Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line - PubMed (original) (raw)

Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line

Keiichi Ohshima et al. PLoS One. 2010.

Abstract

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 down-regulation 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 exosomes from AZ-P7a cells may reflect their oncogenic characteristics including tumorigenesis and metastasis. Since let-7 miRNAs generally play a tumor-suppressive role as targeting oncogenes such as RAS and HMGA2, our results suggest that AZ-P7a cells release let-7 miRNAs via exosomes into the extracellular environment to maintain their oncogenesis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Characterization of exosomes.

A. Morphological characterization of exosomes derived from AZ-P7a, AZ-521, and SBC-3 cells by transmission electron microscopy. 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). C. 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).

Figure 2

Figure 2. Total RNAs in exosomes and culture media from various cancer cell lines.

A. Amounts of total RNAs recovered from exosomes (lower panel) and culture media (upper panel). The amount of total RNAs per cell was shown. 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.

Figure 3

Figure 3. miRNA profiling in the intra- and extra-cellular fractions from AZ-P7a, AZ-521, NCI-H69, SBC-3, DMS53, SW480 and SW620 cells.

miRNA profiles in cells (C), exosomes (Ex), and culture media (CM) were obtained by miRNA microarray analysis. Y axis represents log2 of hybridization signals, shown by bars and filled boxes. The arrows on the left side from the bunch of signals represent signals corresponding to let-7 miRNA family including let-7a (red), let-7b (yellow), let-7c (light green), let-7d (dark green), let-7e (sky blue), let-7f (blue), let-7g (navy blue), and let-7i (purple).

Figure 4

Figure 4. RT-PCR analyses of intra- and extra-cellular let-7 miRNA family.

A, B. Levels of mature let-7 miRNA family including let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i in cells (shaded bars), exosomes (filled bars), and culture medium (open bars) from AZ-P7a cells (A) and AZ-521 cells (B). Each miRNA level was analyzed using quantitative RT-PCR. The cycle threshold (Ct) value is presented as the mean ± SD (n = 4). C. Levels of U6 snRNA in cells (shaded bars), exosomes (filled bars), and culture medium (open bars) from AZ-P7a and AZ-521 cells detected by quantitative RT-PCR analysis. The cycle threshold (Ct) value is presented as the mean ± SD (n = 4). D. Relative amounts of let-7 miRNA family in cells (shaded bars), exosomes (filled bars), and culture medium (open bars) from AZ-521 cells versus AZ-P7a cells. The dotted line and arrow represents the levels of let-7 miRNA family in AZ-P7a cells, shown as 100%. The levels of U6 snRNA in each sample were used as an internal standard for normalization amounts of let-7 miRNAs. Except let-7f and let-7g, the levels of let-7 miRNAs were reduced in the extracellular fractions. E. Relative amounts of let-7a in cells (shaded bars) and exosomes (filled bars) from 7 cancer cell lines including AZ-P7a, AZ-521, NCI-H69, SBC-3, DMS53, SW480 and SW620 cells. The dotted line represents the levels in AZ-P7a cells, shown as 1. The levels of U6 snRNA in each sample were used for normalized amounts of let-7a. The value of fold change is presented as the mean ± SD (n = 3) from samples independently prepared from cell culture. F. Relative amounts of exosomal let-7a in 7 cancer cell lines. The dotted line represents the level in AZ-P7a cells, shown as 100%.

Figure 5

Figure 5. Models on difference in localization of let-7 miRNA family.

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 (Figure 4E). 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 (Figure 4E).

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