Biliary tract cancer (BTC) is an aggressive type of malignant tumour. Even after radical resection, the risk of recurrence is still high, resulting in a poor prognosis. Here, we investigated the usefulness of serum miRNAs as predictive markers of recurrence and prognosis for patients with BTC after radical surgery using 66 serum samples that were collected at three time points from 22 patients with BTC who underwent radical surgery. Using microarray analysis, we successfully identified six specific miRNAs (miR-1225-3p, miR-1234-3p, miR1260b, miR-1470, miR-6834-3p, and miR-6875-5p) associated with recurrence and prognosis of BTC after radical surgery. In addition, using a combination of these miRNAs, we developed a recurrence predictive index to predict recurrence in patients with BTC after operation with high accuracy. Patients having higher index scores (≥ cut-off) had significantly worse recurrence-free survival (RFS) and overall survival (OS) than those with lower index scores (

The D40 gene encodes a kinetochore protein that plays an essential role in kinetochore formation during mitosis. Short inhibitory RNA against D40, D40 siRNA, has been shown to deplete the D40 protein in the human cancer cell line HeLa, which harbors wild-type p53, and this activity was followed by the significant inhibition of cell growth and induction of apoptotic cell death. The p53-null cancer cell line, PC-3M-luc, is also sensitive to the significant growth inhibition and cell death induced by D40 siRNA. The growth of PC-3M-luc tumors transplanted into nude mice was inhibited by the systemic administration of D40 siRNA and the atelocollagen complex. Furthermore, D40 siRNA significantly inhibited growth and induced apoptotic cell death in a cell line with a gain-of-function (GOF) mutation in p53, MDA-MB231-luc, and also inhibited the growth of tumors transplanted into mice when administered as a D40 siRNA/atelocollagen complex. These results indicated that D40 siRNA induced apoptotic cell death in human cancer cell lines, and inhibited their growth in vitro and in vivo regardless of p53 status. Therefore, D40 siRNA is a potential candidate anti-cancer reagent.

Adhesion of cancer cells to vascular endothelial cells in target organs is an initial step in cancer metastasis. Our previous studies revealed that amphoterin-induced gene and open reading frame 2 (AMIGO2) promotes the adhesion of tumor cells to liver endothelial cells, followed by the formation of liver metastasis in a mouse model. However, the precise mechanism underlying AMIGO2-promoted the adhesion of tumor cells and liver endothelial cells remains unknown. This study was conducted to explore the role of cancer cell-derived AMIGO2-containing extracellular vesicles (EVs) in the adhesion of cancer cells to human hepatic sinusoidal endothelial cells (HHSECs). Western blotting indicated that AMIGO2 was present in EVs from AMIGO2-overexpressing MKN-28 gastric cancer cells. The efficiency of EV incorporation into HHSECs was independent of the AMIGO2 content in EVs. When EV-derived AMIGO2 was internalized in HHSECs, it significantly enhanced the adhesion of HHSECs to gastric (MKN-28 and MKN-74) and colorectal cancer cells (SW480), all of which lacked AMIGO2 expression. Thus, we identified a novel mechanism by which EV-derived AMIGO2 released from AMIGO2-expressing cancer cells stimulates endothelial cell adhesion to different cancer cells for the initiate step of liver metastasis.

Recent studies have shown that extracellular vesicles (EVs) can be utilized as appropriate and highly specific biomarkers in liquid biopsy for the diagnosis and prognosis of serious illness. However, there are few methods that can collect and isolate miRNA in EVs simply, quickly and efficiently using general equipment such as a normal centrifuge. In this paper, we developed an advanced glass membrane column (AGC) device incorporating a size-controlled macro-porous glass (MPG) membrane with a co-continuous structure to overcome the limitations of conventional EV collection and miRNA extraction from the EVs. The size of macro-pores in the MPG membrane could be accurately controlled by changing the heating temperature and time on the basis of spinodal decomposition of B2O3, Na2O, and SiO2 in phase separation. The AGC device with an MPG membrane could collect the EVs simply and quickly (< 10 min) from cell culture supernatant, serum and urine. This AGC device could extract miRNA from the EVs captured in the MPG membrane with high efficiency when combined with a miRNA extraction solution. We suggest that the AGC device with an MPG membrane can be useful for the diagnosis and prognosis of serious illness using of EVs in various kinds of body fluids.

