki Saijo, Yuki Kuwano, Shoichiro Tange, Kazuhito Rokutan and Kensei Nishida : A novel long non-coding RNA from the HOXA6-HOXA5 locus facilitates colon cancer cell growth., BMC Cancer, Vol.19, No.1, 2019.
(Summary)
Our results suggested that HOXA5 short RNA, a novel lncRNA, may play a crucial role in colon tumor growth through activation of EGF signaling.
Diversification of transcriptomic and epigenomic states may occur during the expansion of colorectal cancers. Certain cancer cells lose their epithelial characters and gain mesenchymal properties, known as epithelial-mesenchymal transition (EMT), and they aggressively migrate into the non-tumorigenic extracellular matrix. In this study, we isolated a subpopulation with accelerated baseline motility (MG cells) and an immotile one (non-MG cells) from a colon cancer cell line (HCT116). Gene expression signatures of the MG cells indicated that this subpopulation was likely an EMT hybrid. The MG cells substantially lost their migratory properties after treatment with a methyltransferase inhibitor, 5-azacytidine, suggesting a role of DNA methylation in this process. Global transcriptome assays of both types of cells with or without 5-azacytidine treatment identified 640 genes, whose expression might be methylation-dependently down-regulated in the MG cells. Global methylation analysis revealed that 35 out of the 640 genes were hyper-methylated in the MG cells. Among them, we focused on the anti-oncogene ZNF350, which encodes a zinc-finger and BRCA1-interacting protein. Notably, ZNF350 knockdown accelerated migration of the non-MG cells, while overexpression of ZNF350 in the MG cells significantly impaired their migration. Finally, pyrosequence analysis together with dual luciferase assays of serially truncated fragments of the ZNF350 promoter (-268 to +49 bp) indicated that three hyper-methylated sites were possibly responsible for the basal promoter activity of ZNF350. Taken together, our results suggest that hyper-methylation of the ZNF350 proximal promoter may be one of the crucial determinants for acquiring increased migratory capabilities in colon cancer cells.
Kiyoshi Masuda and Yuki Kuwano : Diverse roles of RNA-binding proteins in cancer traits and their implications in gastrointestinal cancers., Wiley Interdisciplinary Reviews. RNA, Vol.10, No.3, 2018.
(Summary)
Gene expression patterns in cancer cells are strongly influenced by posttranscriptional mechanisms. RNA-binding proteins (RBPs) play key roles in posttranscriptional gene regulation; they can interact with target mRNAs in a sequence- and structure-dependent manner, and determine cellular behavior by manipulating the processing of these mRNAs. Numerous RBPs are aberrantly deregulated in many human cancers and hence, affect the functioning of mRNAs that encode proteins, implicated in carcinogenesis. Here, we summarize the key roles of RBPs in posttranscriptional gene regulation, describe RBPs disrupted in cancer, and lastly focus on RBPs that are responsible for implementing cancer traits in the digestive tract. These evidences may reveal a potential link between changes in expression/function of RBPs and malignant transformation, and a framework for new insights and potential therapeutic applications. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
Satake Yuzuru, Yuki Kuwano, Tatsuya Nishikawa, Fujita Kinuyo, Saijo Saki, Miki Itai, Tanaka Hiroki, Kensei Nishida and Kazuhito Rokutan : Nucleolin facilitates nuclear retention of an ultraconserved region containing TRA24 and accelerates colon cancer cell growth., Oncotarget, Vol.9, No.42, 26817-26833, 2018.
(Summary)
Transcribed-ultraconserved regions (T-UCRs), which contain conserved sequences with 100% identity across human, rat and mouse species, are a novel category of functional RNAs. The human gene () encodes a UCR that spans exon 2 (276 bp) and its neighboring introns. Among five spliced RNA variants () transcribed from the gene, only contains the conserved exon 2. is overexpressed in colon cancer cells and accelerates cell growth by blocking the transcription of . However, the mechanisms underlying the overexpression of in colon cancer cells are unknown. Using biotinylated RNA pull-down assays followed by liquid chromatography-mass spectrometric analysis, we identified nucleolin as a -binding protein. Knockdown of nucleolin reduced the nuclear retention of and accelerated its degradation in the cytoplasm, whereas nucleolin overexpression increased levels and its mitogenic activity. Nucleolin directly bound to exon 2 via the glycine/arginine-rich (GAR) domain. Overexpression of GAR-deficient nucleolin failed to increase expression and growth of colon cancer cells. RNA fluorescence hybridization showed that co-localized with nucleolin in nuclei but not with the mutant lacking GAR. Our results suggest that specific interactions between nucleolin and UCR-containing may be associated with abnormal growth of colon cancer cells.
Miki Itai, Yuki Kuwano, Tatsuya Nishikawa, Kazuhito Rokutan and Kensei Nishida : Geranylgeranylacetone prevents stress-induced decline of leptin secretion in mice., The Journal of Medical Investigation : JMI, Vol.65, No.1.2, 103-109, 2018.
(Summary)
Geranylgeranylacetone (GGA) is a chaperon inducer that protects various types of cell and tissue against stress. We examined whether GGA modulated energy intake and expenditure under stressful conditions. After mice were untreated or treated orally with GGA (0.16 g per kg body weight per day) for 10 days, they were subjected to 2-h restraint stress once or once a day for 5 consecutive days. GGA administration did not affect corticosterone response to the stress. Restraint stress rapidly decreased plasma leptin levels in control mice. GGA significantly increased circulating leptin levels without changing food intake and prevented the stress-induced decline of circulating leptin. However GGA-treated mice significantly reduced food intake during the repeated stress, compared with control mice. GGA prevented the stress-induced decline of leptin mRNA and its protein levels in epidydimal adipose tissues. We also found that GGA decreased ghrelin mRNA expression in gastric mucosa before the stress, whereas GGA-treated mice recovered the ghrelin mRNA expression to the baseline level after the repeated stress. Leptin and ghrelin are now recognized as regulators of anxiety and depressive mood. Our results suggest that GGA may regulate food intake and relief stress-induced mood disturbance through regulating leptin and ghrelin secretions. J. Med. Invest. 65:103-109, February, 2018.
Mai Takada, Kensei Nishida, Y. Gondo, H. Kikuchi-Hayakawa, H. Ishikawa, K. Suda, M. Kawai, R. Hoshi, Yuki Kuwano, K. Miyazaki and Kazuhito Rokutan : Beneficial effects of Lactobacillus casei strain Shirota on academic stress-induced sleep disturbance in healthy adults: a double-blind, randomised, placebo-controlled trial., Beneficial Microbes, Vol.8, No.2, 153-162, 2017.
(Summary)
The present study examined whether Lactobacillus casei strain Shirota (LcS) improves sleep quality under psychological stress. A double-blind, placebo-controlled trial was conducted in healthy 4(th) year medical students exposed to academic examination stress. The trial was repeated over two consecutive years in different groups of students, and the data were pooled. For 8 weeks prior to and 3 weeks after a national standardised examination, a total of 48 and 46 subjects received a daily dose of 100 ml of LcS-fermented milk or non-fermented placebo milk, respectively. Study measures included subjective anxiety, overnight single-channel electroencephalography (EEG) recordings, and the Oguri-Shirakawa-Azumi (OSA) sleep inventory scores of subjective sleep quality. Total OSA scores were significantly lower than baseline on the day before the exam and recovered after the exam, indicating a stress-induced decline in sleep quality. There was a significant positive effect of LcS treatment on OSA factors for sleepiness on rising and sleep length. Sleep latency measured by EEG lengthened as the exam approached in the placebo group but was significantly suppressed in the LcS group. The percentage of stage 3 non-REM (N3) sleep decreased in the placebo group as the exam approached, whereas it was maintained in the LcS group throughout the trial. Delta power during the first sleep cycle, measured as an index of sleep intensity, increased as the exam approached in the LcS group and was significantly higher than in the placebo group. These findings suggest that daily consumption of LcS may help to maintain sleep quality during a period of increasing stress. The observed retention of N3 sleep and increased delta power in the LcS group may have contributed to higher perceived sleep satisfaction.
Daisuke Sawada, Tomoko Kawai, Kensei Nishida, Yuki Kuwano, Shigeru Fujiwara and Kazuhito Rokutan : Daily intake of Lactobacillus gasseri CP2305 improves mental, physical, and sleep quality among Japanese medical students enrolled in a cadaver dissection course., Journal of Functional Foods, Vol.31, 188-197, 2017.
Tomoko Kawai, Yuki Kuwano, Kiyoshi Masuda, Kinuyo Fujita, Hiroki Tanaka, Tatsuya Nishikawa, Kazuhito Rokutan and Kensei Nishida : Adverse parenting is associated with blunted salivary cortisol awakening response and altered expression of glucocorticoid receptor and 2-adrenergic receptor mRNAs in leukocytes in Japanese medical students., Stress, Vol.20, No.2, 159-166, 2017.
