br Initially miR p was
Initially, miR-126-3p was noted to be poorly expressed, whereas ADAM9 was highly expressed in pancreatic cancer cell lines, with our results suggesting that miR-126-3p could directly inhibit the LDN193189 of ADAM9. Studies have indicated that miR-126-3p is poorly expressed in various tumors, including hepatocellular carci-noma, a finding which was consistent with the results of our study.19 The downregulation of miR-126-3p has also been flagged in cervical cancer tissues when compared with normal cervical tissues,28–30 whereas its level in cervical mucus was found to be increased among patients with cervical cancer.31 Furthermore, decreased miR-126 expression has been previously linked with pancreatic cancer.24,32
Figure 5. miR-126-3p Inhibited Biological Function in Pancreatic Cancer Cells by Negatively Regulating ADAM9
(A) The effect of ADAM9 on the proliferation of PANC-1 detected by EDU assay (original magnification 400). (B) The effect of ADAM9 on migration ability of PANC-1 using Transwell assay (original magnification 200). (C) The effect of ADAM9 on the invasiveness of PANC-1 using Transwell assay (original magnification 200). (D) The effect of ADAM9 on the apoptosis rate of PANC-1 determined by flow cytometry. (E–G) The mRNA and protein expression of ADAM9 gene measured using qRT-PCR (E) and western blot analysis (F and G), respectively. The data were all measured and expressed by mean ± SD. The independent sample t test was used for statistical analysis between two groups, and the experiment was repeated three times. ADAM9, a disintegrin and metalloprotease-9; EdU, 5-ethynyl-20-deoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR-126-3p, microRNA-126-3p; NC, negative control.
level in BMSCs
level in Exosome
ADAM9 protein Relative expression
Figure 7. miR-126-3p Could Be Carried by BMSC-Derived Exosomes
(A) The ultrastructure of exosomes observed by TEM (original magnification 400). (B) Size distribution of exosomes detected by NanoSight particle size analysis. (C) Surface markers of exosomes (CD63, Hsp70) detected by western blot analysis. (D) The exosomes absorbed by PANC-1 cells under confocal fluorescence microscope at different time points. Red: miR-126-Cy3; green: pCDNA3.1-GFP; blue; DAPI. (E) The expression of miR-126-3p in BMSCs, exosomes, and co-cultured pancreatic cancer cells determined by qRT-PCR. (F) mRNA expression of ADAM9 in pancreatic cancer cells determined by qRT-PCR. (G and H) Protein expression of ADAM9 in pancreatic cancer cells determined by western blot analysis. The data in the chart were analyzed using an independent sample t test, and the experiment was repeated three times. *p < 0.5. BMSC, bone marrow mesenchymal stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR-126-3p, microRNA-126-3p; NC, negative control; TEM, transmission electron microscopy.
Molecular Therapy: Nucleic Acids
Figure 8. GW4869 Inhibited the Release of BMSC-Derived Exosomes to Inhibit the Expression of miR-126-3p
(A) The effect of GW4869 on the release of exosomes detected by AChE activity assay. (B) The effect of GW4869 on the expression of miR-126-3p in pancreatic cancer cells determined using qRT-PCR. (C and E) The effect of GW4869 on migration ability of PANC-1 detected by Transwell assay (photograph of migration cell, C, and number of migration cell, E). (D and F) The effect of GW4869 on invasion of PANC-1 determined by Transwell assay (photograph of invasion cell, D, and number of migration cell, F). The data were measured by the mean ± SD. Comparison between two groups was conducted using an independent sample t test for statistical analysis; *p < 0.05, compared with the DMSO group. The experiment was repeated three times. miR-126-3p, microRNA-126-3p.
Existing literature has suggested that ADAM9 is overexpressed dur-ing the progression of cancer in addition to indicating that silencing ADAM9 could promote both the radio-sensitivity and chemosensi-tivity of cancer cells to therapeutic drugs.33 Statistical evidence has demonstrated that pancreatic cancer cells exhibit a significantly higher level of ADAM9, which was also observed in the current study.34 Liu et al.20 concluded that miR-126 could suppress the expression of ADAM9 and act to further inhibit the growth of the ESCC through the epidermal growth factor receptor (EGFR)-AKT pathway. Various reports have pointed to the fact that pancreatic can-cer cells exhibit downregulated levels of miR-126-3p and upregulated ADAM9, in addition to revealing that ADAM9 is a target gene of miR-126-3p, which acts to directly inhibit ADAM9.