br to surgery in addition to
to surgery, in addition to being pathologically diagnosed postopera-tively independently by two pathologists. Tissue samples were obtained and immediately preserved in liquid nitrogen and stored at 70 C.
Plasmids and Lentivirus Transfection
Pancreatic cancer LLY507 were inoculated into six-well plates (2 105 cells/well) 1 day prior to transfection. Transfection was performed when cell confluence was confirmed to have reached 60%–80%. The cells were transfected with miR-126-3p/NC mimic, miR-126-3p/NC inhibitor, and shADAM9/NC plasmids using Lipofectamine 2000 (Carlsbad, CA, USA) in accordance with the instructions pro-vided. In an attempt to reduce toxicity, the transfection complex was replaced with fresh medium after 6 h had elapsed. The cells were analyzed and further examined 48 h after transfection.
Lentivirus packaging was performed in a 60-mm culture vessel that contains 1 mm pMD2G, 3 mg psPAX2, and 4 mg Prutou3-mChely/ LuxIFEase/mimic NC/miR-126-3p using the transient co-transfec-tion system of HEK293T cells. 24 h after transfection, supernatant was collected, following the addition of a fresh medium for the second supernatant collection after 24 h. The two aforementioned superna-tants were then mixed for target cell infection purposes. Lentivirus-transfected BMSCs were cultured overnight in 24-well plates at a density of 5 104 cells/well. Medium containing 500 mL lentivirus supernatant, 500 mL fresh culture medium, and 8 mg polyacrylamide
miR-126-3p exosomes ADAM9
Figure 11. miR-126-3p Transferred by BMSC-Derived Exosomes Entered Pancreatic Cancer Cells
miR-126-3p can enter BMSC-derived exosomes and then be transferred to pancreatic cancer cells. In pancreatic cancer cells, miR-126-3p, by downregulating ADAM9 gene, decreases the expression of Ki67, VEGF, COX-2, and MMP-14, thus inhibiting proliferation, migration, and invasion and promoting apoptosis of pancreatic cancer cells. ADAM9, a disintegrin and metalloprotease-9; BMSC, bone marrow mesenchymal stem cell; COX-2, cyclooxygenase-2; miR-126-3p, microRNA-126-3p; MMP-14, matrix metalloproteinase-14; VEGF, vascular endothelial growth factor.
(Sigma, St. Louis, MO, USA) was used to facilitate the uptake of virus particles. Then the plate was centrifuged (7,890 g) at 37 C, then permitted to react for 1 h and replaced with fresh medium. The cells were screened by 7 g/mL rodenticidin postinfection, and the DsRed fluorescence was observed under the fluorescence microscope (MICHE) 7 days later.
Dual-Luciferase Reporter Gene Assay
Bioinformatics database (microRNA.org; http://www.microrna.org/ microrna/home.do) was used to provide information predicting whether ADAM9 was indeed a target gene of miR-126-3p. Human embryo kidney HEK293T cells were cultured in DMEM containing 10% fetal bovine serum (FBS) at 37 C with 5% CO2. The cDNA frag-ment in ADAM9 30UTR containing miR-126-3p binding sites was in-serted into pmirGLO vector. The cDNA fragments of ADAM9 30 UTR with binding site mutation were inserted into pmirGLO vector. The recombinant vector of pmirGLO-ADAM9 or pmirGLO-mutADAM9 was co-transfected with miR-126-3p mimic (miR-126-3p overexpression sequence) or miR-NC (NC sequence) into HEK293T cells by liposome transfection, respectively. Subsequently, the cells were then cultured for 48 h, collected, and lysed. A total volume of 100 mL lysate supernatant was added with 100 mL renilla luciferase detection fluid in order to determine the renilla luciferase activity. In addition, 100 mL lysate supernatant was mixed with 100 mL firefly luciferase detection reagent in order to detect the firefly luciferase activity. The renilla luciferase and firefly luciferase activities were detected by the multifunctional enzyme analyzer SpectraMaxM5 for 10 s at 2-s interval.
5-Ethynyl-20-Deoxyuridine Cells exhibiting logarithmic growth were inoculated into 96-well plates at a density of 4 103 to 1 105 cells/well and cultured until to normal cell development stage. The cell culture medium was used to dilute 5-ethynyl-20-deoxyuridine (EdU) solution at a ratio of 1000:1, and 50 mM EdU culture medium was prepared accordingly.
The cells were incubated with 100 mL EdU (50 mM) culture medium for 2 h and then abandoned. The cells were fixed with 50 mL cell fixa-tive solution (PBS containing 4% paraformaldehyde) for 30 min; then the fixative solution was discarded. After 50 mL (2 mg/mL) glycine was added, cells were incubated in the decolorized shaker for 5 min, after which the glycine solution was discarded. The cells were then incubated for 10 min in a decolorized shaker with
100 mL 1 Apollo staining reaction liquid was added to the cells and incubated in a decolorized shaker under conditions void of light for 30 min followed by the removal of the reactive liquid. The cells were then washed with 100 mL infiltrator (PBS containing 0.5% Triton X-100) in decolorized shaker two to three times, each time for 10 min, followed by the removal of infiltrator. Reagent F was diluted by deion-