Cell lines and cell culture
The human NSCLC cell lines A549, HLF-A (human lung epithelial cells), and MSCs (human bone marrow mesenchymal stem cells) were obtained from the Fenghbio Biological Technology (China). A549 cells were maintained in Ham’s F12K (F12K) medium, HLF-A were maintained in alpha-minimal essential medium (α-MEM) medium and MSCs were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco BRL, USA), containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin solution (Hyclone), at 37 °C with 5% CO2.
Exosome isolation and characterization
MSCs were cultured in media with 10% exosome-free FBS for 48h. Cell culture media were collected, and exosomes were isolated from the supernatant by differential centrifugation using a ultracentrifuge as described by Liu et al(18). Briefly, the medium was collected and centrifuged at 1000 g for 15 min, at 3000 g for 30 min, at 10,000 g for 1h, at 100,000 g for 4 h. All centrifugal steps were performed at 4°C. The final pellet containing exosomes was re-suspended in PBS. The representative marker of MSCs, CD44, CD29, CD34 and CD45 were assayed by immunofluorescence staining and flow cytometry analysis.
According to the manufacturer’s instructions for Invitrogen Lipofectamine 2000 (Thermo Fisher Scientific, USA), A549 cells were transiently transfected with miRNA-486-5p mimic/inhibitor, pcDNA3.1-MIER3/ASO-MIER3, or the corresponding NC ((NC mimic/inhibitor, empty vector (pcDNA3.1) and ASO-NC)) (RiboBio, Guangzhou, China). The level of RNA or protein was measured using Western blot at 48 h after transfection.
Co-culture experiments and exosomes culture
MSCs and A549 cells (or HLF-A cells) were co-cultured by the Transwell chamber (0.4 μm, 6-well plates, Corning, USA). MSCs (1.0 × 103 cells in 500 µL medium) were seeded in the upper chamber, and A549 cells (1.0 × 104 cells in 1500 µL medium) were seeded in the lower chamber, using DMEM and F12K medium containing 10% exosome-free FBS for two chambers, respectively. MSCs and A549 cells were co-cultured for 48 h and then proceeded to follow-up experiments. MSC-Exos (100 mg/mL) were added to A549 cells/HLF-A.
The MTT assay was performed following the manufacturer’s instructions. Briefly, A549 cells were cultured in 96-well plates (5× 103 cells per well). After incubation for 48h, MTT solution (5 mg/mL) was added into each well and incubated with the cells at 37 °C for 4 h. The medium was then removed, and 150 μL DMSO was added to dissolve the crystal adequately. The absorbance was measured at 570 nm using a microplate reader (Perlong, China).
Cell cycle analysis
A549 cells were seeded in 6-well plates (200,000 cells/well), transfected or co-cultured. At the end of transfection and/or treatments, the cells were collected by trypsinization, washed twice with cold PBS, and fixed with 75% ethanol overnight at 4˚C. Following fixation, the cells were washed again with PBS, incubated with PI at 4˚C for 30min. Cell cycle phase distribution was analysed using a BD FACSCantoII and ModFit LT software (BD, USA).
Colony formation assay
The A549 cells (1× 103 cells per well) were plated into a six-well plate (Corning, USA) continuously cultured for about 2 weeks. After that, the colonies were successively fixed with absolute ethanol for 15 min and stained with crystal violet (0.5% w/v) for 30 min. Finally, the colonies per well were counted and imaged.
Luciferase reporter assays
Luciferase reporter plasmids that contained the wild-type and mutant of the MIER3 promoter was constructed. The wild-type (pmirGLO-lncRNA MIER3 3’UTR wt) and mutant (pmirGLO-lncRNA MIER3 3’UTR mut) of luciferase reporter plasmids were co-transfected with miRNA-486-5p mimic or NC into A549 cells for 48 h. Luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega, USA).
