circRNA: A New Biomarker and Therapeutic Target for Esophageal Cancer.

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.

Multiple G-quadruplexes in the LMNA promoter regulate LMNA variant 6 transcription and promote colon cancer cell growth.

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.

Overexpression of the transcribed ultraconserved region Uc.138 accelerates colon cancer progression.

Ultraconserved regions (UCRs) are 481 genomic sequences with 100% identity across humans, rats, and mice. Increasing evidence suggests that non-coding RNAs transcribed from UCRs are involved in various diseases, especially cancers. The human transformer 2ß gene (TRA2B) encodes a UCR (uc.138) that spans exon 2 and its neighboring introns. TRA2B4 RNA is the only transcript that contains the whole exon 2 among five spliced TRA2B RNA variants (TRA2B1-5). TRA2B4 is upregulated in colon cancer cell lines, although it is not translated to Tra2ß protein because of its nuclear retention. Nevertheless, the clinical significance and biological functions of uc.138 in colon cancer cells remain unclear. In this study, RNA in situ hybridization showed that TRA2B4 was predominantly overexpressed in the nucleus of colon adenocarcinoma and adenoma. Overexpression of TRA2B4 in colon cancer HCT116 cells promoted cell proliferation by changing the expression of G2/M-related cell cycle regulators. Moreover, TRA2B4 increased migration and cell viability in a uc.138 sequence-dependent manner. TRA2B4 significantly enhanced tumorigenesis in vivo. Taken together, uc.138 encoded in TRA2B4 plays an oncogenic role in tumor progression and may become a potential biomarker and therapeutic target in colon cancer.

The MicroRNA-23b/27b/24 Cluster Facilitates Colon Cancer Cell Migration by Targeting FOXP2.

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 miR-23b/27b/24 cluster increased in accordance with the increased expression of the short C9orf3 transcript, a host gene of the miR-23b/27b/24 cluster. E2F1-binding sequences were involved in the basic transcription activity of the short C9orf3 expression, and E2F1-small-interfering (si)RNA treatment reduced the expression of both the C9orf3 and miR-23b/27b/24 clusters. Overexpression experiments showed that miR-23b and miR-27b promoted cell migration, but the opposite effect was observed with miR-24. Forkhead box P2 (FOXP2) mRNA and protein levels were reduced by both/either miR-23b and miR-27b. Furthermore, FOXP2 siRNA treatment significantly promoted cell migration. Our findings demonstrated a novel role of the miR-23b/27b/24 cluster in cell migration through targeting FOXP2, with potential implications for the development of microRNA-based therapy targeted at inhibiting cancer migration.

HnRNPA1 interacts with G-quadruplex in the TRA2B promoter and stimulates its transcription in human colon cancer cells.

The human TRA2B gene consists of 10 exons and 9 introns and produces 5 splice isoforms (TRA2ß1 to TRA2ß5). TRA2B exon 2 encodes multiple premature termination codons. TRA2ß1 lacks exon 2 and is translated into a functional transformer 2ß (Tra2ß) protein, whereas TRA2ß4 contains 10 exons and works as a functional RNA. Overexpressed Tra2ß and ectopic expression of TRA2ß4 may be oncogenic. We found that heterogeneous nuclear ribonucleoprotein (hnRNP)A1 and hnRNPU interacted with TRA2ß4 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 TRA2ß1 and TRA2ß4 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.

A novel long non-coding RNA from the HOXA6-HOXA5 locus facilitates colon cancer cell growth.

BACKGROUND: Homeobox A5 (HOXA5), a member of the HOX family, plays an important role in tumor development and morphogenesis, although opposite effects on tumorigenesis have been observed, depending on the tissue type. In this study, we aimed to investigate the role of a novel transcript from the HOXA6-HOXA5 locus in colon cancer tumorigenesis. METHODS: Human colon cancer cell lines were analyzed using next generation sequencing-based targeted mRNA capture. The effects of overexpression and silencing of HOXA5 transcripts were evaluated in vitro and using a xenograft nude mouse model. RESULTS: We identified three novel transcripts (HOXA5 short, long 1, and long 2) transcribed from the HOXA6-HOXA5 locus in HCT116 colon cancer cells using next generation sequencing-based targeted mRNA capture. Knockdown of HOXA5 long 1 and long 2 transcripts did not affect cell growth, while selective silencing of HOXA5 short RNA inhibited cell growth independent of HOXA5 expression. Stable overexpression of HOXA5 short RNA promoted proliferation and migration of colon cancer cell lines HCT116, DLD1, and HT-29 and accelerated tumor growth in the xenograft mouse model. In vitro translation assays suggested HOXA5 short RNA was a functional long non-coding RNA (lncRNA). Consistent with these observations, expression of HOXA5 short RNA was upregulated in advanced colon cancer tissues. Ingenuity Pathway Analysis of differentially expressed genes between HOXA5 short RNA overexpressed and silenced HCT116 cells revealed that HOXA5 short RNA preferentially modified expression of epidermal growth factor (EGF) signal-related genes. Western blot analysis demonstrated that stable overexpression of HOXA5 short RNA increased EGF receptor levels and facilitated its phosphorylation in both HCT116 cells and xenograft tumors. CONCLUSIONS: Our results suggested that HOXA5 short RNA, a novel lncRNA, may play a crucial role in colon tumor growth through activation of EGF signaling.

