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BMSC-Derived Exosomes Carry miR-126-3p
In order to isolate and subsequently identify the BMSC-derived exo-somes, exosomes with the diameter of 60–160 nm were collected by means of ultracentrifugation, exhibiting circular or oval shape with complete membrane structure and low-density substance under transmission electron microscopy (TEM) (Figure 7A). Moreover, in
Figure 3. Overexpressed miR-126-3p Contributed to Inhibited Proliferation, Migration, and Invasion of Pancreatic Cancer Cells and Promoted Their Apoptosis
(A) The effect of miR-126-3p on the proliferation of pancreatic cancer cell line PANC-1 detected by EdU assay (original magnification 400). (B) The effect of miR-126-3p on migration ability of PANC-1 detected by Transwell assay (original magnification 200). (C) The effect of miR-126-3p on the invasion of PANC-1 detected by Transwell assay (original magnification 200). (D) The effect of miR-126-3p on the apoptosis rate of PANC-1 detected by flow cytometry. The data in the chart were all measured and expressed as mean ± SD. The independent samples t test was used for statistical analysis between two groups, and the experiment was repeated three times. EdU, 5-ethynyl-20-deoxyuridine; miR-126-3p, microRNA-126-3p; NC, negative control.
order to determine their respective size distribution, the NanoSightNS300 nanoparticle tracking analysis tool was used. As illustrated in Figure 7B, exosome particles were located predomi-nately around 100 nm. Western blot analysis results revealed that the exosome surface marker proteins CD63 and Hsp70 were specif-ically detected in BMSC-derived exosomes (Figure 7C). Cy3-labeled exosomes in BMSCs were co-cultured with PANC-1 cell and tagged with GFP for 6 h. Under the confocal fluorescence microscope, slight red fluorescence of Cy3-exosomes could be observed in the PANC-1 LDN-193189 (Figure 7D), which was indicative of the entrance of a small num-ber of Cy3-exosomes into PANC-1 cells. After a 12-h period of co-culturing, a small number of PANC-1 cells was observed to have exhibited red fluorescence, with the fluorescence mainly concentrated
in the cytoplasm, indicating that Cy3-exosomes were mainly located in the cytoplasm of PANC-1 cells. With the prolongation of co-cul-ture time, more PANC-1 cells exhibited red fluorescence, indicating that the number of Cy3-exosomes absorbed by PANC-1 cells increased gradually. At 48 h of co-culture, Cy3-exosomes were obviously absorbed by PANC-1 cells. Notably, the expression of miR-126-3p was found to be markedly higher in the BMSCs, exo-somes, and co-cultured pancreatic cancer cells when compared with the NC group (Figure 7E), which suggested that miR-126-3p could be carried by BMSC-derived exosomes. The levels of ADAM9 were further detected and the results demonstrated there to be diminished levels of ADAM9 after the pancreatic cancer cells had absorbed the exosomal miR-126-3p (Figures 7F–7H).
Molecular Therapy: Nucleic Acids
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of ADAM9 protein
(A) The targeting relationship between miR-126-3p and ADAM9 predicted in the biological prediction website microRNA.org (http://www.microrna.org/microrna/home.do).
(B) The target relationship between miR-126-3p and ADAM9 verified by dual-luciferase reporter gene assay. (C) mRNA expression of ADAM9 in each group determined by qRT-PCR. (D) Protein expression of ADAM9 in each group measured using western blot analysis. *p < 0.05, compared with the ADAM9-mut group. The data were measured and expressed as mean ± SD. Independent sample t test was used for statistical analysis, and the experiment was repeated three times. ADAM9, a disintegrin and metalloprotease-9; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR-126-3p, microRNA-126-3p; MUT, mutant; NC, negative control; WT, wild-type.
GW4869 Inhibits the Release of BMSC-Derived Exosomes, Thus Suppressing miR-126-3p Expression
In order to further investigate whether miR-126-3p carried by exosomes influences the biological functions of pancreatic cancer cells, the exo-somes’ secretory-specific inhibitor GW4869 was added to the co-culture system in the Transwell chamber to elucidate the effect of GW4869 on release of exosomes and the expression of miR-126-3p in pancreatic cancer cells. Compared with the NC DMSO group, the activity of acetyl-cholinesterase (AChE) in the GW4869 group was decreased, suggesting that the release of the exosomes and the miR-126-3p expression in the exosomes were reduced (p < 0.05; Figures 8A and 8B). Furthermore, the migration and invasion ability of pancreatic cancer cells was higher in the GW4869 group when compared with that of the DMSO group (p < 0.05; Figures 8C–8F). Based on the aforementioned results, it was concluded that GW4869 inhibited the release of BMSC-derived exosomes, resulting in the suppression of miR-126-3p expression.