J. Youn

MicroRNA and gene expression analysis of melatonin-exposed human breast cancer cell lines indicating involvement of the anticancer effect.

Abstract:  MicroRNAs (miRNAs) are small, noncoding RNAs that play a crucial role in regulation of gene expression. Recent studies have shown that miRNAs implicated in initiation and progression of various human cancers, including breast cancer and also analysis of miRNA expression profiles in cancer provide new insights into potential mechanisms of carcinogenesis. Melatonin, N‐acetyl‐5‐methoxytryptamine, is synthesized by the pineal gland in response to the dark/light cycle and has been known to act as a synchronizer of the biological clock. Melatonin has a variety of therapeutic effects, such as immunomodulatory actions, anti‐inflammatory effects, and antioxidant actions. Furthermore, melatonin is reported to have an anticancer function including suppression of the metabolism of tumor cells and induction of tumor suppressor genes in cancer cells, including breast cancer cells. In this study, we determined whether miRNAs play a role in regulation of various gene expression responses to melatonin in MCF‐7 human breast cancer cells. We examined whole‐genome miRNA and mRNA expression and found that 22 miRNAs were differentially expressed in melatonin‐treated MCF‐7 cells. We further identified a number of mRNAs whose expression level shows a high inverse correlation with miRNA expression. The Gene Ontology (GO) enrichment analysis and pathways analysis were performed for identification of the signaling pathways and biological processes affected by differential expression of miRNA and miRNA‐related genes. Our findings suggested that melatonin may modulate miRNA and gene expression as an anticancer mechanism in human breast cancer cells.

NF-κB Activation Pathway is Essential for the Chemokine Expression in Intestinal Epithelial Cells Stimulated with Clostridium difficile Toxin A

Intestinal epithelial cells are known to upregulate the expression of several chemokines in response to stimulation with bacterial toxin. However, the cellular mechanisms of Clostridium difficile toxin A‐induced mucosal inflammation have not yet been fully elucidated. In this study, we investigated whether nuclear factor‐kappa B (NF‐κB) could regulate chemokine expression in intestinal epithelial cells. Toxin A increased the levels of NF‐κB complexes containing p65/p50 heterodimers and p65/p65 homodimers. Concurrently, toxin A decreased the levels of IκBα. Toxin A stimulation also increased the signals of phosphorylated IκB kinase (IKK)α/β and NF‐κB‐inducing kinase (NIK). In the toxin A‐stimulated HT‐29 cells, the suppression of IKK or NIK inhibited the upregulation of downstream target genes of NF‐κB such as IL‐8 and monocyte‐chemotactic protein (MCP)‐1 and similarly, inhibition of NF‐κB also downregulated the expression of IL‐8, growth‐related oncogene‐α, and MCP‐1. These results suggest that NF‐κB signalling events may be involved in the inflammatory responses to toxin A produced by toxigenic C. difficile.

Induction of proinflammatory mediators requires activation of the TRAF, NIK, IKK and NF-κB signal transduction pathway in astrocytes infected with Escherichia coli

Escherichia coli is associated with inflammation in the brain. To investigate whether astrocytes are involved in E. coil‐induced inflammation, we assessed the levels of expression of proinflammatory mediators produced by E. coli‐infected astrocytes. E. coli infection in primary human astrocytes and cell lines increased expression of the CXC chemokine IL‐8/GRO‐α, the CC chemokine MCP‐1, TNF‐α, and iNOS. E. coli infection activated p65/p50 heterodimeric NF‐κB and concurrently decreased the signals of IκBα. Blocking the NF‐κB signals by IκBα‐superrepressor‐containing retrovirus or antisense p50 oligonucleotide transfection resulted in down‐regulation of expression of the proinflammatory mediators. Furthermore, superrepressors of IκBα, IκB kinase (IKK) or NF‐κB inducing kinase (NIK) inhibited the up‐regulated expression of the downstream target genes of NF‐κB such as IL‐8 and MCP‐1, and superrepressors of TNF receptor‐associated factor (TRAF)2 and TRAF5 also inhibited expression of the E. coli‐induced target genes of NF‐κB. These results indicate that proinflammatory mediators such as the CXC chemokine IL‐8/GRO‐α, the CC chemokine MCP‐1, TNF‐α, and iNOS can be expressed in E. coli‐infected astrocytes via an NF‐κB pathway, suggesting that these mediators may contribute to inflammation in the brain, including infiltration of inflammatory cells.

Escherichia coli up-regulates proinflammatory cytokine expression in granulocyte/macrophage lineages of CD34+ stem cells via p50 homodimeric NF-κB

Umbilical cord blood has emerged as an alternative source of haematopoietic CD34+ cells for allogeneic stem cell transplantation. Although bacteraemia induced by Escherichia coli is considered one of the complications of transplantation, expression of proinflammatory cytokines is poorly understood. In this study, we report the altered expression of proinflammatory cytokines in CD34+ cells and their in vitro cultured cells following E. coli infection. CD34+ stem cells and their cultured cells up‐regulated expression of proinflammatory cytokines such as interleukin (IL)‐1α, IL‐6, IL‐8 and tumour necrosis factor (TNF)‐α after infection with E. coli. Expression of the proinflammatory cytokines was generated mainly by the granulocyte‐macrophage lineages. E. coli infection activated the signals of p50/p50 nuclear factor‐kappaB (NF‐κB) homodimers and IκB kinase. Furthermore, inhibition of NF‐κB activation lowered the up‐regulated expression of the proinflammatory cytokines. These results suggest that CD34+ cells and their cultured cells infected with E. coli induce the expression of proinflammatory cytokines via the NF‐κB pathway.

