Identification of lung adenocarcinoma-specific exosome RNAs in peripheral blood by RNA-Seq analysis.

OBJECTIVE: Several plasma-derived exosome RNAs have been identified as key regulators in cancer development. They have been considered as potential biomarkers for a non-invasive "liquid biopsy" to diagnose and assess the progression of cancer. This study aimed to identify human lung adenocarcinoma-specific exosome RNAs in peripheral blood, while assessing the feasibility and efficiency of this recently developed deep-sequencing technology for transcriptome profiling. PATIENTS AND METHODS: Plasma-derived exosome RNAs were isolated from 13 lung adenocarcinoma patients, 3 patients with benign lung diseases, and 15 healthy volunteers. RNA-seq analysis of ribosomal RNA-depleted total RNA was performed. RNAs differentially expressed between lung adenocarcinoma and benign lung diseases or healthy volunteers were identified, followed by GO and KEGG pathway enrichment analyses for the identification of key exosome RNAs associated with lung adenocarcinomas. RESULTS: Significant differentially expressed RNAs, such as UDP glucuronosyltransferase family 1 member A1 (UGT1A1) and BAI1-associated protein 2 like 1 (BAIAP2L1), were identified as differentially expressed between lung adenocarcinoma patients and patients with benign lung diseases. Eight pseudogenes, including Tropomyosin 1 (Alpha) Pseudogene (LOC100129096), Prothymosin, Alpha Pseudogene 2 (PTMAP2), Cell Division Cycle 14C, Pseudogene (CDC14C), Tropomyosin 1 (Alpha) Pseudogene (LOC643634), Ferritin Heavy Chain 1 Pseudogene 2 (FTH1P2), Actin Related Protein 2/3 Complex Subunit 3 Pseudogene 3 (ARPC3P3), Ferritin Heavy Chain 1 Pseudogene 11 (FTH1P11), and Prothymosin Alpha Pseudogene 5 (PTMAP5) were identified from plasma-derived exosomes in lung adenocarcinoma patients, who were more abundant/detectable than healthy volunteers. CONCLUSIONS: Our data indicate that plasma-derived exosome RNAs, UGT1A1, and BAIAP2L1, as well as the eight isolated pseudogenes could serve as diagnostic and prognostic biomarkers for an effective non-invasive "liquid biopsy" of lung adenocarcinomas.

Dietary omega-3, omega-6, and omega-9 unsaturated fatty acids and growth factor and cytokine gene expression in unstimulated and stimulated monocytes. A randomized volunteer study.

Dietary omega-3 fatty acids retard coronary atherosclerosis. Previously, we demonstrated that dietary omega-3 fatty acids reduce platelet-derived growth factor (PDGF)-A and PDGF-B mRNA levels in unstimulated, human mononuclear cells (MNCs). In a randomized, investigator-blinded intervention trial, we have now compared the effect of ingestion of 7 g/d omega-3, omega-6, or omega-9 fatty acids for 4 weeks versus no dietary intervention on PDGF-A, PDGF-B, heparin-bound epidermal growth factor (HB-EGF), monocyte chemoattractant protein-1 (MCP-1), and interleukin-10 gene expression in unstimulated MNCs and in monocytes that were adherence-activated ex vivo in a total of 28 volunteers. In unstimulated MNCs, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25+/-10%, 31+/-13%, and 40+/-14%, respectively, after omega-3 fatty acid ingestion, as assessed by quantitative polymerase chain reaction (all P<0.05). In monocytes that were adherence-activated ex vivo for 4 and 20 hours, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25+/-13%, 20+/-15%, and 30+/-8%, respectively (all P<0.05). Interleukin-10 and HB-EGF mRNA steady-state levels were not influenced by omega-3 fatty acid ingestion. Expression of all respective mRNAs in control volunteers or in those ingesting omega-6 or omega-9 fatty acids were not altered. We conclude that human gene expression for PDGF-A, PDGF-B, and MCP-1, factors thought relevant to atherosclerosis, is constitutive, is constant, and can be reduced only by dietary omega-3 fatty acids in unstimulated and adherence-activated monocytes.

The human leukotriene A4 hydrolase gene is expressed in two alternatively spliced mRNA forms.

We identified a novel short (s-) mRNA of the human leukotriene-A4 (LTA4) hydrolase using sequential reverse transcriptase PCR mapping. The s-mRNA is generated by skipping an 83 bp exon located in the 3' coding region and suggests the expression of an LTA4 hydrolase isoform with a calculated molecular mass of 59 kDa and a distinct C-terminus. Both LTA4 hydrolase mRNAs are constitutively expressed in blood cells, endothelial cells, smooth muscle cells, fibroblasts and tumour cells. The ratios of their mRNA expression levels are cell specific, with relatively high s-form mRNA expression in reticulocytes. Our data strongly suggest that the novel mRNA codes for the structurally related but distinct LTA4 hydrolase isoenzyme that has been postulated.

n-3 fatty acids and cysteinyl-leukotriene formation in humans in vitro, ex vivo, and in vivo.

In humans, dietary n-3 fatty acids ameliorate some chronic inflammatory diseases. In contrast, however, dietary n-3 fatty acids had no effect in patients with bronchial asthma. In bronchial asthma, the cysteinyl-leukotrienes C4, D4, and E4, formed from arachidonic acid, are considered important mediators. They are as vasoconstrictive and bronchoconstrictive as leukotrienes C5, D5, and E5, cysteinyl-leukotrienes derived from eicosapentaenoic acid. Whether and how n-3 fatty acids affect human cysteinyl-leukotriene metabolism is largely unknown. We therefore investigated human cysteinyl-leukotriene metabolism in vitro, ex vivo, and in vivo in the absence and presence of dietary n-3 fatty acids. We demonstrate formation of leukotrienes C5, D5, and E5 from eicosapentaenoic acid in vitro and ex vivo in stimulated human granulocytes. Proof of formation relies on cochromatography with authentic standards on reverse-phase high-performance liquid chromatography, ultraviolet-absorbance spectra, and radioactive tracer studies. In vitro, amounts of leukotrienes C5, D5, and E5 formed depended on the amount of exogenous eicosapentaenoic acid; leukotrienes C4, D4, and E4 formed from endogenous arachidonic acid, however, remained unaltered. A randomized, controlled, observer-blind study in 14 human volunteers, seven of whom supplemented their diet with 7 gm/day of an 85% n-3 fatty acid concentrate for 6 weeks was subsequently performed. Ex vivo, levels of leukotriene E5 almost equaled those of leukotriene E4. Moreover, urinary excretion of leukotriene E4 was assessed to estimate formation of cysteinyl leukotrienes from arachidonic acid in vivo. Urinary excretion of leukotriene E4 was reduced by 35% after dietary supplementation with n-3 fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)