Lymphovascular invasion (LVI) is a fundamental step toward the spread of cancer. Extracellular vesicles (EVs) promote cellular communication by shuttling cargo, such as microRNAs (miRNAs). However, whether EV-associated miRNAs serve as biomarkers for LVI remains unclear. This study aimed to identify EV-associated miRNAs related to LVI and validate the miRNA levels from patients with early-stage lung adenocarcinoma (LADC). Blood samples were collected from patients undergoing pulmonary resection for stage I LADC before surgery. The patients were classified into three groups according to the presence of LVI and postoperative recurrence. Serum-derived EVs in the derivation cohort were used for small RNA sequencing, while the selected LVI miRNA candidates were validated via real-time quantitative polymerase chain reaction using 44 patient and 16 healthy donor samples as the validation cohorts. Five miRNAs (miR-99b-3p, miR-26a-5p, miR-93-5p, miR-30d-5p, and miR-365b-3p) were assessed, and miR-30d-5p (p = 0.036) levels were significantly downregulated in the LVI-positive group. miR-30d-5p levels in healthy donors were lower than those in LADC patients. Patients with high miR-30d-5p levels had favorable survival compared to those with low miR-30d-5p levels. miR-30d-5p level in EVs may serve as a promising biomarker for detecting LVI in patients with early-stage LADC.

Extracellular vesicles (EVs) play an important role in the transfer of biomolecules between cells. To elucidate the intercellular transfer fate of EVs in vivo, we generated a new transgenic (Tg) rat model using green fluorescent protein (GFP)-tagged human CD63. CD63 protein is highly enriched on EV membranes via trafficking into late endosomes and is often used as an EV marker. The new Tg rat line in which human CD63-GFP is under control of the CAG promoter exhibited high expression of GFP in various body tissues. Exogenous human CD63-GFP was detected on EVs isolated from three body fluids of the Tg rats: blood serum, breast milk and amniotic fluid. In vitro culture allowed transfer of serum-derived CD63-GFP EVs into recipient rat embryonic fibroblasts, where the EVs localized in endocytic organelles. These results suggested that this Tg rat model should provide significant information for understanding the intercellular transfer and/or mother-child transfer of EVs in vivo.

It is well known that natural products are a rich source of compounds for applications in medicine, pharmacy, and biology. However, the exact molecular mechanisms of natural agents in human health have not been clearly defined. Here, we demonstrate for the first time that the polyphenolic phytoalexin resveratrol promotes expression and activity of Argonaute2 (Ago2), a central RNA interference (RNAi) component, which thereby inhibits breast cancer stem-like cell characteristics by increasing the expression of a number of tumour-suppressive miRNAs, including miR-16, -141, -143, and -200c. Most importantly, resveratrol-induced Ago2 resulted in a long-term gene silencing response. We also found that pterostilbene, which is a natural dimethylated resveratrol analogue, is capable of mediating Ago2-dependent anti-cancer activity in a manner mechanistically similar to that of resveratrol. These findings suggest that the dietary intake of natural products contributes to the prevention and treatment of diseases by regulating the RNAi pathway.

There are many subtypes of dementia, and identification of diagnostic biomarkers that are minimally-invasive, low-cost, and efficient is desired. Circulating microRNAs (miRNAs) have recently gained attention as easily accessible and non-invasive biomarkers. We conducted a comprehensive miRNA expression analysis of serum samples from 1348 Japanese dementia patients, composed of four subtypes-Alzheimer's disease (AD), vascular dementia, dementia with Lewy bodies (DLB), and normal pressure hydrocephalus-and 246 control subjects. We used this data to construct dementia subtype prediction models based on penalized regression models with the multiclass classification. We constructed a final prediction model using 46 miRNAs, which classified dementia patients from an independent validation set into four subtypes of dementia. Network analysis of miRNA target genes revealed important hub genes, SRC and CHD3, associated with the AD pathogenesis. Moreover, MCU and CASP3, which are known to be associated with DLB pathogenesis, were identified from our DLB-specific target genes. Our study demonstrates the potential of blood-based biomarkers for use in dementia-subtype prediction models. We believe that further investigation using larger sample sizes will contribute to the accurate classification of subtypes of dementia.