Rebamipide attenuates autoimmune arthritis severity in SKG mice via regulation of B cell and antibody production.

Oxidative stress is involved in the pathophysiology of rheumatoid arthritis (RA). We investigated the therapeutic potential of rebamipide, a gastroprotective agent with a property of reactive oxygen species scavenger, on the development of inflammatory polyarthritis and the pathophysiological mechanisms by which rebamipide might confer anti‐arthritic effects in SKG mice, an animal model of RA. Intraperitoneal (i.p.) injection of rebamipide attenuated the severity of clinical and histological arthritis. Rebampide treatment reduced the number of T helper type 1 (Th1), Th2, Th17, inducible T cell co‐stimulator (ICOS)+ follicular helper T (Tfh) transitional type (T2) and mature B cells in the spleen, but increased the number of regulatory T (Treg), CD19+ CD1dhigh CD5high, CD19+ CD25high forkhead box protein 3 (FoxP3)+ regulatory B (Breg) cells, memory B cells, and transitional type 1 (T1) B cells. In addition, flow cytometric analysis revealed significantly decreased populations of FAS+GL‐7+ germinal centre B cells and B220− CD138+ plasma cells in the spleens of rebamipide‐treated SKG mice compared to controls. Rebamipide decreased germinal centre B cells and reciprocally induced Breg cells in a dose‐dependent manner in vitro. Rebamipide‐induced Breg cells had more suppressive capacity in relation to T cell proliferation and also inhibited Th17 differentiation from murine CD4+ T cells. Together, these data show that i.p. administration of rebamipide suppresses arthritis severity by inducing Breg and Treg cells and suppressing Tfh and Th17 cells in a murine model of RA.

Bacillus-derived poly-γ-glutamic acid reciprocally regulates the differentiation of T helper 17 and regulatory T cells and attenuates experimental autoimmune encephalomyelitis

Forkhead box protein 3 (FoxP3+) regulatory T (Treg) cells and interleukin (IL)‐17‐producing T helper 17 (Th17) cells have opposing effects on autoimmunity, as the former are crucial for maintaining self‐tolerance while the latter play a key role in precipitating inflammatory autoimmune diseases. Here we report that Bacillus‐derived poly‐γ‐glutamic acid (γ‐PGA) signals naive CD4+ T cells to promote the selective differentiation of Treg cells and to suppress the differentiation of Th17 cells. The γ‐PGA inducibility of FoxP3 expression was due partially to transforming growth factor (TGF)‐β induction through a Toll‐like receptor (TLR)‐4/myeloid differentiating factor 88 (MyD88)‐dependent pathway. However, this pathway was dispensable for γ‐PGA suppression of Th17 differentiation. γ‐PGA inhibited IL‐6‐driven induction of Th17‐specific factors including signal transducer and activator of transcription‐3 (STAT‐3) and retinoic acid‐related orphan receptor γt (RORγt) while up‐regulating the STAT‐3 inhibitor suppressor of cytokine signalling 3 (SOCS3). Importantly, in vivo administration of γ‐PGA attenuated the symptoms of experimental autoimmune encephalomyelitis and at the same time reduced Th17 cell infiltrates in the central nervous system. Thus, we have identified the microbe‐associated molecular pattern, γ‐PGA, as a novel regulator of autoimmune responses, capable of promoting the differentiation of anti‐inflammatory Treg cells and suppressing the differentiation of proinflammatory Th17 cells. These findings draw attention to the potential of γ‐PGA for treating Th17 cell‐mediated autoimmune diseases.

New application methods for chromosomal abnormalities screening test using digital PCR

The probability of hereditary diseases occurring in the fetus and the childbearing age of the recently-married population have both rapidly increased over the last 10 years. That occupies a large part of such genetic diseases was greater than or equal to the number of chromosomes and genes, the probability of these chromosomal abnormalities occurring increases as the mother’s age increases. Regarding these genetic diseases that are caused by chromosomal abnormalities, it has been possible to reduce the pain of the mother and child if the abnormalities are detected early because schemes can be prepared to address them; therefore, early diagnosis is important. Recently, as part of an analysis that involved the use of very small samples, a digital PCR that allowed for the simultaneous processing of a number of samples was actively developed due to the corresponding advantage. Digital-PCR technology means that a real-time diagnosis with a high sensitivity can be obtained, and it enables the development of a variety of diagnostic techniques-without requiring the use of NGS deep sequencing- through advantages such as digitized measurement results. After extracting the circulating nucleic acid from maternal blood for the screening of fetal DNA, after first confirming that the blood contained fetal DNA, the nucleic acid was selectively applied to the digital PCR for the detection of chromosomal abnormalities, thereby developing a new detection application. The results of 43 samples were analyzed, and the experimental results of the newly developed analytical method show that 9 samples are “high risk,” whereas 34 samples are “low risk.” We consider the accuracy of each test as very reliable. The non-invasive prenatal-screening methods that use digital PCR were developed in this research study due to their lower monetary costs that are derived from an experimental testing time that is less than that of the conventional NGS; provided that the mother is easily accessible, this method can confirm the health status of a fetus. Also, the biomarker knowledge and the analysis methods that were developed in this research study can be applied in chromosomal-abnormality tests. It is expected that the development of a non-invasive examination method to replace the existing invasive testing method will be useful in the improvement of maternal and fetal safety.