(Summary)
Adverse parenting is associated with an increased risk for the development of mood and behavioral disorders. In this study, we assessed the perceived parental bonding of 232 medical students using the parental bonding instrument (PBI) and extracted 22 students who reported their parents' rearing attitudes as affectionless control (LOW; low care, high overprotection). Using the 28-item general health questionnaire, the Zung self-rating depression scale (Zung-SDS), the hospital anxiety and depression scale (HADS), and the Spielberger state-trait-anxiety-inventory (STAI), physical and mental state of the LOW students were compared with those of 30 students who reported their parental bonding as optimal (OPT; high care and low overprotection). These questionnaire measurements demonstrated significantly higher anxiety and depressive mood in the LOW students versus the OPT students. Compared with the OPT students, the LOW students also exhibited a significantly reduced salivary cortisol awakening response (CAR) without changes across the rest of the diurnal salivary cortisol profile. Among glucocorticoid-related genes examined (GR, ADRB2, IκBα, IL10, IL1R2, IL1RN, MR, MC2R, TGFB1, TGFB2 and FASLG), real-time reverse transcription-PCR showed that the LOW students significantly increased expression of a dominant negative glucocorticoid receptor β (GRβ) mRNA and decreased β2-adrenergic receptor (ADRB2) mRNA levels in circulating leukocytes. These results suggest that negative perception of parents' child-rearing attitudes may be associated with anxiety and depressive mood and altered glucocorticoid signaling even in healthy young adults.
K. Nobutani, Daisuke Sawada, Shiferu Fujiwara, Yuki Kuwano, Kensei Nishida, J. Nakayama, H. Kutsumi, T. Azuma and Kazuhito Rokutan : The effects of administration of the Lactobacillus gasseri strain CP2305 on quality of life, clinical symptoms and changes in gene expression in patients with irritable bowel syndrome., Journal of Applied Microbiology, Vol.122, No.1, 212-224, 2017.
(Summary)
To clarify the effects of Lactobacillus gasseri CP2305 (CP2305) on quality of life and clinical symptoms and its functional mechanisms in patients with irritable bowel syndrome (IBS). After the patients were administered CP2305 daily for 4 weeks, the IBS-severity index score was significantly improved compared with that of the placebo group, and this improvement was accompanied by a reduction in health-related worry and changes in intestinal microbiota. The gene expression profiling of the peripheral blood leucocytes showed that CP2305 treatment significantly up-regulated genes related to eukaryotic initiation factor 2 (EIF2) signalling. Eighty-two genes were down-regulated in IBS patients compared with healthy controls. The expression of 23 of these genes exhibited a CP2305-dependent increase associated with an improvement in IBS severity. The majority of the restored genes were related to EIF2 signalling. CP2305 administration is a potential candidate therapeutic option for patients with IBS. Although probiotics have been proposed to benefit IBS patients, objective clinical evidence and elucidation of the functional mechanism remain insufficient. Our study demonstrated that CP2305 administration beneficially influences IBS patients in both subjective and objective evaluations, and gene expression profiling provided insights into the functional mechanism.
(Keyword)
Adult / Female / Humans / Irritable Bowel Syndrome / Lactobacillus gasseri / Male / Middle Aged / Probiotics / Quality of Life / Treatment Outcome
Yuki Kuwano, Kensei Nishida, Yoko Akaike, Ken Kurokawa, Tatsuya Nishikawa, Kiyoshi Masuda and Kazuhito Rokutan : Homeodomain-Interacting Protein Kinase-2: A Critical Regulator of the DNA Damage Response and the Epigenome., International Journal of Molecular Sciences, Vol.17, No.10, 2016.
(Summary)
Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase that phosphorylates and activates the apoptotic program through interaction with diverse downstream targets including tumor suppressor p53. HIPK2 is activated by genotoxic stimuli and modulates cell fate following DNA damage. The DNA damage response (DDR) is triggered by DNA lesions or chromatin alterations. The DDR regulates DNA repair, cell cycle checkpoint activation, and apoptosis to restore genome integrity and cellular homeostasis. Maintenance of the DDR is essential to prevent development of diseases caused by genomic instability, including cancer, defects of development, and neurodegenerative disorders. Recent studies reveal a novel HIPK2-mediated pathway for DDR through interaction with chromatin remodeling factor homeodomain protein 1. In this review, we will highlight the molecular mechanisms of HIPK2 and show its functions as a crucial DDR regulator.
Akito Kato-Kataoka, Kensei Nishida, Mai Takada, Mitsuhisa Kawai, Hiroko Kikuchi-Hayakawa, Kazunori Suda, Hiroshi Ishikawa, Yusuke Gondo, Kensuke Shimizu, Takahiro Matsuki, Akira Kushiro, Ryoutaro Hoshi, Osamu Watanabe, Tomoki Igarashi, Kouji Miyazaki, Yuki Kuwano and Kazuhito Rokutan : Fermented Milk Containing Lactobacillus casei Strain Shirota Preserves the Diversity of the Gut Microbiota and Relieves Abdominal Dysfunction in Healthy Medical Students Exposed to Academic Stress., Applied and Environmental Microbiology, Vol.82, No.12, 3649-3658, 2016.
(Summary)
A novel clinical trial was conducted with healthy medical students under examination stress conditions. It was demonstrated that the daily consumption of lactic acid bacteria provided health benefits to prevent the onset of stress-associated abdominal symptoms and a good change of gut microbiota in healthy medical students.
K Kajita, Yuki Kuwano, Y Satake, S Kano, K Kurokawa, Y Akaike, Kiyoshi Masuda, Kensei Nishida and Kazuhito Rokutan : Ultraconserved region-containing Transformer 24 controls senescence of colon cancer cells., Oncogenesis, Vol.5, 2016.
M Takada, Kensei Nishida, A Kataoka-Kato, Y Gondo, H Ishikawa, K Suda, M Kawai, R Hoshi, O Watanabe, T Igarashi, Yuki Kuwano, K Miyazaki and Kazuhito Rokutan : Probiotic Lactobacillus casei strain Shirota relieves stress-associated symptoms by modulating the gut-brain interaction in human and animal models., Neurogastroenterology and Motility, 2016.
Saki Saijo, Yuki Kuwano, Kiyoshi Masuda, Tatsuya Nishikawa, Kazuhito Rokutan and Kensei Nishida : Serine/arginine-rich splicing factor 7 regulates p21-dependent growth arrest in colon cancer cells., The Journal of Medical Investigation : JMI, Vol.63, No.3-4, 219-226, 2016.
(Summary)
Serine/arginine-rich splicing factors (SRSFs) play wide-ranging roles in gene expression through post-transcriptional regulation as well as pre-mRNA splicing. SRSF7 was highly expressed in colon cancer tissues, and its knockdown inhibited cell growth in colon cancer cells (HCT116) in association with altered expression of 4,499 genes. The Ingenuity Pathway Analysis revealed that cell cycle-related canonical pathways were ranked as the highly enriched category in the affected genes. Western blotting confirmed that p21, a master regulator in cell cycle, was increased without any induction of p53 in SRSF7 knockdown cells. Furthermore, cyclin-dependent kinase 2 and retinoblastoma protein were remained in the hypophosphorylated state. In addition, the SRSF7 knockdown-induced cell growth inhibition was observed in p53-null HCT116 cells, suggesting that p53-independent pathways were involved in the SRSF7 knockdown-induced cell growth inhibition. The reduction of SRSF7 stabilized cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNA without any activation of the CDKN1A promoter. Interestingly, SRSF7 knockdown also blocked p21 degradation. These results suggest that the reduction of SRSF7 post-transcriptionally regulates p21 induction at the multistep processes. Thus, the present findings disclose a novel, important role of SRSF7 in cell proliferation through regulating p21 levels. J. Med. Invest. 63: 219-226, August, 2016.
A Kato-Kataoka, Kensei Nishida, M Takada, K Suda, M Kawai, K Shimizu, A Kushiro, R Hoshi, O Watanabe, T Igarashi, K Miyazaki, Yuki Kuwano and Kazuhito Rokutan : Fermented milk containing Lactobacillus casei strain Shirota prevents the onset of physical symptoms in medical students under academic examination stress., Beneficial Microbes, Vol.7, No.2, 153-156, 2016.
(Summary)
This pilot study investigated the effects of the probiotic Lactobacillus casei strain Shirota (LcS) on psychological, physiological, and physical stress responses in medical students undertaking an authorised nationwide examination for promotion. In a double-blind, placebo-controlled trial, 24 and 23 healthy medical students consumed a fermented milk containing LcS and a placebo milk, respectively, once a day for 8 weeks until the day before the examination. Psychophysical state, salivary cortisol, faecal serotonin, and plasma L-tryptophan were analysed on 5 different sampling days (8 weeks before, 2 weeks before, 1 day before, immediately after, and 2 weeks after the examination). Physical symptoms were also recorded in a diary by subjects during the intervention period for 8 weeks. In association with a significant elevation of anxiety at 1 day before the examination, salivary cortisol and plasma L-tryptophan levels were significantly increased in only the placebo group (P<0.05). Two weeks after the examination, the LcS group had significantly higher faecal serotonin levels (P<0.05) than the placebo group. Moreover, the rate of subjects experiencing common abdominal and cold symptoms and total number of days experiencing these physical symptoms per subject were significantly lower in the LcS group than in the placebo group during the pre-examination period at 5-6 weeks (each P<0.05) and 7-8 weeks (each P<0.01) during the intervention period. Our results suggest that the daily consumption of fermented milk containing LcS may exert beneficial effects preventing the onset of physical symptoms in healthy subjects exposed to stressful situations.
