Human umbilical vein endothelial cells (HUVECs) (#GDC166, CCTCC) were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 U/ml of penicillin, and 100 μg/ml of streptomycin in a 5% CO2 humidified atmosphere at 37 °C. The miR-31-5p mimic, miRNA-31-5p inhibitor, and the relevant negative controls (mimic NC and inhibitor NC) were obtained from Ribobio company (Guangzhou, China). The sequences were presented in Table S1 . HUVECs were transfected using riboFECT™CP Reagent according to the manufacturer’s instruction. The plasmids for overexpressing were purchased from Genomeditech Biotechnology (Shanghai, China). Cell transfection was performed with the NEOFECT™ DNA transfection reagent according to the manufacturer’s protocol. At 48 h after transfection, the cells were processed for in vitro assays.
Mir 31 5p mimic
Manufactured by RiboBio
Sourced in China
The MiR-31-5p mimic is a synthetic RNA molecule designed to mimic the natural microRNA MiR-31-5p. MicroRNAs are small, non-coding RNA molecules that play a role in regulating gene expression. The MiR-31-5p mimic can be used in research applications to study the biological functions and effects of this specific microRNA.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mimic nc, by RiboBio (1 mentions)
H1299, by Cobioer Biosciences (1 mentions)
H1975, by Cobioer Biosciences (1 mentions)
Ribofect cp transfection kit, by RiboBio (1 mentions)
Si timp3, by GenePharma (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pkh67, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
4 protocols using mir 31 5p mimic
1
Transfection of HUVECs with miR-31-5p
2
Lentiviral Transduction of H1299 and H1975 Cells
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H1299 and H1975 cells were purchased from COBIOER BIOSCIENCES (Nanjing, Jiangsu, China). They were cultured in RPMI-1640 medium supplemented with 10% FBS at 37 ˚C, 5% CO2. The lentivirus expressing empty lenti-vector alone and HSD17B6 were purchased from HanBio (Shanghai, China). The cells were infected with lentivirus supplemented with 5 μg/ml polybrene for 24 h, then selected with puromycin (2 μg/ml) for 14 days. The miR-31-5p mimic, miR-31-5p inhibitor, scramble sequence (negative control, NC), and the riboFECT CP transfection kit were supplied by Ribobio (Guangzhou, Guangdong, China). Transfection was performed according to the manufacturer’s instruction.
3
NSCLC Cell Lines and MEG3 Regulation
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NSCLC cell lines H522 and A549 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and human bronchial epithelial cells 16HBE were purchased from Chuan Qiu Biotechnology (Shanghai, China). All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco) and 0.05% penicillin/streptomycin.
MEG3 overexpression vector (MEG3) and its negative control pcDNA, small interfering RNA (siRNA) targeting MEG3 (si-MEG3), siRNA targeting TIMP3 (si-TIMP3) and siRNA negative control (si-NC) were synthesized by Genepharma (Shanghai, China). MiR-31-5p mimics, miR-31-5p inhibitor (in-miR-31-5p), miRNA mimics negative control (miR-NC) and miRNA inhibitor negative control inhibitor (anti-NC) were purchased from RIBOBIO (Guangzhou, China). All the vectors were transfected in H522 and A549 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
MEG3 overexpression vector (MEG3) and its negative control pcDNA, small interfering RNA (siRNA) targeting MEG3 (si-MEG3), siRNA targeting TIMP3 (si-TIMP3) and siRNA negative control (si-NC) were synthesized by Genepharma (Shanghai, China). MiR-31-5p mimics, miR-31-5p inhibitor (in-miR-31-5p), miRNA mimics negative control (miR-NC) and miRNA inhibitor negative control inhibitor (anti-NC) were purchased from RIBOBIO (Guangzhou, China). All the vectors were transfected in H522 and A549 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
4
Visualizing Exosome Internalization in LUAD Cells
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SATB2 siRNA, miR-31-5p mimics and inhibitors were obtained from RiboBio Company (Guangzhou, China). Cells were seeded into 6-well plates for 24 h before transfection. When the cells reached 60 % confluence, they were transfected with SATB2 siRNA, miRNA mimics or inhibitors using Lipofectamine 3000. To determine whether the exosomes could be internalized by LUAD cells, the exosomes were labeled with PKH67 (Sigma) as the manufacturer described. Briefly, purified exosomes were suspended in a mixture consisting of 500 µL of Diluent C and 4 µL of PKH67, and then incubated in the dark for 4 min at room temperature. Next, 2 mL of 0.5 % bovine serum albumin (BSA) was added to stop staining, and the mixture was subsequently centrifuged at 100,000 g for 1 h. The labeled exosomes were suspended with PBS and then used immediately or stored at 20℃. LUAD cells were treated with labeled exosomes for 8 h at 37℃, and the nuclei were stained by Dapi (Sigma) for 30 min. After staining, the samples were washed twice with PBS and observed under a fluorescence microscope.
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