Let-7 MicroRNA Family Is Selectively Secreted into the Extracellular Environment via Exosomes in a Metastatic Gastric Cancer Cell Line

Background: Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene downregulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well. Methodology/Principal Findings: Exosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity. Conclusions/Significance: The enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosome

Placenta-specific novel splice variants of Rho GDP dissociation inhibitor β are highly expressed in cancerous cells

Abstract Background Alternative splicing of pre-mRNA transcripts not only plays a role in normal molecular processes but is also associated with cancer development. While normal transcripts are ubiquitously expressed in normal tissues, splice variants created through abnormal alternative splicing events are often expressed in cancer cells. Although the Rho GDP dissociation inhibitor β (ARHGDIB) gene has been found to be ubiquitously expressed in normal tissues and involved in cancer development, the presence of splice variants of ARHGDIB has not yet been investigated. Results Validation analysis for the presence of and exon structures of splice variants of ARHGDIB, performed using reverse-transcriptase polymerase chain reaction and DNA sequencing, successfully identified novel splice variants of ARHGDIB, that is, 6a, 6b, and 6c, in colon, pancreas, stomach, and breast cancer cell lines. Quantitative real-time polymerase chain reaction analysis showed that these variants were also highly expressed in normal placental tissue but not in other types of normal tissue. Conclusions Expression of ARHGDIB variants 6a, 6b, and 6c appears to be restricted to cancer cells and normal placental tissue, suggesting that these variants possess cancer-specific functions and, as such, are potential cancer-related biomarkers.</p

Models on difference in localization of let-7 miRNA family.

<p>Based on the results obtained by microarray and RT-PCR analyses, these models were drawn. In comparison between AZ-P7a cells (left) and AZ-521cells (center), normalized by the amount of U6 snRNA (open triangles), the amount of exosomal let-7a miRNA (filled triangles) in AZ-521 cells was approximately 3% of that in AZ-P7a cells while the intracellular amount in AZ-521 cells was rather 1.4 times greater than that in AZ-P7a cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013247#pone-0013247-g004" target="_blank">Figure 4E</a>). These models are applied to six of eight let-7 miRNA family tested, including let-7a, let-7b, let-7c, let-7d, let-7e, and let-7i. In SW620 cells (right), the amount of let-7a was 3.5 times greater than that in AZ-P7a cells while the amount of exosomal let-7a was 0.7 times less than in AZ-P7a cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013247#pone-0013247-g004" target="_blank">Figure 4E</a>).</p

Total RNAs in exosomes and culture media from various cancer cell lines.

<p><b>A.</b> Amounts of total RNAs recovered from exosomes (lower panel) and culture media (upper panel). The amount of total RNAs per cell was shown. <b>B.</b> Distribution in length of RNA. Isolated total RNAs from the exosomes and culture medium of AZ-P7a cells were detected using Bioanalyzer. The result of cellular total RNAs was shown for comparison.</p

Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome.

International audienceOleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (β-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga

Characterization of exosomes.

<p><b>A.</b> Morphological characterization of exosomes derived from AZ-P7a, AZ-521, and SBC-3 cells by transmission electron microscopy. <b>B.</b> Immunoelectron micrographs of AZ-P7a exosomes labeled with immunogold anti-CD63. Exosomes with black colloidal gold particles on the capsular membranes were observed as positive CD 63 staining under the transmission electron microscope (arrowheads). <b>C.</b> Molecular characterization of exosomes derived from AZ-P7a, AZ-521, and SBC-3 cells by western blot analysis. Protein extracts (10 µg) prepared from cells (C) or exosomes (Ex) were assessed using antibodies against exosomal protein markers (CD29/β1-integrin, Aip1/Alix, and Tsg101) and an endoplasmic reticulum marker (Bip/Grp78).</p

Ranking of let-7 miRNA family in the intra- and extra-cellular fractions based on signal intensities from microarray data.

<p>Ex = exosomes. CM = culture medium. miRNAs = the total number of miRNAs detected. A dash (-) indicates no detection.</p