Yuki Kuwano, Kensei Nishida, Keisuke Kajita, Yuzuru Satake, Yoko Akaike, Kiyuyo Fujita, Shizuka Kano, Kiyoshi Masuda and Kazuhito Rokutan : Transformer 2 and miR-204 regulate apoptosis through competitive binding to 3 UTR of BCL2 mRNA, Cell Death and Differentiation, Vol.22, No.5, 815-825, 2015.
(Summary)
RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2β (Tra2β) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2β antibody and microarray analysis identified a subset of Tra2β-associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including Bcl-2 (B-cell CLL/lymphoma 2). Tra2β knockdown in HCT116 cells decreased Bcl-2 expression and induced apoptosis. Tra2β knockdown accelerated the decay of BCL2α mRNA that encodes Bcl-2 and full-length 3' UTR, while it did not affect the stability of BCL2β mRNA having a short, alternatively spliced 3' UTR different from BCL2α 3' UTR. RIP assays with anti-Tra2β and anti-Argonaute 2 antibodies, respectively, showed that Tra2β bound to BCL2α 3' UTR, and that Tra2β knockdown facilitated association of miR-204 with BCL2α 3' UTR. The consensus sequence (GAA) for Tra2β-binding lies within the miR-204-binding site of BCL2 3' UTR. Mutation of the consensus sequence canceled the binding of Tra2β to BCL2 3' UTR without disrupting miR-204-binding to BCL2 3' UTR. Transfection of an anti-miR-204 or introduction of three-point mutations into the miR-204-binding site increased BCL2 mRNA and Bcl-2 protein levels. Inversely, transfection of precursor miR-204 reduced their levels. Experiments with Tra2β-silenced or overexpressed cells revealed that Tra2β antagonized the effects of miR-204 and upregulated Bcl-2 expression. Furthermore, TRA2β mRNA expression was significantly upregulated in 22 colon cancer tissues compared with paired normal tissues and positively correlated with BCL2 mRNA expression. Tra2β knockdown in human lung adenocarcinoma cells (A549) increased their sensitivity to anticancer drugs. Taken together, our findings suggest that Tra2β regulates apoptosis by modulating Bcl-2 expression through its competition with miR-204. This novel function may have a crucial role in tumor growth.
Y Akaike, Yuki Kuwano, Kensei Nishida, K Kurokawa, K Kajita, S Kano, Kiyoshi Masuda and Kazuhito Rokutan : Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1., Oncogene, Vol.34, No.26, 3463-3473, 2014.
(Summary)
Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1γ pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor.
Yoko Akaike, Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida, Keisuke Kajita, Ken Kurokawa, Yuzuru Satake, Katsutoshi Shoda, Issei Imoto and Kazuhito Rokutan : HuR Regulates Alternative Splicing of the TRA2 Gene in Human Colon Cancer Cells under Oxidative Stress., Molecular and Cellular Biology, Vol.34, No.15, 2857-2873, 2014.
(Summary)
Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2 gene encodes splicing factor transformer 2 (Tra2) and generates 5 mRNA isoforms (TRA21 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells to sodium arsenite stimulated checkpoint kinase 2 (Chk2)- and mitogen-activated protein kinase p38 (p38(MAPK))-mediated phosphorylation of HuR at positions S88 and T118. This induced an association between HuR and the 39-nucleotide (nt) proximal region of TRA2 exon 2, generating a TRA24 mRNA that includes exon 2, which has multiple premature stop codons. HuR knockdown or Chk2/p38(MAPK) double knockdown inhibited the arsenite-stimulated production of TRA24 and increased Tra2 protein, facilitating Tra2-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38(MAPK) double knockdown were also confirmed using a TRA2 minigene spanning exons 1 to 4, and the effects disappeared when the 39-nt region was deleted from the minigene. In endogenous HuR knockdown cells, the overexpression of a HuR mutant that could not be phosphorylated (with changes of serine to alanine at position 88 [S88A], S100A, and T118A) blocked the associated TRA24 interaction and TRA24 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA24-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress.
Ken Kurokawa, Yoko Akaike, Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida, Naoko Yamagishi, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells, Oncogene, Vol.33, No.11, 1407-1417, 2014.
(Summary)
Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth.
Shizuka Kano, Kensei Nishida, Hiroyuki Kurebe, Chihiro Nishiyama, Kentaro Kita, Yoko Akaike, Keisuke Kajita, Ken Kurokawa, Kiyoshi Masuda, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Oxidative stress-inducible truncated serine/arginine-rich splicing factor 3 regulates interleukin-8 production in human colon cancer cells, American Journal of Physiology, Cell Physiology, Vol.306, No.3, C250-C262, 2014.
(Summary)
Serine/arginine-rich splicing factor 3 (SRSF3) is a member of the SR protein family and plays wide-ranging roles in gene expression. The human SRSF3 gene generates two alternative splice transcripts, a major mRNA isoform (SRSF3-FL) encoding functional full-length protein and a premature termination codon (PTC)-containing isoform (SRSF3-PTC). The latter is degraded through nonsense-mediated mRNA decay (NMD). Treatment of a human colon cancer cell line (HCT116) with 100 μM sodium arsenite increased SRSF3-PTC mRNA levels without changing SRSF3-FL mRNA levels. A chemiluminescence-based NMD reporter assay system demonstrated that arsenite treatment inhibited NMD activity and increased SRSF3-PTC mRNA levels in the cytoplasm, facilitating translation of a truncated SRSF3 protein (SRSF3-TR) from SRSF3-PTC mRNA. SRSF3-TR lacked two-thirds of the Arg/Ser-rich (RS) domain whose phosphorylation state is known to be crucial for subcellular distribution. SRSF3-FL was localized in the nucleus, while overexpressed SRSF3-TR was diffusely distributed in the cytoplasm and the nucleus. A part of SRSF3-TR was also associated with stress granules in the cytoplasm. Interestingly, treatment of HCT116 cells with a small interference RNA specifically targeting SRSF3-PTC mRNA significantly attenuated arsenite-stimulated induction of c-JUN protein, its binding activity to the AP-1 binding site (-126 to 120 bp) in the interleukin (IL)-8 gene promoter, and AP-1 promoter activity, resulting in significant reduction of arsenite-stimulated IL-8 production. Our results suggest that SRSF3-TR may function as a positive regulator of oxidative stress-initiated inflammatory responses in colon cancer cells.
Keisuke Kajita, Yuki Kuwano, Naruka Kitamura, Yuzuru Satake, Kensei Nishida, Ken Kurokawa, Yoko Akaike, Manami Honda, Kiyoshi Masuda and Kazuhito Rokutan : Ets1 and heat shock factor 1 regulate transcription of the Transformer 2β gene in human colon cancer cells, Journal of Gastroenterology, Vol.48, No.11, 1222-1233, 2013.
(Summary)
Transformer (Tra) 2β is a member of the serine/arginine-rich (SR)-like protein family that regulates alternative splicing of numerous genes in a concentration-dependent manner. Several types of cancer cells up-regulate Tra2β expression, while the regulatory mechanism of Tra2β expression remains to be elucidated. In this study, we examined the transcriptional regulation and possible functions of Tra2β in human colon cancer cells. We cloned 959 bp-upstream of the human TRA2β 5'-flank into luciferase constructs. Chromatin immunoprecipitation (ChIP) was employed to identify crucial cis element(s) and trans activator(s) of the TRA2β promoter. Tra2β expression in the human colon and colon cancer tissues was examined by immunohistochemistry. In response to sodium arsenite, colon cancer cells (HCT116) increased levels of TRA2β1 mRNA encoding a functional, full-length Tra2β with a peak around 6 h without changing its mRNA stability. Transient expression assays using a reporter gene driven by serially truncated TRA2β promoters and Chip assay demonstrated that an Ets1-binding site present at -64 to -55 bp was crucial for basal transcription, while three heat shock elements (HSEs) located at -145 to -99 bp mediated the oxidant-induced transactivation of TRA2β. Tra2β knockdown caused apoptosis of HCT116 cells. Tra2β were preferentially expressed in proliferative compartment of normal human colonic glands and adenocarcinomas, where Ets1 and heat shock factor 1 were also highly expressed. Our results suggest that oxidative stress-responsive Tra2β may play an important role in colon cancer growth.
Manami Honda, Yuki Kuwano, Sakurako Katsuura-Kamano, Kinuyo Fujita, Yoko Akaike, Shizuka Kano, Kensei Nishida, Kiyoshi Masuda and Kazuhito Rokutan : Chronic academic stress increases a group of microRNAs in peripheral blood, PLoS ONE, Vol.8, No.10, e75960, 2013.
(Summary)
MicroRNAs (miRNAs) play key roles in regulation of cellular processes in response to changes in environment. In this study, we examined alterations in miRNA profiles in peripheral blood from 25 male medical students two months and two days before the National Examination for Medical Practitioners. Blood obtained one month after the examination were used as baseline controls. Levels of seven miRNAs (miR-16, -20b, -26b, -29a, -126, -144 and -144*) were significantly elevated during the pre-examination period in association with significant down-regulation of their target mRNAs (WNT4, CCM2, MAK, and FGFR1 mRNAs) two days before the examination. State anxiety assessed two months before the examination was positively and negatively correlated with miR-16 and its target WNT4 mRNA levels, respectively. Fold changes in miR-16 levels from two days before to one month after the examination were inversely correlated with those in WNT4 mRNA levels over the same time points. We also confirmed the interaction between miR-16 and WNT4 3'UTR in HEK293T cells overexpressing FLAG-tagged WNT4 3'UTR and miR-16. Thus, a distinct group of miRNAs in periheral blood may participate in the integrated response to chronic academic stress in healthy young men.