MSCs were fixed with 4% paraformaldehyde for 15 min at room temperature. Following blocking with 5% FBS for 1 h at room temperature. Then the cells were incubated with primary antibody at 4˚C overnight. Then, the MSCs were stained with secondary antibody at 37˚C for 1 h. The primary antibody (1:200 dilution) and secondary antibody (1:400 dilution) were purchased from Abcam (UK).DAPI was used to stain the nucleus at room temperature for 3-5 min. Images were captured using a laser confocal microscope (Olympus, Japan).
RNA fluorescence in situ hybridization (FISH) assay
FISH assay was performed on the tissue according to the manufacturer's instructions (Boster, USA). Briefly, the dewaxed sections were fixed with 4% paraformaldehyde for 20 minutes and dehydrated in an ascending series of ethanol solutions. Then, the dewaxed sections were prehybridized in hybridization buffer (formamide, 50 mM Tris-HCl, 5 mol NaCl, and 0.05% sodium dodecyl sulfate). The dewaxed sections were hybridized overnight at 42 ℃ with probe (probe of miRNA-486-5:)(Servicebio, China). Finally, the nucleus were stained with DAPI (Solarbio, China) and the images were acquired using a confocal microscope (Olympus, Japan).
HE staining was performed on the tissue according to the manufacturer's instructions (Solarbio, China). Briefly, the dewaxed sections were firstly incubated with hematoxylin to stain the nucleus for 5 min, then 1% ethanol-hydrochloric acid for 30 s and eosin solution for 3 min. Finally, the sections were dehydrated in graded alcohol following by clearing in xylene. Images were acquired on an Olympus BH2 microscope (Olympus Corporation, Japan).
Sections were deparaffinized, rehydrated, retrieved the antigens, then incubated with 1% H2O2 in methanol for 15-20 min to block endogenous peroxidase. After blocked with 5% bovine serum albumin, sections were incubated overnight with antibody (1:100 dilution) at 4°C. Then, incubating sequentially with a biotinylated secondary antibody and horseradish peroxidase conjugated streptavidin (1:400 dilution) for 30 min at 37℃. Immunoreactivity was visualized using diaminobenzidine (DAB). Then a light hematoxylin counterstain was applied. Images were captured by Olympus BX61 microscope (Olympus, Japan).
Quantitative real-time polymerase chain reactions (qRT-PCR)
Cultured cells were lysed by TRIzol (Invitrogen), and RNA was extracted according to the manufacturer’s instruction. 1 μg of total RNA from each sample was reverse transcribed using M-MLV (Takara) in a final volume of 20 μL. qRT-PCR was performed by the kitspecification of [email protected] Premix Ex Taqm II (TakaRa, Japan) on iQ5 Real-Time PCR System (BioRad, USA). All quantitative real-time PCR (qRT-PCR) results were carried out in duplicate and normalized to U6.
This experiment was accordance with previous study(19). In briefly, tissue or A549 cells lysates were prepared in Protein Lysis Buffer (Beyotime, China) according to the manufacturer’s directions. The primary antibody (1:1000 dilution) and secondary antibody (1:8000 dilution) were purchased from Abcam (UK). GAPDH was used as an endogenous control to normalize the protein expression data.
All animal procedures were performed according to national guidelines and approved by the Animal Care Ethics Committee. Twenty male mice (4–6 weeks old, Laboratory Animal Center of Shanghai, Academy of Science) were procured. All mice received subcutaneous injections of A549 cells in the right armpit (1×107 cells in 200 μL of PBS per mouse). Twenty mice were randomly divided into two groups (the control group and MSCs-exosome group) when the tumors reached a volume of 50–100 mm3 (3 weeks after subcutaneous injections of tumor cells); exosomes (100 μg of total protein) were injected into the implanted tumors every 2 days for 10 times; the control group was injected with equal volume PBS. The mice were examined every 2 days, and all mice were sacrificed by cervical dislocation under general anesthesia with chloral hydrate (5%, 100 μL/10 g).
All data were analyzed using the InStat software (GraphPad, CA, USA) and displayed as mean ± SD. Two-tailed Student’s t-test was used for statistical analysis, and significance was defined at *** p<0.001, ** p<0.01, * p<0.05.