The lack of useful biomarkers is a crucial problem for patients with soft tissue sarcomas (STSs). Emerging evidence has suggested that circulating microRNAs (miRNAs) in body fluids have novel impact as biomarkers for patients with malignant diseases, but their significance in synovial sarcoma (SS) patients remains unknown. Initial global miRNA screening using SS patient serum and SS cell culture media identified a signature of four upregulated miRNAs. Among these candidates, miR-92b-3p secretion from SS cells was confirmed, which was embedded within tumour-derived exosomes rather than argonaute-2. Animal experiments revealed a close correlation between serum miR-92b-3p levels and tumour dynamics. Clinical relevance was validated in two independent clinical cohorts, and we subsequently identified that serum miR-92b-3p levels were significantly higher in SS patients in comparison to that in healthy individuals. Moreover, serum miR-92b-3p was robust in discriminating patients with SS from the other STS patients and reflected tumour burden in SS patients. Overall, liquid biopsy using serum miR-92b-3p expression levels may represent a novel approach for monitoring tumour dynamics of SS.

Dysfunction of the p53 network is a major cause of cancer development, and selective elimination of p53-inactivated cancer cells therefore represents an ideal therapeutic strategy. In this study, we performed a microRNA target screen that identified NEK9 (NIMA-related kinase 9) as a crucial regulator of cell-cycle progression in p53-inactivated cancer cells. NEK9 depletion selectively inhibited proliferation in p53-deficient cancer cells both in vitro and in vivo. The resultant cell-cycle arrest occurred predominantly in G1 phase, and exhibited senescence-like features. Furthermore, NEK9 repression affected expression of a broad range of genes encoding cell-cycle regulators and factors involved in mRNA processing, suggesting a novel role for NEK9 in p53-deficient cells. Lung adenocarcinoma patients with positive staining for NEK9 and mutant p53 proteins exhibited significantly poorer prognoses, suggesting that expression of both proteins promotes tumor growth. Our findings demonstrate that a novel NEK9 network regulates the growth of cancer cells lacking functional p53.

Mutant p53 (mtp53) gain of function (GOF) contributes to various aspects of tumor progression including cancer stem cell (CSC) property acquisition. A key factor of GOF is stabilization and accumulation of mtp53. However, the precise molecular mechanism of the mtp53 oncogenic activity remains unclear. Here, we show that ribophorin II (RPN2) regulates CSC properties through the stabilization of mtp53 (R280K and del126-133) in breast cancer. RPN2 stabilized mtp53 by inactivation of glycogen synthase kinase-3β (GSK3β) which suppresses Snail, a master regulator of epithelial to mesenchymal transition. RPN2 knockdown promoted GSK3β-mediated suppression of heat shock proteins that are essential for mtp53 stabilization. Furthermore, our study reveals that high expression of RPN2 and concomitant accumulation of mtp53 were associated with cancer tissues in a small cohort of metastatic breast cancer patients. These findings elucidate a molecular mechanism for mtp53 stabilization and suggest that RPN2 could be a promising target for anti-CSC therapy.

Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma worldwide. However, the strategy of HBV to escape from the host immune system remains largely unknown. In this study, we examined extracellular vesicles (EVs) secreted from human hepatocytes infected with HBV. EVs includeing exosomes are nano-size vesicles with proteins, mRNAs, and microRNAs (miRNAs), which can be transmitted to different cells. We found that 104 EV associated miRNAs were increased in hepatocytes more than 2-fold by HBV infection. We then selected those that were potentially implicated in immune regulation. Among them, five HBV-induced miRNAs were found to potentially target multiple sequences in the 3'UTR of IL-21, a cytokine that induces anti-viral immunity. Moreover, expression of a reporter gene with the 3' UTR of human IL-21 mRNA was suppressed by the five miRNAs individually. Finally, IL-21 expression in cloned human T cells was down-regulated by the five miRNAs. Collectively, this study identified the novel 3' UTR sequences of human IL-21 mRNA and potential binding sites of HBV-induced EV-miRNAs.