(Keyword)
Adult / Carrier Proteins / Humans / Male / MicroRNAs / Protein-Serine-Threonine Kinases / Receptor, Fibroblast Growth Factor, Type 1 / Stress, Psychological / Teaching / Wnt4 Protein
Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida, Kazuhito Rokutan and Issei Imoto : NF90 in Posttranscriptional Gene Regulation and MicroRNA Biogenesis., International Journal of Molecular Sciences, Vol.14, No.8, 17111-17121, 2013.
(Summary)
Gene expression patterns are effectively regulated by turnover and translation regulatory (TTR) RNA-binding proteins (RBPs). The TTR-RBPs control gene expression at posttranscriptional levels, such as pre-mRNA splicing, mRNA cytoplasmic export, turnover, storage, and translation. Double-stranded RNA binding proteins (DSRBPs) are known to regulate many processes of cellular metabolism, including transcriptional control, translational control, mRNA processing and localization. Nuclear factor 90 (NF90), one of the DSRBPs, is abundantly expressed in vertebrate tissue and participates in many aspects of RNA metabolism. NF90 was originally purified as a component of a DNA binding complex which binds to the antigen recognition response element 2 in the interleukin 2 promoter. Recent studies have provided us with interesting insights into its possible physiological roles in RNA metabolism, including transcription, degradation, and translation. In addition, it was shown that NF90 regulates microRNA expression. In this review, we try to focus on the function of NF90 in posttranscriptional gene regulation and microRNA biogenesis.
Naoko Yamagishi, Shigetada Kondo, Kiyoshi Masuda, Kensei Nishida, Yuki Kuwano, Duyen T Dang, Long H Dang, Takeshi Nikawa and Kazuhito Rokutan : Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells, BMC Cancer, Vol.13, 229, 2013.
(Summary)
Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.
Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida and Kazuhito Rokutan : Application of DNA microarray technology to gerontological studies, Methods in Molecular Biology, Vol.1048, 285-308, 2013.
(Summary)
Gene expression patterns change dramatically in aging and age-related events. The DNA microarray is now recognized as a useful device in molecular biology and widely used to identify the molecular mechanisms of aging and the biological effects of drugs for therapeutic purpose in age-related diseases. Recently, numerous technological advantages have led to the evolution of DNA microarrays and microarray-based techniques, revealing the genomic modification and all transcriptional activity. Here, we show the step-by-step methods currently used in our lab to handling the oligonucleotide microarray and miRNA microarray. Moreover, we introduce the protocols of ribonucleoprotein [RNP] immunoprecipitation followed by microarray analysis (RIP-chip) which reveal the target mRNA of age-related RNA-binding proteins.
Shizuka Kano, Kensei Nishida, Chihiro Nishiyama, Yoko Akaike, Keisuke Kajita, Ken Kurokawa, Kiyoshi Masuda, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Truncated serine/arginine-rich splicing factor 3 accelerates cell growth through up-regulating c-Jun expression, The Journal of Medical Investigation : JMI, Vol.60, No.3-4, 228-235, 2013.
(Summary)
Serine/arginine-rich splicing factor 3 (SRSF3), a member of the SRSF family, plays a wide-ranging role in gene expression. The human SRSF3 gene generates a major mRNA isoform encoding a functional, full-length protein and a PTC-containing isoform (SRSF3-PTC). The latter is expected to be degraded through the nonsense-mediated mRNA decay system. However, it was reported that SRSF3-PTC mRNA was produced under stressful conditions and translated into a truncated SRSF3 protein (SRSF3-TR). To disclose unknown functions of SRSF3-TR, we established Flp-In-293 cells stably expressing SRSF3-TR. The SRSF3-TR-expressing cells increased mRNA and protein levels of positive regulators for G1 to S phase transition (cyclin D1, cyclin D3, CDC25A, and E2F1) and accelerated their growth. c-Jun is required for progression through the G1 phase, the mechanism by which involves transcriptional control of the cyclin D1 gene. We also found that the JUN promoter activity was significantly increased in the Flp-In-293 cells stably expressing SRSF3-TR, compared with mock-transfected control cells. The SRSF3-TR-expressing cells increased c-Jun and Sp-1 levels, which are important for the positive autoregulation and basal transcription of JUN, respectively. Our results suggest that stress-inducible SRSF3-TR may participate in the acceleration of cell growth through facilitating c-Jun-mediated G1 progression under stressful conditions.
(Keyword)
Alternative Splicing / Base Sequence / Cell Line / Cell Proliferation / DNA / G1 Phase Cell Cycle Checkpoints / Genes, bcl-1 / Genes, jun / Humans / Molecular Sequence Data / Oxidative Stress / Peptide Fragments / Promoter Regions, Genetic / RNA-Binding Proteins / Up-Regulation
Yoshiko Kamezaki, Sakurako Katsuura, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Circulating cytokine signatures in healthy medical students exposed to academic examination stress., Psychophysiology, Vol.49, No.7, 991-997, 2012.
(Summary)
Stress-induced production of proinflammatory cytokines in the brain and periphery is associated with mental distress. In this study, we measured changes in levels of salivary cortisol and 50 circulating immune mediators in 28 4th-grade medical students (19 males and 9 females) 7 weeks before, 1 day before, immediately after, and 1 week after an authorized nationwide examination for promotion. Repeated measures ANOVA with multiple testing correction and post hoc tests revealed that the examination significantly increased levels of proinflammatory cytokines (granulocyte colony-stimulating factor, interferon-, interleukin (IL)-1, and tumor necrosis factor-), Th2 cytokines (IL-4, IL-5, and IL-13), and -nerve growth factor in association with significant decreases in salivary cortisol levels and anxiety after the examination. These mediators may have a negative impact on the mental state of healthy young adults exposed to naturalistic stressors.
Sakurako Katsuura, Yuki Kuwano, Naoko Yamagishi, Ken Kurokawa, Keisuke Kajita, Yoko Akaike, Kensei Nishida, Kiyoshi Masuda, Toshihito Tanahashi and Kazuhito Rokutan : MicroRNAs miR-144/144* and miR-16 in peripheral blood are potential biomarkers for naturalistic stress in healthy Japanese medical students., Neuroscience Letters, Vol.516, No.1, 79-84, 2012.
(Summary)
Non-coding microRNAs (miRNAs) are suggested to serve fundamental roles in cellular stress responses and in coping with sudden environmental changes in experimental animals. We examined whether naturalistic stressor-responsive miRNAs were detectable in whole blood. Blood and saliva were collected between 16:00 and 17:00 from 10 healthy medical students (5 males and 5 females; aged 22.4±0.8 years, mean±SD) 7 weeks before, one day before, immediately after, and one week after a nationally administered examination for academic promotion. Samples obtained one week after the examination were used as baseline controls. State anxiety and salivary cortisol levels reached maximum levels the day before the examination. Eleven candidate miRNAs (miR-144, -144*, -16, -15a, -19a, -19b, -26b, -30b, -106b, -126, and -142-3p) were extracted using a human miRNA microarray, and quantitative real-time reverse transcription PCR confirmed significant elevation of miR-144/144* and miR-16 levels immediately after finishing the examination. miR-16 levels in individual students were positively correlated with those of serum tumor necrosis factor (TNF)- measured immediately after the examination. Percentage changes in miR-144* and miR-16 levels from immediately after to one week after the examination were significantly correlated with percentage changes in circulating interferon- and/or TNF- levels over the same time points. Our results suggest that miR-144/144* and miR-16 may constitute a part of an integrated response to naturalistic stressors in healthy young adults.
(Keyword)
Biological Markers / Female / Humans / Interferon-gamma / Japan / Male / MicroRNAs / Prevalence / Stress, Psychological / Students, Medical / Tumor Necrosis Factor-alpha / Young Adult
Ken Kurokawa, Toshihito Tanahashi, Tsutomu Iima, Yuta Yamamoto, Yoko Akaike, Kensei Nishida, Kiyoshi Masuda, Yuki Kuwano, Yoshiki Murakami, Masakazu Fukushima and Kazuhito Rokutan : Role of miR-19b and its target mRNAs in 5-fluorouracil resistance in colon cancer cells., Journal of Gastroenterology, Vol.47, No.8, 883-895, 2012.
