Cell culture
Adipose tissues were respectively obtained from patients undergoing liposuction and GC patients peritoneam and healthy donors according to procedures approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. A-MSCs were isolated and culture-expanded as previously discribed[16]. The cells were incubated in DMEM/F-12 (Gibco,USA ) supplemented with 10% FBS (Gibco,USA ), 1% penicillin and streptomycin (Solarbio, China), 2 mmol/L glutamine and 10 ng/mL epidermal growth factor (PeproTech). A-MSCs at passage 3-5 were choosed for the folowing research. To inducing A-MSCs-adipogenesis, A-MSCs were remaining under adipogenic differentiation medium. (DMEM/F-12 1μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 0.5 μg/ml insulin, 0.5 mM isobutylmethylxanthine) with 10% FBS.
SGC7901, MGC803 (human gastric adenocarcinoma cell) were bought from cell bank of Chinese Academy of Sciences (Shanghai, China). SGC7901 and MGC803 cells were cultured in DMEM medium (Gibco, USA) supplemented with 10% FBS。
exosomes isolation and identification
Exosomes in medium was isolated according to previous report. GC cells culture media were collected at 300g and 3,000g eliminate cells and debris. Next, the supernatant was centrifuged at 100000g for 70 min at 4℃ to abtain exosomes. Exosomes was dropped onto the copper grid and negatively stained with 3% (w/V) sodium phosphotungstate solution (pH6.8) for 5 min and washed gently with double distilled water. Exosomes were observed and photographed by transmission electron microscopy (TEM). Western Blot was used to detected exosomes specific markers, including Alix, Tgs101 and CD63. selected randomly to measure the mean diameterusing the Nanosight NS 300 system (NanoSight technology, Malvern, UK).
RNA isolation and quantitative RT-PCR
Total RNA was isolated from the cultured cells, exosomes, and tissues using TRIzol Reagent (Invitrogen), according to the manufacturer's instruction. cDNA was obtained by using avian myeloblastosis virus (AMV) reverse transcriptase (TaKaRa). Quantitis of miR-155 was detected via TaqMan miRNA probes (Applied Biosystems) and normalized to U6 snRNA expression. mRNA expression levels of C/EPBβ, C/EPBα and PPARγ was performed with the SYBR Green PCR Master Mix (Qiagen) through ABI PRISM 7900 (AppliedBiosystems). The mRNA expression was normalized to GADPH. All reactions were performed at least in triplicate. Relative levels of genes were calculated with the 2-ΔCT method. The primer sequences were as follows:
5’GGGCCCTGAATCGCTTA A.3’(C/EPBβ, sense);
5’ATCAACAGCAACAAGCCCG.3’(C/EPBβ, anti-sense);
5’- GAAGTTGGTGGAGCTGTCGG-3’ (C/EPBα, sense);
5’- TGAGGTATGGGTCGTTGGGA-3’ (C/EPBα, anti-sense);
5’- AGCCTCATGAAGAGCCTTCCA-3’ (PPARγ, sense);
5’- ACCCTTGCATAATTCACAAGC-3’ (PPARγ, anti-sense);
5’-AGAAGGCTGGGGCTCATTTG-3’ (GAPDH, sense);
5’-AGGGGCCATCCACAGTCTTC-3’ (GAPDH, anti-sense).
Western blotting
The total protein was extracted using RIPA buffer. The protein concentration was quantified by the BCA Protein Assay kit (Thermo). Protein (30 μg) of each sample was loaded into a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel(SDS-PAGE gels) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking with 5% BSA at room temperature for 1 h, membranes were incubated with primary antibodies (1/1000 dilution) at 4℃ overnight . After membranes were incubated with corresponding secondary antibodies (1/5000 dilution) for 1 h at room temperature. Blots were detected using enhanced chemiluminescence solution (Invitrogen) and visualized with the ImageQuantLAS-4010 (GE).
Oil red O staining
The cells were washed there times with phosphate-buffered saline (PBS) gentally and then fixed with 10% formaldehyde for 10 min at room temperature. After fixation, cells were treated with filtered 0.25% oil red O solution for 40 minutes. red-stained lipid droplets in cells were observed and photographed by light microscopy.
Luciferase assay
The miRNA target prediction and analysis were performed with the algorithms from TargetScan (http://www.targetscan.org/vert_72/), PicTar(https://pictar.mdc-berlin.de/), and miRanda (http://miranda.org.uk/).
The reporter plasmid p-MIR-C/EPBβ was designed by Genescript (Nanjing, China) containing the predicted miR-155 binding site. Part of the wild-type and mutated 3’-UTR of C/EPBβ was cloned into the firefly luciferase reporter. For the luciferase reporter assays, 2 mg firefly luciferase reporter plasmid; 2 mg b-galactosidase vector; and equal doses(200 pmol) of mimics, inhibitors, or scrambled negative control RNA were transfected into AMSCs. At 24h after transfection, luciferase activity of cells were analysed by using the Dual Luciferase Assay Kit (Promega) according to the manufacturer’s instructions.
Immunofluorescence
Cells were cultured in 12-well plates with slides. At harvest, the cells were first fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100 for 10 min, followed by confinement for 1 h. Then, the cells were incubated with C/EPBβ, C/EPBα, and PPARγ (1:100; Abcam) at 4 °C overnight and FITC goat anti-rabbit IgG (Invitrogen) for 1h in the dark at RT. DAPI (Solarbio, China) was used for nuclear staining. For confocal microscopy, a Nikon C2 Plus confocal microscope was used.
Immunohistochemistry
The inguinal adipose tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and then stained with anti-FABP4 antibodies (Abcam). Quantitative analysis was conducted by quantifying the fluorescence intensity from at least five sections.
Establishment of tumor in nude mice
lentiviral expression plasmids that were used to increase or decrease the expression of miR-155 and control lentivirus were purchased from Shanghai Genechem. Puromycin (Sigma-Aldrich, USA) was used to successfully obtain stably infected SGC7901 cells. Cells infected with lentivirus above were injected into nude mice by orthotopic implantation. Mice were sacrificed and tumors were removed at the 28th Day.
Statistics
All experiments were performed in triplicate, and the results are presented as the mean value ± standard deviation. The data were statistically analyzed using Student’s
t-test in SPSS statistical software, with p < 0.05 considered statistically significant. *
indicates p< 0.05; ** indicates p< 0.01 and *** indicates p<0.001