Certain dietary agents, such as natural products, have been reported to show anti-cancer effects. However, the underlying mechanisms of these substances in human cancer remain unclear. We recently found that resveratrol exerts an anti-cancer effect by upregulating tumour-suppressor microRNAs (miRNAs). In the current study, we aimed to identify new dietary products that have the ability to activate tumour-suppressor miRNAs and that therefore may serve as novel tools for the prevention and treatment of human cancers. We describe the generation and use of an original screening system based on a luciferase-based reporter vector for monitoring miR-200c tumour-suppressor activity. By screening a library containing 139 natural substances, three natural compounds - enoxolone, magnolol and palmatine chloride - were identified as being capable of inducing miR-200c expression in breast cancer cells at 10 μM. Moreover, these molecules suppressed the invasiveness of breast cancer cells in vitro. Next, we identified a molecular pathway by which the increased expression of miR-200c induced by natural substances led to ZEB1 inhibition and E-cadherin induction. These results indicate that our method is a valuable tool for a fast identification of natural molecules that exhibit tumour-suppressor activity in human cancer through miRNA activation.

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are liver originated malignant tumors. Of the two, ICC has the worse prognosis because it has no reliable diagnostic markers and its carcinogenic mechanism is not fully understood. The aim of this study was to integrate metabolomics and transcriptomics datasets to identify variances if any in the carcinogenic mechanism of ICC and HCC. Ten ICC and 6 HCC who were resected surgically, were enrolled. miRNA and mRNA expression analysis were performed by microarray on ICC and HCC and their corresponding non-tumor tissues (ICC_NT and HCC_NT). Compound analysis was performed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Principle component analysis (PCA) revealed that among the four sample groups (ICC, ICC_NT, HCC, and HCC_NT) there were 14 compounds, 62 mRNAs and 17 miRNAs with two distinct patterns: tumor and non-tumor, and ICC and non-ICC. We accurately (84.38%) distinguished ICC by the distinct pattern of its compounds. Pathway analysis using transcriptome and metabolome showed that several pathways varied between tumor and non-tumor samples. Based on the results of the PCA, we believe that ICC and HCC have different carcinogenic mechanism therefore knowing the specific profile of genes and compounds can be useful in diagnosing ICC.

Small interfering RNA (siRNA)-based therapeutics have been used in humans and offer distinct advantages over traditional therapies. However, previous investigations have shown that there are several technical obstacles that need to be overcome before routine clinical applications are used. Currently, we are launching a novel class of RNAi therapeutic agents (PnkRNA™, nkRNA) that show high resistance to degradation and are less immunogenic, less cytotoxic, and capable of efficient intracellular delivery. Here, we develop a novel platform to promote naked RNAi approaches administered through inhalation without sophisticated delivery technology in mice. Furthermore, a naked and unmodified novel RNAi agent, such as ribophorin II (RPN2-PnkRNA), which has been selected as a therapeutic target for lung cancer, resulted in efficient inhibition of tumor growth without any significant toxicity. Thus, this new technology using aerosol delivery could represent a safe, potentially RNAi-based strategy for clinical applications in lung cancer treatment without delivery vehicles.

Gene targeting in embryonic stem cells (ESCs) has become the principal technology for generating knockout models. Although numerous studies have predicted that the disruption of p53 leads to increased developmental anomalies and malignancies, most p53 knockout mice develop normally. Therefore, the role of p53 in animal development was examined using rat knockout models. Conventionally generated homozygous KO males developed normally, whereas females rarely survived due to neural tube defects. Mutant chimeras generated via blastocyst injection with p53-null ESCs exhibited high rates of embryonic lethality in both sexes. This phenotype could be observed in one month by the use of zinc-finger nucleases. The p53-null ESCs were resistant to apoptosis and differentiation, and exhibited severe chromosome instabilities in the chimera-contributed cells, suggesting an essential role for p53 in maintaining ESC quality and genomic integrity. These results demonstrate that p53 functions as a guardian of embryogenesis in the rats.