(Summary)
Drug resistance in colorectal cancers is assumed to be mediated by changes in the expression of microRNAs, but the specific identities and roles of microRNAs are largely unclear. We examined the effect of 5-fluorouracil (5-FU) resistance on microRNA expression. Two types of 5-FU-resistant colon cancer cells were derived from the DLD-1 and KM12C cell lines. The expressions of microRNAs were profiled with a microarray containing 723 microRNAs and validated by quantitative real-time polymerase chain reaction (qRT-PCR). To survey the downstream mediators of microRNA, we used a microRNA:mRNA immunoprecipitation (RIP)-Chip and pathway analysis tool to identify potential direct targets of microRNA. In response to 5-FU, miR-19b and miR-21 were over-expressed in 5-FU-resistant cells. Of note, miR-19b was up-regulated 3.47-fold in the DLD-1 resistant cells, which exhibited no alteration in cell cycle profiles despite exposure to 5-FU. After transfection of miR-19b, specific mRNAs were recruited to microRNA:mRNA complexes isolated with Ago2 antibody and subjected to whole-genome transcriptional analysis. In this analysis, 66 target mRNAs were enriched by at least 5.0-fold in the microRNA:mRNA complexes from DLD-1 resistant cells. Ingenuity pathway analysis of mRNA targets significantly (P < 0.05) indicated the category "Cell Cycle" as a probable area of the molecular and cellular function related with 5-FU resistance. Among candidate mRNA targets, SFPQ and MYBL2 have been linked to cell cycle functions. We revealed up-regulation of miR-19b in response to 5-FU and potential targets of miR-19b mediating the cell cycle under treatment with 5-FU. Our study provides an important insight into the mechanism of 5-FU resistance in colorectal cancers.
Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida and Kazuhito Rokutan : General RBP expression in human tissues as a function of age., Ageing Research Reviews, Vol.11, No.4, 423-431, 2012.
(Summary)
Gene expression patterns vary dramatically in a tissue-specific and age-dependent manner. RNA-binding proteins that regulate mRNA turnover and/or translation (TTR-RBPs) critically affect the subsets of expressed proteins. Although many proteins implicated in age-related processes are encoded by mRNAs that are targets of TTR-RBPs, very little is known regarding the tissue- and age-dependent expression of TTR-RBPs in humans. Recent analysis of TTR-RBPs expression using human tissue microarray has provided us interesting insight into their possibly physiologic roles as a function of age. This analysis has also revealed striking discrepancies between the levels of TTR-RBPs in senescent human diploid fibroblasts (HDFs), widely used as an in vitro model of aging, and the levels of TTR-RBPs in tissues from individuals of advancing age. In this article, we will review our knowledge of human TTR-RBP expression in different tissues as a function of age.
Yuki Kuwano, Yoko Kamio, Tomoko Kawai, Sakurako Katsuura, Naoko Inada, Akiko Takaki and Kazuhito Rokutan : Autism-associated gene expression in peripheral leucocytes commonly observed between subjects with autism and healthy women having autistic children., PLoS ONE, Vol.6, No.9, e24723, 2011.
(Summary)
Autism spectrum disorder (ASD) is a severe neuropsychiatric disorder which has complex pathobiology with profound influences of genetic factors in its development. Although the numerous autism susceptible genes were identified, the etiology of autism is not fully explained. Using DNA microarray, we examined gene expression profiling in peripheral blood from 21 individuals in each of the four groups; young adults with ASD, age- and gender-matched healthy subjects (ASD control), healthy mothers having children with ASD (asdMO), and asdMO control. There was no blood relationship between ASD and asdMO. Comparing the ASD group with control, 19 genes were found to be significantly changed. These genes were mainly involved in cell morphology, cellular assembly and organization, and nerve system development and function. In addition, the asdMO group possessed a unique gene expression signature shown as significant alterations of protein synthesis despite of their nonautistic diagnostic status. Moreover, an ASD-associated gene expression signature was commonly observed in both individuals with ASD and asdMO. This unique gene expression profiling detected in peripheral leukocytes from affected subjects with ASD and unaffected mothers having ASD children suggest that a genetic predisposition to ASD may be detectable even in peripheral cells. Altered expression of several autism candidate genes such as FMR-1 and MECP2, could be detected in leukocytes. Taken together, these findings suggest that the ASD-associated genes identified in leukocytes are informative to explore the genetic, epigenetic, and environmental background of ASD and might become potential tools to assess the crucial factors related to the clinical onset of the disorder.
Yoko Akaike, Ken Kurokawa, Keisuke Kajita, Yuki Kuwano, Kiyoshi Masuda, Kensei Nishida, Seung Kang Wan, Toshihito Tanahashi and Kazuhito Rokutan : Skipping of an alternative intron in the srsf1 3' untranslated region increases transcript stability., The Journal of Medical Investigation : JMI, Vol.58, No.3-4, 180-187, 2011.
(Summary)
The srsf1 gene encodes serine/arginine-rich splicing factor 1 (SRSF1) that participates in both constitutive and alternative splicing reactions. This gene possesses two ultraconserved elements in the 3' untranslated region (UTR). Skipping of an alternative intron between the two elements has no effect on the protein-coding sequence, but it generates a premature stop codon (PTC)-containing mRNA isoform, whose degradation is considered to depend on nonsense-mediated mRNA decay (NMD). However, several cell lines (HCT116, RKO, HeLa, and WI38 cells) constitutively expressed significant amounts of the srsf1 PTC variant. HCT116 cells expressed the PTC variant nearly equivalent to the major isoform that includes the alternative intron in the 3' UTR. Inhibition of NMD by silencing a key effecter UPF1 or by treatment with cycloheximide failed to increase amounts of the PTC variant in HCT116 cells, and the PTC variant was rather more stable than the major isoform in the presence of actinomycin D. Our results suggest that the original stop codon may escape from the NMD surveillance even in skipping of the alternative intron. The srsf1 gene may produce an alternative splice variant having truncated 3' UTR to relief the microRNA- and/or RNA-binding protein-mediated control of translation or degradation. J. Med. Invest. 58: 180-187, August, 2011.
Subramanya Srikantan, Kotb Abdelmohsen, Kyung Eun Lee, Kumiko Tominaga, S Sarah Subaran, Yuki Kuwano, Ritu Kulshrestha, Rohit Panchakshari, Ho Hyeon Kim, Xiaoling Yang, L Jennifer Martindale, S Bernard Marasa, M Mihee Kim, P Robert Wersto, E Fred Indig, Dipanjan Chowdhury and Myriam Gorospe : Translational control of TOP2A influences doxorubicin efficacy., Molecular and Cellular Biology, Vol.31, No.18, 3790-3801, 2011.
(Summary)
The cellular abundance of topoisomerase II (TOP2A) critically maintains DNA topology after replication and determines the efficacy of TOP2 inhibitors in chemotherapy. Here, we report that the RNA-binding protein HuR, commonly overexpressed in cancers, binds to the TOP2A 3'-untranslated region (3'UTR) and increases TOP2A translation. Reducing HuR levels triggered the recruitment of TOP2A transcripts to RNA-induced silencing complex (RISC) components and to cytoplasmic processing bodies. Using a novel MS2-tagged RNA precipitation method, we identified microRNA miR-548c-3p as a mediator of these effects and further uncovered that the interaction of miR-548c-3p with the TOP2A 3'UTR repressed TOP2A translation by antagonizing the action of HuR. Lowering TOP2A by silencing HuR or by overexpressing miR-548c-3p selectively decreased DNA damage after treatment with the chemotherapeutic agent doxorubicin. In sum, HuR enhances TOP2A translation by competing with miR-548c-3p; their combined actions control TOP2A expression levels and determine the effectiveness of doxorubicin.
(Keyword)
3' Untranslated Regions / Antibiotics, Antineoplastic / Antigens, Neoplasm / Antigens, Surface / DNA / DNA Damage / DNA Topoisomerases, Type II / DNA-Binding Proteins / Doxorubicin / Gene Expression Regulation / HeLa Cells / Humans / MicroRNAs / Protein Biosynthesis / RNA Interference / RNA, Messenger / RNA, Small Interfering / RNA-Binding Proteins / RNA-Induced Silencing Complex
Sakurako Katsuura, Yoshiko Kamezaki, Naoko Yamagishi, Yuki Kuwano, Kensei Nishida, Kiyoshi Masuda, Toshihito Tanahashi, Tomoko Kawai, Kokichi Arisawa and Kazuhito Rokutan : Circulating vascular endothelial growth factor is independently and negatively associated with trait anxiety and depressive mood in healthy Japanese university students., International Journal of Psychophysiology, Vol.81, No.1, 38-43, 2011.
(Summary)
Anxiety and depressive mood are sometimes accompanied by modulation of neuroendocrine and immune functions. The aim of this study was to identify circulating immune mediators reflecting anxiety and depressive mood in healthy young adults. Anxiety and depressive mood in 209 healthy medical students (125 males and 84 females, aged 20.7±2.7years (mean±SD)) were assessed by the Spielberger state-trait anxiety inventory (STAI) and the Zung self-rating depression scale (Zung-SDS), respectively. Cortisol and chromogranin A (CgA) levels in saliva were measured using enzyme immunoassay kits, and 50 different mediators in sera were measured by a multiplex-suspension array system. The level of statistical significance was set at =0.05. Forty-four mediators were measurable in sera, and each mediator showed substantial individual variations. After determining Pearson correlation coefficients, we selected candidate cytokines whose levels were associated with STAI-state (2 cytokines), STAI-trait (8 cytokines), or SDS scores (8 cytokines). The candidate cytokines plus interleukin (IL)-1, IL-6, tumor necrosis factor-, and macrophage migration inhibitory factor were then subjected to multiple regression analysis adjusted for gender, BMI, and salivary concentrations of cortisol and CgA. Vascular endothelial growth factor (VEGF) was independently and negatively associated with both trait anxiety (p<0.05) and depressive mood (p<0.01). IL-1 showed independently positive association with depressive mood (p<0.05). Interactions between these two cytokines and gender or BMI were not observed. Besides IL-1, circulating VEGF may be a potential biomarker for negative mood states in healthy young adults.
