Opti mem

All the U2-OS cells were starved for 12 h before transfection and divided into 4 groups: (1) miR-586 group: 10 μl Lipofectamine TM 2000 (Invitrogen, Carlsbad, Calif., USA), 4 g pLVX-shRNA1-miR-586 recombinant plasmid (provided by the surgery research center, Second Hospital of Hebei Medical University) and 240 μl Opti-MEM (Solarbio, Beijing, China); (2) anti-miR-586 group: 10 μl Lipofectamine 2000, 4 g pLVX-shRNA1anti-miR-586, and 240 μl Opti-MEM; (3) control group: 10 μl Lipofectamine 2000, 4 g pLVX-shRNA1-miR empty control plasmid, and 240 μl Opti-MEM, and (4) blank group: no plasmid; the rest is the same as in the other groups. When entering the exponential phase, cells were transfected according to the specification of Lipofectamine 2000. After transfection for 48 h, an Olympus IX-71 inverted fluorescence microscope (Olympus Deutschland GmbH, Hamburg, Germany) was used to observe green fluorescent protein (GFP)-positive expression. Flow cytometry was used to sort GFP-positive cells and continue to cultivate the cells. RFQ-PCR was used to sort and identify miR-586 expressions in the cells.