Diverse roles of RNA-binding proteins in cancer traits and their implications in gastrointestinal cancers.

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.

Nucleolin facilitates nuclear retention of an ultraconserved region containing TRA2ß4 and accelerates colon cancer cell growth.

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 transformer 2ß gene (TRA2B) encodes a UCR that spans exon 2 (276 bp) and its neighboring introns. Among five spliced RNA variants (TRA2ß1-5) transcribed from the TRA2B gene, only TRA2ß4 contains the conserved exon 2. TRA2ß4 is overexpressed in colon cancer cells and accelerates cell growth by blocking the transcription of CDKN1A. However, the mechanisms underlying the overexpression of TRA2ß4 in colon cancer cells are unknown. Using biotinylated RNA pull-down assays followed by liquid chromatography-mass spectrometric analysis, we identified nucleolin as a TRA2ß4-binding protein. Knockdown of nucleolin reduced the nuclear retention of TRA2ß4 and accelerated its degradation in the cytoplasm, whereas nucleolin overexpression increased TRA2ß4 levels and its mitogenic activity. Nucleolin directly bound to TRA2ß4 exon 2 via the glycine/arginine-rich (GAR) domain. Overexpression of GAR-deficient nucleolin failed to increase TRA2ß4 expression and growth of colon cancer cells. RNA fluorescence in situ hybridization showed that TRA2ß4 co-localized with nucleolin in nuclei but not with the mutant lacking GAR. Our results suggest that specific interactions between nucleolin and UCR-containing TRA2ß4 may be associated with abnormal growth of colon cancer cells.

ZNF350 promoter methylation accelerates colon cancer cell migration.

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.

Homeodomain-Interacting Protein Kinase-2: A Critical Regulator of the DNA Damage Response and the Epigenome.

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 1γ. In this review, we will highlight the molecular mechanisms of HIPK2 and show its functions as a crucial DDR regulator.

Serine/arginine-rich splicing factor 7 regulates p21-dependent growth arrest in colon cancer cells.

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.

HuR regulates alternative splicing of the TRA2ß gene in human colon cancer cells under oxidative stress.

Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2ß gene encodes splicing factor transformer 2ß (Tra2ß) and generates 5 mRNA isoforms (TRA2ß1 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 TRA2ß exon 2, generating a TRA2ß4 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 TRA2ß4 and increased Tra2ß protein, facilitating Tra2ß-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38(MAPK) double knockdown were also confirmed using a TRA2ß 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 TRA2ß4 interaction and TRA2ß4 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA2ß4-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress.

Oxidative stress-inducible truncated serine/arginine-rich splicing factor 3 regulates interleukin-8 production in human colon cancer cells.

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.

Truncated serine/arginine-rich splicing factor 3 accelerates cell growth through up-regulating c-Jun expression.

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.

NF90 in posttranscriptional gene regulation and microRNA biogenesis.

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.

Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.

Ets1 and heat shock factor 1 regulate transcription of the Transformer 2ß gene in human colon cancer cells.

BACKGROUND: Transformer (Tra) 2ß 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 Tra2ß expression, while the regulatory mechanism of Tra2ß expression remains to be elucidated. In this study, we examined the transcriptional regulation and possible functions of Tra2ß in human colon cancer cells. METHODS: We cloned 959 bp-upstream of the human TRA2ß 5'-flank into luciferase constructs. Chromatin immunoprecipitation (ChIP) was employed to identify crucial cis element(s) and trans activator(s) of the TRA2ß promoter. Tra2ß expression in the human colon and colon cancer tissues was examined by immunohistochemistry. RESULTS: In response to sodium arsenite, colon cancer cells (HCT116) increased levels of TRA2ß1 mRNA encoding a functional, full-length Tra2ß with a peak around 6 h without changing its mRNA stability. Transient expression assays using a reporter gene driven by serially truncated TRA2ß 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 TRA2ß. Tra2ß knockdown caused apoptosis of HCT116 cells. Tra2ß were preferentially expressed in proliferative compartment of normal human colonic glands and adenocarcinomas, where Ets1 and heat shock factor 1 were also highly expressed. CONCLUSIONS: Our results suggest that oxidative stress-responsive Tra2ß may play an important role in colon cancer growth.

MicroRNAs miR-144/144* and miR-16 in peripheral blood are potential biomarkers for naturalistic stress in healthy Japanese medical students.

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)-α 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-γ and/or TNF-α 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.

Circulating cytokine signatures in healthy medical students exposed to academic examination stress.

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-γ, interleukin (IL)-1ß, and tumor necrosis factor-α), Th2 cytokines (IL-4, IL-5, and IL-13), and ß-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.

Role of miR-19b and its target mRNAs in 5-fluorouracil resistance in colon cancer cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.