(Keyword)
Adolescent / Anxiety / Body Mass Index / cytokinesis / Depressive Disorder / Endothelial Growth Factors / female / Humans / Hydrocortisone / Japan / male / Psychiatric Status Rating Scales / Questionnaires / Regression Analysis / Saliva / Sex Factors / Students / Universities / Vascular Endothelial Growth Factor A / Young Adult
Ken Kurokawa, Toshihito Tanahashi, Akiho Murata, Yoko Akaike, Sakurako Katsuura, Kensei Nishida, Kiyoshi Masuda, Yuki Kuwano, Tomoko Kawai and Kazuhito Rokutan : Effects of chronic academic stress on mental state and expression of glucocorticoid receptor α and β isoforms in healthy Japanese medical students., Stress, Vol.14, No.4, 431-438, 2011.
(Summary)
Chronic academic stress responses were assessed by measuring mental state, salivary cortisol levels, and the glucocorticoid receptor (GR) gene expression in healthy Japanese medical students challenging the national medical license examination. Mental states of 17 male and 9 female medical undergraduates, aged 25.0 ± 1.2 years (mean ± SD), were assessed by the State and Trait Anxiety Inventory (STAI) and the Self-Rating Depression Scale (SDS) 2 months before, 2 days before, and 1 month after the examination. At the same time points, saliva and blood were collected. STAI-state scores peaked 2 days before the examination. Scores on STAI-trait and SDS, and salivary cortisol levels were consistently higher during the pre-examination period. One month after the examination, all these measures had significantly decreased to baseline levels. Real-time reverse transcription PCR showed that this chronic anxious state did not change the expression of the functional GRα mRNA isoform in peripheral leukocytes, while it resulted in reduced expression of the GRβ isoform 2 days before the examination. Our results replicate and extend a significant impact of chronic academic stressors on the mental state of healthy Japanese medical students and suggest a possible association of GRβ gene in response to psychological stress.
(Keyword)
Adult / Asian Continental Ancestry Group / Female / Humans / Hydrocortisone / Male / Protein Isoforms / RNA, Messenger / Receptors, Glucocorticoid / salivary glands / Stress, Psychological / Students, Medical
Ken Kurokawa, Toshihito Tanahashi, Kiyoshi Masuda, Yuki Kuwano and Kazuhito Rokutan : Regulation of gene expression by alternative splicing, Shikoku Acta Medica, Vol.66, No.5,6, 157-162, 2010.
(Summary)
The human genome sequence has been decoded, and the more complicated regulation of gene function is revealed in the post-genome era. In the various mechanisms of epigenome, RNA dramatically controls gene expression through the various post-transcriptional processing including transcription, splicing, cap addition, polyadenylation, nuclear export, translation. Especially, the alternative splicing is involved in all of those post-transcriptional regulations, as well as splicing of pre-mRNA. However, there were few reports, how the alternative splicing contributes to the regulations of cellular functions because of its difficulty of the analysis. This review discusses the molecular mechanism of alternative splicing and its regulator ; Serine/arginine-rich splicing factor (SRSF). We also discuss how the SRSF genes sustain their own proper expressions and functions.
Yuki Kuwano, Sakurako Katsuura, Kiyoshi Masuda, Toshihito Tanahashi and Kazuhito Rokutan : A novel biomental tool for assessing psychological stress response, Shikoku Acta Medica, Vol.66, No.5,6, 119-122, 2010.
(Summary)
Stress plays an important role in both mental and physical problems. Stressful life events initiate a coordinated physiological process that is produced by interactions between the hypothalamuspituitary- adrenal axis, sympathetic nervous system, and immune system. The response to psychological stress varies considerably and depends on a wide range of environmental and genetic factors. Establishment of a new biomental tool for objectively assessing stress response is required. We focus on high-throughput analysis of gene expression using microarray system to study the complex stress responses. Alternative splicing(AS)regulates the gene expression program in response to surrounding environment. However, acute psychological stress-initiated AS events have not been documented yet. Academic examinations are one of the brief naturalistic stressors and have been shown to change gene expression in peripheral leukocytes, which is postulated to be involved in the psychological response. Using the GeneChip human exon1．0ST array, AS events of 27 genes with splicing indices >1．0could be detected immediately after the examination among healthy university students. Real-time reverse transcription PCR validated the stress-initiated skipping of exon 63 of SMG -1 that encodes a phosphatidylinositol 3-kinase-related protein kinase crucial for activations of p 53-dependent pathways and mRNA decay system. These results indicate that AS mediated regulation of gene expression in response to brief naturalistic stressors in peripheral leukocytes, and suggest the SMG -1 splice variant as a potential biomarker for acute psychological stress.
(Keyword)
microarray / gene expression / psychological stress / alternative splicing
Ken Kurokawa, Yuki Kuwano, Kumiko Tominaga, Tomoko Kawai, Sakurako Katsuura, Naoko Yamagishi, Yuzuru Satake, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : Brief naturalistic stress induces an alternative splice variant of SMG-1 lacking exon 63 in peripheral leukocytes., Neuroscience Letters, Vol.484, No.2, 128-132, 2010.
(Summary)
Alternative splicing (AS) not only regulates the gene expression program in response to surrounding environment, but also produces protein isoforms with unique properties under stressful conditions. However, acute psychological stress-initiated AS events have not been documented in human studies. After assessments of changes in salivary cortisol levels and anxiety among 28 fourth-grade medical students 7 weeks prior to, 1 day before, immediately after, and 1 week after an examination for promotion, we selected 5 male students, who showed a typical stress response, and screened AS events in their circulating leukocytes using the GeneChip human exon 1.0 ST array. AS events of 27 genes with splicing indices >1.0 could be detected between immediately after and either 7 weeks before, 1 day before, or 1 week after the examination. The examination stress preferentially caused skipping rather than inclusion: 21 out of the 27 pre-mRNAs underwent skipping of exons, and skipping in 3'UTR was observed in 8 genes. Among the candidate genes, real-time reverse transcription PCR validated the stress-initiated skipping of exon 63 of SMG-1 that encodes a phosphatidylinositol 3-kinase-related protein kinase crucial for activations of p53-dependent pathways and nonsense-mediated mRNA decay. Our results indicate a significant impact of brief naturalistic stressors on AS-mediated regulation of gene expression in peripheral leukocytes, and suggest the SMG-1 splice variant as a potential biomarker for acute psychological stress.
(Keyword)
Alternative Splicing / Analysis of Variance / Exons / Female / Gene Expression Profiling / Gene Expression Regulation / Humans / Hydrocortisone / Leukocytes / Male / Oligonucleotide Array Sequence Analysis / Phosphatidylinositol 3-Kinases / Psychiatric Status Rating Scales / Salvia / Stress, Psychological / Young Adult
Kotb Abdelmohsen, Emmette R. Hutchison, Eun Kyung Lee, Yuki Kuwano, Mihee M. Kim, Kiyoshi Masuda, Subramanya Srikantan, Sarah S. Subaran, Bernard S. Marasa, Mark P. Mattson and Myriam Gorospe : miR-375 inhibits differentiation of neurites by lowering HuD levels., Molecular and Cellular Biology, Vol.30, No.17, 4197-4210, 2010.
(Summary)
Neuronal development and plasticity are maintained by tightly regulated gene expression programs. Here, we report that the developmentally regulated microRNA miR-375 affects dendrite formation and maintenance. miR-375 overexpression in mouse hippocampus potently reduced dendrite density. We identified the predominantly neuronal RNA-binding protein HuD as a key effector of miR-375 influence on dendrite maintenance. Heterologous reporter analysis verified that miR-375 repressed HuD expression through a specific, evolutionarily conserved site on the HuD 3' untranslated region. miR-375 overexpression lowered both HuD mRNA stability and translation and recapitulated the effects of HuD silencing, which reduced the levels of target proteins with key functions in neuronal signaling and cytoskeleton organization (N-cadherin, PSD-95, RhoA, NCAM1, and integrin alpha1). Moreover, the increase in neurite outgrowth after brain-derived neurotrophic factor (BDNF) treatment was diminished by miR-375 overexpression; this effect was rescued by reexpression of miR-375-refractory HuD. Our findings indicate that miR-375 modulates neuronal HuD expression and function, in turn affecting dendrite abundance.
Yuki Kuwano, Kumiko Tominaga, Tsukasa Kawahara, Hidekazu Sasaki, Keiko Takeo, Kensei Nishida, Kiyoshi Masuda, Tomoko Kawai, Shigetada Teshima-Kondo and Kazuhito Rokutan : Tumor necrosis factor alpha activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells., Free Radical Biology and Medicine, Vol.45, No.12, 1642-1652, 2008.
