Exosomes are microvesicles that are released from various cells into the extracellular space. It has been reported that the components within exosomes vary according to the type of secreted cell. In the present study, we investigated the tetraspanin family proteins CD63, CD9 and CD81 as useful collection markers of exosomes derived from the three colorectal cancer (CRC) cell lines HCT-15, SW480 and WiDr. In addition, we aimed to detect the mRNAs, microRNAs and natural antisense RNAs within the exosomes secreted from the three CRC cell lines. Furthermore, we examined whether exosomes containing their RNAs were transferred into the hepatoma cell line HepG2 and lung cancer cell line A549. CD81 was detected in exosomes secreted from the three CRC cell lines. This result indicates that CD81 can be a collection marker of exosomes derived from the three CRC cell lines. When the RNA species within exosomes derived from the three CRC cell lines were examined, the mRNAs of housekeeping genes such as ACTB and GAPDH, the microRNAs such as miR-21, miR-192 and miR-221, and the natural antisense RNAs of LRRC24, MDM2 and CDKN1A genes, were detected. We discovered their natural antisense RNAs within exosomes for the first time in the present study. Furthermore, PKH67-labeled exosomes derived from the CRC cell lines were taken up into HepG2 and A549 cells. These findings indicate that the intracellular RNAs enclosed within exosomes are secreted to the outside, and exosomes derived from the CRC cell lines are transferred into HepG2 and A549 cells. In conclusion, we reveal that exosomes derived from the CRC cell lines contain mRNAs, microRNAs and natural antisense RNAs, and can be delivered into HepG2 and A549 cells. These findings indicate that exosomal RNAs can shuttle between cells, and may be involved in the regulation of gene expression in recipient cells.

Pancreatic cancer has the lowest 5 year survival rate among all cancers. Several extracellular factors are involved in the development and metastasis of pancreatic cancer to distant organs. Exosomes are lipid-bilayer, membrane-enclosed nanoparticles that are recognised as important mediators of cell-to-cell communications. However, the role of exosomes released from pancreatic cancer cells in tumour micro-environment remains unknown. Here, we show that exosomes released from pancreatic cancer PK-45H cells activate various gene expressions in human umbilical vein endothelial cells (HUVECs) byin vitroanalyses. In addition, these exosomes released from PK-45H cells promote phosphorylation of Akt and ERK1/2 signalling pathway molecules and tube formation via dynamin-dependent endocytosis in HUVECs. Our findings suggested that exosomes released from pancreatic cancer cells may act as a novel angiogenesis promoter.

Hepatocyte-specific Phosphatase and tensin homolog (Pten)-knockout (KO) mice exhibit hepatic lesions analogous to non-alcoholic steatohepatitis (NASH). 1,8-cineole is a monoterpene oxide and it has several biological effects including hepatoprotective effects. In this study we revealed that 1,8-cineole ameliorates NASH of Pten KO mice. Pten KO mice were assigned to a control group without any medication or to a 1,8-cineole group injected with 50 mg/kg i.p. twice per week for eight weeks. At eight weeks, livers from each group were processed to measure triglyceride (TG) content, gene expression analysis, western blot analysis, and histological examination including Oil red O staining. 1,8-cineole ameliorated hepatic steatosis in Pten KO mice, revealed by TG content and Oil red O staining. Moreover, 1,8-cineole downregulated collagen 1a1 expression and improved liver fibrosis. Thus, 1,8-cineole has potential as a candidate to treat NASH by inactivating the Akt/PI3-kinase pathway.

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A recent study demonstrated that intracellular small/microRNAs are released from cells, and some of these extracellular RNAs are embedded in vesicles, such as ceramide-rich exosomes, on lipid-bilayer membranes. In the present study, we examined the effects of sphingomyelin phosphodiesterase 3 (SMPD3), which generates ceramide from sphingomyelin, on the release of small/microRNAs from intracellular to extracellular spaces. In these experiments, SW480 human colorectal and HuH-7 human hepatocellular cancer cells were cultured for 48 h in serum-free media. Culture supernatants were then collected, and floating cells and debris were removed by centrifugation and filtration through a 0.22-µm filter. Extracellular small RNAs in purified culture supernatants were stable for 4 weeks at room temperature, after 20 freeze-thaw cycles and exposure to pH 2.0, and were resistant to ribonuclease A degradation. Amino acid sequence analyses of SMPD3 showed high homology between mammals, indicating evolutionary conservation. Therefore, to investigate the mechanisms of cellular small/microRNA export, SW480 and HuH-7 cells were treated with the SMPD3 inhibitor GW4869 in serum-free media. Culture supernatants were collected for microarray and/or reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments. The number of microRNAs in culture supernatants was decreased following treatment with GW4869. Among these, extracellular and intracellular miR-638 were dose-dependently decreased and increased, respectively. These data suggest that SMPD3 plays an important role in the release of microRNAs into extracellular spaces.

Exposure to high-doses of ionizing radiation (IR) leads to development of a strong acute radiation syndrome (ARS) in mammals. ARS manifests after a latency period and it is important to develop fast prognostic biomarkers for its early detection and assessment. Analysis of chromosomal aberrations in peripheral blood lymphocytes is the gold standard of biological dosimetry, but it fails after high doses of IR. Therefore, it is important to establish novel biomarkers of exposure that are fast and reliable also in the high dose range. Here, we investigated the applicability of miRNA levels in mouse serum. We found significantly increased levels of miR-375-3p following whole body exposure to 7 Gy of X-rays. In addition, we analyzed their levels in various organs of control mice and found them to be especially abundant in the pancreas and the intestine. Following a dose of 7 Gy, extensive cell death occurred in these tissues and this correlated negatively with the levels of miR-375-3p in the organs. We conclude that high expressing tissues of miR-375-3p may secrete this miRNA in serum following exposure to 7 Gy. Therefore, elevated miR-375-3p in serum may be a predictor of tissue damage induced by exposure to a high radiation dose.