Alzheimer's disease (AD) is characterized by the accumulation of β-amyloid peptide (Aβ) in the brain because of an imbalance between Aβ production and clearance. Neprilysin (NEP) is the most important Aβ-degrading enzyme in the brain. Thus, researchers have explored virus-mediated NEP gene delivery. However, such strategies may entail unexpected risks, and thus exploration of a new possibility for NEP delivery is also required. Here, we show that human adipose tissue-derived mesenchymal stem cells (ADSCs) secrete exosomes carrying enzymatically active NEP. The NEP-specific activity level of 1 μg protein from ADSC-derived exosomes was equivalent to that of ~ 0.3 ng of recombinant human NEP. Of note, ADSC-derived exosomes were transferred into N2a cells, and were suggested to decrease both secreted and intracellular Aβ levels in the N2a cells. Importantly, these characteristics were more pronounced in ADSCs than bone marrow-derived mesenchymal stem cells, suggesting the therapeutic relevance of ADSC-derived exosomes for AD.

Since liver metastasis is the main cause of death in cancer patients, we attempted to identify the driver gene involved. QRsP-11 fibrosarcoma cells were injected into the spleens of syngeneic mice to isolate tumour sub-populations that colonize the liver. Cells from liver metastatic nodules were established and subsequently injected intrasplenically for selection. After 12 cycles, the cell subline LV12 was obtained. Intravenous injection of LV12 cells produced more liver metastases than QRsP-11 cells, whereas the incidence of lung metastases was similar to that of QRsP-11 cells. LV12 cells adhered to liver-derived but not to lung-derived endothelial cells. DNA chip analysis showed that amphoterin-induced gene and open reading frame 2 (Amigo2) was overexpressed in LV12 cells. siRNA-mediated knockdown of Amigo2 expression in LV12 cells attenuated liver endothelial cell adhesion. Ex vivo imaging showed that suppression of Amigo2 in luciferase-expressing LV12 cells reduced attachment/metastasis to liver to the same level as that observed with QRsP-11 cells. Forced expression of Amigo2 in QRsP-11 cells increased liver endothelial cell adhesion and liver metastasis. Additionally, Amigo2 expression in human cancers was higher in liver metastatic lesions than in primary lesions. Thus, Amigo2 regulated tumour cell adhesion to liver endothelial cells and formation of liver metastases.

Hepatocellular carcinoma (HCC) is a typical hyper-vascular tumor, so the understanding the mechanisms of angiogenesis in HCC is very important for its treatment. However, the influence of the exosomes secreted from HCC cells (HCC-exosomes) on angiogenesis remains poorly understood. We herein examined the effects of the exosomes secreted from HepG2 cells (HepG2-exosomes) on the lumen formation of human umbilical vein endothelial cells (HUVECs) by the imaging of angiogenesis. The degree of lumen formation of HUVECs was dependent on the number of HepG2-exosomes. The HepG2-exosomes expressed NKG2D, an activating receptor for immune cells, and HSP70, a stress-induced heat shock protein associated with angiogenesis through the vascular endothelial growth factor (VEGF) receptor. In addition, the HepG2-exosomes contained several microRNAs (miRNAs) reported to exist in the serum of HCC patients. These results suggest that the HCC-exosomes play an important role in angiogenesis. Further studies on the function of HCC-exosomes may provide a new target for HCC treatment.

Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs acquire drug resistance with the upregulation of P-glycoprotein (P-gp, ABCB1), contrary to traditional assumptions. Furthermore, P-gp expression was higher in TECs of highly metastatic tumors than in those of low metastatic tumors. However, the detailed mechanism of differential P-gp expression in TECs remains unclear. miRNA was identified in highly metastatic tumor extracellular vesicles (EVs) and the roles of miRNA in endothelial cell resistance were analyzed in vitro and in vivo. In the present study, we found that treatment of highly metastatic tumor-conditioned medium induced resistance to 5-fluorouracil (5-FU) with interleukin-6 (IL-6) upregulation in endothelial cells (ECs). Among the soluble factors secreted from highly metastatic tumors, we focused on EVs and determined that miR-1246 was contained at a higher level in highly metastatic tumor EVs than in low metastatic tumor EVs. Furthermore, miR-1246 was transported via the EVs into ECs and induced IL-6 expression. Upregulated IL-6 induced resistance to 5-FU with STAT3 and Akt activation in ECs in an autocrine manner. These results suggested that highly metastatic tumors induce drug resistance in ECs by transporting miR-1246 through EVs.