(Summary)
NADPH oxidase 1 (Nox1) is a multicomponent enzyme consisting of p22(phox), Nox organizer 1 (NOXO1), Nox1 activator 1, and Rac1. Interleukin-1beta, flagellin, interferon-gamma, and tumor necrosis factor alpha (TNF-alpha) similarly induced Nox1 in a colon cancer cell line (T84), whereas only TNF-alpha fully induced NOXO1 and upregulated superoxide-producing activity by ninefold. This upregulation was canceled by knockdown of NOXO1 with small interfering RNAs. TNF-alpha rapidly phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase 1/2, followed by phosphorylation of c-Jun and c-Fos and appearance of an AP-1 binding activity within 30 min. We cloned the 5' flank of the human NOXO1 gene (-3888 to +263 bp), and found that the region between -585 and -452 bp, which contains consensus elements of YY-1, AP-1, and Ets, and the GC-rich region encoding three putative binding sites for SP-1, was crucial for TNF-alpha-dependent promoter activity. Serial mutation analysis of the elements identified an AP-1 binding site (from -561 to -551 bp, agtAAGtcatg) as a crucial element for TNF-alpha-stimulated transcription of the human NOXO1 gene, which was also confirmed by the AP-1 decoy experiments. Thus, TNF-alpha acts as a potent activator of Nox1-based oxidase in colon epithelial cells, suggesting a potential role of this oxidase in inflammation of the colon.
Kazuhito Rokutan, Tsukasa Kawahara, Yuki Kuwano, Kumiko Tominaga, Keisei Nishida and Shigetada Teshima-Kondo : Nox enzymes and oxidative stress in the immunopathology of the gastrointestinal tract., Seminars in Immunopathology, Vol.30, No.3, 315-327, 2008.
(Summary)
Chronic inflammation caused by Helicobacter pylori infection or inflammatory bowel disease (IBD) is closely linked to cancer development. Innate immune abnormalities and enhanced production of reactive oxygen species through a phagocyte NADPH oxidase (Nox2) are key issues in understanding the pathogenesis of inflammation-dependent carcinogenesis. Besides Nox2, functionally distinct homologues (Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2) have been identified. Nox1 and Duox2 are highly expressed in the gastrointestinal tract. Although the functional roles of Nox/Duox in the gastrointestinal tract are still unclear, we will review their potential roles in the gastrointestinal immunopathology, particularly in H. pylori-induced inflammation, IBD, and malignancy.
Norihiro Kubota, Shuichi Suzuki, Yuki Kuwano, Sayaka Kakiyama, Naomi Harima-Mizusawa and Kensei Nishida : IDO1 and FOXP3: Possible immune-regulating genes alleviating cedar pollinosis via L. plantarum YIT 0132., Allergy, Vol.79, No.7, 1966-1969, 2024.
Katsutoshi Shoda, Yuki Kuwano, Daisuke Ichikawa and Kiyoshi Masuda : circRNA: A New Biomarker and Therapeutic Target for Esophageal Cancer, Biomedicines, Vol.10, No.7, 1643, Jul. 2022.
(Summary)
Circular RNAs (circRNAs) comprise a large class of endogenous non-coding RNA with covalently closed loops and have independent functions as linear transcripts transcribed from identical genes. circRNAs are generated by a "back-splicing" process regulated by regulatory elements in cis and associating proteins in trans. Many studies have shown that circRNAs play important roles in multiple processes, including splicing, transcription, chromatin modification, miRNA sponges, and protein decoys. circRNAs are highly stable because of their closed ring structure, which prevents them from degradation by exonucleases, and are more abundant in terminally differentiated cells, such as brains. Recently, it was demonstrated that numerous circRNAs are differentially expressed in cancer cells, and their dysfunction is involved in tumorigenesis and metastasis. However, the crucial functions of these circRNAs and the dysregulation of circRNAs in cancer are still unknown. In this review, we summarize the recent reports on the biogenesis and biology of circRNAs and then catalog the advances in using circRNAs as biomarkers and therapeutic targets for cancer therapy, particularly esophageal cancer.
Kensei Nishida, Yuki Kuwano, Tatsuya Nishikawa, Kiyoshi Masuda and Kazuhito Rokutan : RNA Binding Proteins and Genome Integrity., International Journal of Molecular Sciences, Vol.18, No.7, Jun. 2017.
(Summary)
Genome integrity can be threatened by various endogenous or exogenous events. To counteract these stressors, the DNA damage response network contributes to the prevention and/or repair of genomic DNA damage and serves an essential function in cellular survival. DNA binding proteins are involved in this network. Recently, several RNA-binding proteins (RBPs) that are recruited to DNA damage sites have been shown to be direct players in the prevention or repair of DNA damage. In addition, non-coding RNAs, themselves, are involved in the RNA-mediated DNA repair system. Furthermore, RNA modification such as m6A methylation might also contribute to the ultraviolet-responsive DNA damage response. Accumulating evidence suggests that RNA metabolism is more deeply involved in diverse cellular functions than previously expected, and is also intricately associated with the maintenance of genome integrity. In this review, we highlight the roles of RBPs in the maintenance of genome integrity.
Yuki Kuwano : Physiological Roles of MicroRNAs in Stress Response, Japanese Journal of Psychosomatic Medicine, Vol.56, No.4, 328-332, Apr. 2016.
(Summary)
Increasing evidences have suggested that the non-protein-coding genome is functionally important for regulation of cellular processes. MicroRNAs (miRNAs) are a class of small non-coding RNAs approximately 22 nucleotides in length. They bind to partially complementary sites within the 3'UTR of target mRNAs and inhibit their translation. More than 2,000 miRNAs are discovered in human cells, and sufficiently expressed miRNAs typically target hundreds of different mRNAs. The important roles of miRNAs are to control and maintain normal physiological functioning of the central nervous system, including neuron maturation, neurogenesis, and synaptic plasticity. Various stressors influence the processing of miRNA, recognition of mRNA targets, and miRNA expression. Recent papers have reported that miRNAs play an essential role in regulation of stress response. Interestingly, experimental animals with mutant miRNAs appear to normally develop, while they cannot cope with stressful conditions. Both acute and chronic stressors change miRNA expression profiles in a brain region-dependent fashion and changes in miRNA expression. We previously investigated that psychological stress-related changes in miRNA expression were also detectable in peripheral tissues, such as peripheral blood leukocytes. Taken together, the stress-mediated miRNA response may activate a program of gene expression that is essential for the production of adaptive response to the stressor.
Keyoumu Nazere, Konoka Tachibana, Yuki Kuwano, Ryosuke Miyamoto, Ryuji Kaji, Yuishin Izumi and Hiroyuki Morino : The identification and functional analysis of novel variants in ADCY5- related movement disorders, 第65回日本神経学会学術大会/AOCN2024, May 2024.
3.
Yuki Kuwano, Keyoumu Nazere and Hiroyuki Morino : The biological role of m6A RNA methylation in cytoplasmic localization of TDP-43, 第65回日本神経学会学術大会/AOCN2024, May 2024.
4.
Yuki Kuwano, Kensei Nishida, 佐竹 譲 and Kazuhito Rokutan : Nuclear retention of transcribed ultraconserved region uc.138 promotes colon cancer cell growth, Cell Symposium-Regulatory RNAs, Berlin, Germany, May 2019.
5.
Saijo Saka, Kensei Nishida, Tatsuya Nishikawa, Yuki Kuwano and Kazuhito Rokutan : A novel role of serine/arginine-rich splicing factor 7 in cell cycle progression, KEYSTONE SYMPOSIA on Molecular and Cellular Biology, Banff, Alberta, Canada, Feb. 2017.
6.
Yuki Kuwano, Satake Yuzuru, Tatsuya Nishikawa, Fujita Kinuyo, Saijo Saki, Kensei Nishida and Kazuhito Rokutan : Ultraconserved region-containing Transformer 2-beta4 associates with nucleolin and regulates cellular proliferation, KEYSTONE SYMPOSIA on Molecular and Cellular Biology, Banff, Alberta, Canada, Feb. 2017.
Yuki Kuwano, K kajita, S Kano, Y Satake, M Fujita, M Itai, Kensei Nishida and Kazuhito Rokutan : Ultraconserved region-containing transformer 24 inhibits senesces of colon cancer cells., Cell symposia-Human genomics, Singapore, Nov. 2015.
9.
Shizuka Kano, Kensei Nishida, Yuki Kuwano, Takuya Naruto and Kazuhito Rokutan : Analysis of functional transcribed-ultraconserved regions in SR protein family., Cold Spring Harbor Laboratory Meeting on Eukaryotic mRNA processing., Sep. 2015.
10.
Saki Saijo, Kensei Nishida, Shizuka Kano, Takuya Naruto, Yuki Kuwano and Kazuhito Rokutan : A role of serine/arginine-rich splicing factor 7 in cell cycle progression, Cold Spring Harbor Laboratory Meeting on Eukaryotic mRNA processing, Aug. 2015.
11.
Kensei Nishida, Saki Saijo, Shizuka Kano, Takuya Naruto, Yuki Kuwano and Kazuhito Rokutan : Analysis of 3 end processing factors expression during epithelial-mesenchymal transition, Cold Spring Harbor Laboratory Meeting on Eukaryotic mRNA processing, Aug. 2015.
12.
Yuki Kuwano, Y Satake, S Kano, K Fujita, M Itai, Kensei Nishida, Takuya Naruto, Kiyoshi Masuda and Kazuhito Rokutan : Transformer 2 and miR-204 regulate cell death through competitive binding to 3 UTR of BCL2 mRNA, Cell Symposia Regulatory RNAs, San Francisco, Oct. 2014.
13.
S Kano, Kensei Nishida, Y Satake, K Fujita, M Itai, Takuya Naruto, Kiyoshi Masuda, Yuki Kuwano and Kazuhito Rokutan : Analysis of function transcribed ultraconserved regions of SR protein family, Cell Symposia Regulatory RNAs, San Francisco, Oct. 2014.
14.
Shizuka Kano, Kensei Nishida, Yuzuru Satake, Yoko Akaike, Kinuyo Fujita, Kiyoshi Masuda, Yuki Kuwano and Kazuhito Rokutan : Arsenite stress-inducible truncated serine/arginine-rich splicing factor 3 regulates interleukin-8 in human colon cancer cells, The 5th EMBO Meeting 2013, Amsterdam, Sep. 2013.
15.
Yoko Akaike, Kiyoshi Masuda, Yuzuru Satake, Kinuyo Fujita, Shizuka Kano, Kensei Nishida, Yuki Kuwano and Kazuhito Rokutan : Homeodomain interacting protein kinase 2 regulates interaction between heterochromatin protein 1 gamma and trimethylated histone H3 Lys9, The 5th EMBO Meeting 2013, Amsterdam, Sep. 2013.
16.
Kinue Fujita, Yuki Kuwano, Shizuka Kano, Yuzuru Satake, Kensei Nishida, Kiyoshi Masuda and Kazuhito Rokutan : Socioeconomic status-related gene expression profiles in peripheral leukocytes from medical staffs, The international conference on social stratification and health, Tokyo, Sep. 2013.
17.
Yuki Kuwano, Sakurako Katsuura-Kamano, Tomoko Kawai, YoKo Kamio and Kazuhito Rokutan : Autism-associated gene expression was commonly observed in peripheral blood leukocytes from subjects with autism and healthy mothers having autistic children., 11th World Congress of Biological Psychiatry, Kyoto, Jun. 2013.
18.
Kiyoshi Masuda, Y Akaike, K Fujita, M Honda, Y Satake, K Kajita, Kensei Nishida, Yuki Kuwano and Kazuhito Rokutan : Hu antigen R (HuR) Regulates an Alternative Splicing of Transformer 2-beta (Tra2beta) and Induces lncRNA (Tra2beta4) under Oxidative Stress, Cell Symposia Regulatory RNAs, Sitges, Spain, Dec. 2012.
19.
Yuki Kuwano, K. Kajita, Y. Satake, Y. Akaike, M. Honda, F. Fujita, Kensei Nishida, Kiyoshi Masuda and Kazuhito Rokutan : The RNA binding protein Transformer-2 beta modulates processing of microRNAs, Cell symposia-Functional RNAs, Dec. 2012.
20.
Y Akaike, Kiyoshi Masuda, K Fujita, M Honda, Y Satake, K Kajita, Kensei Nishida, Yuki Kuwano and Kazuhito Rokutan : Hu antigen R (HuR) Functions as An Alternative Splicing Regulator of Transformer 2-beta (TRA2beta) in response to Oxidative Stress, 4th EMBO meeting 2012, Niece, Sep. 2012.
21.
K Kajita, Yuki Kuwano, Y Satake, Y Akaike, M Honda, K Fujita, Kensei Nishida, Kiyoshi Masuda and Kazuhito Rokutan : Ultraconserved element-containing Transformer 24 mRNA regulates cellular senescence, The 4th EMBO Meeting 2012, Niece, Sep. 2012.
22.
K. Kajita, Yuki Kuwano, Y. Satake, Y. Akaike, M. Honda, K. Fujita, K. Nishida, K. Masuda and K. Rokutan : Ultraconserved element-containing Transformer 24 mRNA regulates cellular senescence., The 4th EMBO Meeting, France, Sep. 2012.
23.
Yuki Kuwano, 神尾 陽子, 河合 智子, Sakurako Katsuura, 稲田 尚子, 高木 晶子 and Kazuhito Rokutan : Autism-associated gene expression signatures in peripheral blood leucocytes, Joint Academic Conference on ASD 2011, Tokyo, Dec. 2011.
24.
Naoko Yamagishi, Shigetada Kondo, Kiyoshi Masuda, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Identification of a novel tumor promoting non-coding RNA encoded in the Vegf gene, Cell Symposia Regulatory RNAs, Chicago, Oct. 2011.
25.
Yuki Kuwano, Keisuke Kajita, Yuzuru Satake, Ken Kurokawa, Naoko Yamagishi, Yoko Akaike, Manami Honda, Kensei Nishida, Kiyoshi Masuda, Toshihito Tanahashi and Kazuhito Rokutan : Transfomer-2beta regulates apoptosis through post-transcriptional regulation of bcl-2, Cell Symposia Regulatory RNAs, Chicago, Oct. 2011.
26.
Kiyoshi Masuda, Naoko Yamagishi, Ken Kurokawa, Yuzuru Satake, Keisuke Kajita, Yoko Akaike, Manami Honda, Kensei Nishida, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Hu antigen R (huR) functions as an alternative pre-mRNA splicing enhancer of transformer 2-beta (Tra2beta) on exon definition under oxidative stress, Cell Symposia Regulatory RNAs, Chicago, Oct. 2011.
Manami Honda, Sakurako Katsuura, Yuki Kuwano, Naoko Yamagishi, Ken Kurokawa, Yuzuru Satake, Keisuke Kajita, Yoko Akaike, Kensei Nishida, Kiyoshi Masuda, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of immunomodulators identifies VEGF as a potential biomarker for trait anxiety and depressive mood in healthy Japanese university students, The International Conference on Social Stratification and Health 2011, Tokyo, Aug. 2011.
30.
Sakurako Katsuura, Kamezaki Yoishiko, Yuki Kuwano, Yamagishi Naoko, Kurokawa Ken, Satake Yuzuru, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of brief naturalistic stress-responsive cytokines in Japanese university students taking examinations, The 7th World Congress on Stress, Leiden, Netherland, Aug. 2010.
31.
Sakurako Katsuura, Yoshiko Kamezaki, Yuki Kuwano, Naoko Yamagishi, Ken Kurokawa, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of immunomodulators identifies VEGF as a potential biomarker for trait anxiety and depressive mood in healthy Japanese university students., The 7th World Congress on Stress, Leiden, Netherlands, Aug. 2010.
32.
Yuki Kuwano, Ken Kurokawa, Sakurako Katsuura, Naoko Yamagishi, Yuzuru Satake, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : Alternative splice variants of SMG-1 as a potential marker for brief naturalistic stressors in peripheral leukocytes., The 7th World Congress on Stress, Leiden, Netherlands, Aug. 2010.
33.
Sakurako Katuura, Yoshiko Kamezaki, Yuki Kuwano, Naoko Yamagishi, Ken Kurokawa, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of immunomodulators identifies VEGF as a potential biomarker for trait anxiety and depressive mood in healthy Japanese university students., The 7th World Congress on Stress, Leiden, Netherland, Aug. 2010.
34.
Sakurako Katuura, Yoshiko Kanmezaki, Yuki Kuwano, Naoko Yamagishi, Ken kurokawa, Yuzuru Satake, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of brief naturalistic stress-responsive cytokines in Japanese university students taking examinations., The 7th World Congress on Stress, Netherland, Aug. 2010.
35.
Ken Kurokawa, Toshihito Tanahashi, Tutomu Iima, Yuta Yamamoto, Kensei Nishida, Kiyoshi Masuda, Yuki Kuwano, Shigetada Kondo, M Fukushima and Kazuhito Rokutan : microRNAs regulate 5-fluorouracil resistance in human colon cancer cells, Digestive Disease Week 2010, New Orleans, May 2010.
36.
Yuki Kuwano, Yuzuru Satake, Kensei Nishida, Kiyoshi Masuda, Ken Kurokawa, Shigetada Kondo, Toshihito Tanahashi and Kazuhito Rokutan : The mechanism of transfomer 2-beta gene expression in response to oxidative stress in human colon epithelial cells, Digestive Disease Week 2010, New Orleans, May 2010.
YAMAUCHI Shoma, Ryosuke Miyamoto, MUTO Kohei, Yuki Kuwano, Keyoumu Nazere, Kensei Nishida, TACHIBANA Konoka, Yuishin Izumi and Hiroyuki Morino : Search for Pathogenic Variant of ALS by Prioritization based on Phenotype, 第64回日本神経学会学術大会, Jun. 2023.
3.
Yuki Kuwano, Kensei Nishida and Hiroyuki Morino : m6A RNA methylation of TRA2B4 suppresses cellular senescence of colon cancer, 第44回日本分子生物学会年会, Dec. 2021.
Tatsuya Nishikawa, Yuki Kuwano, 小玉 美幸, 西條 早希, 田中 裕基, 板井 美樹, 藤田 絹代, Kensei Nishida and Kazuhito Rokutan : Mechanism of alternative splicing for TRA24 and the impact on the regulation of cell cycle, 第39回日本分子生物学会年会, Dec. 2016.
S Kano, Kensei Nishida, Yuki Kuwano, Kiyoshi Masuda, K Kurokawa and Kazuhito Rokutan : Ultraconserved exon-containing SRSF3 mRNA isoform is specifically translated to the truncated SRSF3protein under oxidative stress, 第35回日本分子生物学会, Dec. 2012.
Yuki Kuwano, Manami Honda, Kinuyo Fujita, Yoko Akaike, Shizuka Kano, Yuzuru Satake, Kensei Nishida and Kazuhito Rokutan : Chronic academic stress increases a group of microRNAs in peripheral blood in healthy Japanese students., The international conference on social stratification and health 2013, Sep. 2013.