Tissues samples
Human head and neck tumor tissues and paired adjacent non-tumor tissues were obtained from patients at the Harbin Medical University Cancer Hospital (Harbin, China). Both tumor tissues and non-tumor tissues were histologically evaluated. Written or oral consent was provided by all of the involved patients and the Ethics Committee of Harbin Medical University Cancer Hospital approved all aspects of this research.
Cell lines and cell culture
Human head and neck squamous carcinoma cell lines SCC9, SCC15, FaDu were grown in DMEM/F12 (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco). Human bronchial epithelial cell line BEAS-2B were cultured in DMEM supplemented with 10% LHC-9 medium (Invitrogen) and 10% FBS (Gibco). Human hepatocellular carcinoma cell line HepG2, human colorectal cancer cell line HCT-116 and Breast cancer cell line MCF-7 were cultured in DMEM supplemented with 10% FBS. Laryngeal squamous cell carcinoma cell line SNU899 was maintained in RPMI-1640 (Gibco) with 10% FBS (Gibco). Human monocytic THP-1 cells were maintained in RPMI 1640 containing 10 % of heat inactivated FBS and supplemented with 10 mM Hepes (Gibco), 1 mM pyruvate (Gibco), 2.5 g/l D-glucose (Merck) and 50 pM ß-mercaptoethanol (Gibco). THP-1 monocytes were differentiated into macrophages incubated with 150 nM PMA (Sigma) followed for24 h. These cancerous cell lines were maintained in a humidified incubator at 37 °C with 5% CO2.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted with TRIzol reagent and reverse transcribed to cDNA using a PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green qPCR Master Mix (Takara). The sequences of primers were listed in Table 1
Northern blot
RNA was extracted and heat denatured at 65°C for 15 min. The prepared RNA samples were loaded in the gel in the MOPS running buffer, which was electrophoresed at 5-6 V/cm until the bromophenol blue (the faster-migrating dye) migrated at least 2-3 cm into the gel. The gel was visualized on a UV transilluminator. The 28S rRNA band should be approximately twice as intense as the 18S rRNA band.
Cell cycle analysis
The harvested SCC-9 cells were resuspended with PBS and fixed overnight with 70% ethanol at 4 °C, then incubated with PI staining reagent at room temperature for 30 min. The treated cells were analyzed using a FACS Calibur system (BD Biosciences, San Jose, CA, USA).
Western blot
Proteins were extracted from tissues and cells. The protein concentration was determined by the bicinchoninic acid (BCA) Kit (Beyotime, Shanghai, China). Proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane. After blocking, the membranes were incubated in primary antibodies at 4°C overnight. ANLN-201 and ANLN-210 (1:1000, GeneX health, Beijing), ANLN (1:1000, ab211872, Abcam, Cambridge, MA, USA). Myc (ab32072), F-actin (ab130935), HNRNPC (ab75822), SRSF10 (ab254935), U2AF2 (PA5-30442, Invitrogen), HSP70 (ab2787), CD63 (ab134045), TSG101(ab125011), Calnexin (ab22595), CD206 (ab125028), Arg-1 (ab133543), PTEN (ab267787), Akt (ab8805), p-Akt (ab38449), GAPDH (ab9485), and β-actin (ab8226), GAPDH and β-actin was used as the internal control. Protein bands were visualized by chemiluminescence (ECL, Forevergen, Guangzhou, China). The experiment was conducted for three times.
Immunoprecipitation
To detect ANLN associated with F-actin/Myc, total protein was extracted from SCC-9 cells after treatment. Cell lysates for immunoprecipitation were pre-cleared and incubated with GFP-protein-magnetic beads overnight at 4 °C. The beads were washed and boiled with loading buffer, then for immunoblots with GFP (abcam) antibodies.
Isolation of exosomes
Exosomes were isolated from cell culture medium by ultracentrifugation according to the previous reports [22]. The procedures were performed at 4°C. Briefly, cells were removed by centrifugation at 300g. Other debris were removed by centrifugation at 3,000 g. After that, the supernatants were centrifuged at 10,000 g for 30 min to remove shedding vesicles and others. At last, the supernatants were centrifuged at 110,000 g for 70 min. Exosomes were obtained from the pellets and re-suspended in PBS.
Cell proliferation analysis
Cell proliferation of SCC-9 cells was determined using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. SCC-9 cells transfected as experiments designed were plated in 96-well plate. Cell viability was analyzed for 24, 48, 72, 96h. CCK-8 was added and incubated for 3h according to the time points. The absorbance was measured at 450 nm.
Cell migration and invasion assays
Cell migration and invasion assays were performed using transwell insert chambers (BD Biosciences, USA). SCC-9 cells were transfected as experiments designed. The transfected SCC-9 cells in serum-free medium were seeded into the top chamber. The medium containing 20% FBS was added to the lower chambers. After routinely cultured for the appropriate time, cells on the lower membrane surface of the top chamber were fixed with methanol and stained with DAPI. For invasion analysis, the top chambers were coated with the matrigel (BD Biosciences, USA).
Immunofluorescence
SCC-9 cells were fixed and permeabilized, then incubated with GFP Rabbit antibody (ab290, Abcam) for 3 hours at 4 °C. After washing three times with PBS, the cells were incubated with Alexa Fluor 488 conjugated Goat Anti-Rabbit IgG (H&L) (ab150077; Abcam) for 1 h at room temperature in the dark place. After washing three times with PBS, cells were treated with DAPI (D9564, Sigma) for 10 min at room temperature. Cells were photographed using the confocal microscope (Olympus FV-1000).
Gel electrophoretic mobility shift binding assay (EMSA)
Protein-RNA interactions were evaluated using the LightShift Chemiluminescent RNA EMSA kit (Thermo Scientific). The constructed T7 promoter-ANLN-210 WT or ANLN-210 MUT DNA fragments were used as the templates for in vitro transcription to synthesize plenty of RNA. The 3’end of synthesized RNA was biotinylated using RNA 3’End Biotinylation Kit (Pierce, Rockford, IL, USA), which was used as the probe analyzed for EMSA. The recombinant HNRNPC protein was obtained from Origene (TP315956). Biotin-labeled RNA and a dose-dependent amount of HNRNPC were mixed in the reaction buffer. The samples were electrophoresised on the 5% nondenaturing polyacrylamide gel. The signals were detected using the Luminescent image analyzer (FluorChem M).
Immunohistochemistry (IHC)
Paraffin-embedded HNSCC tissues were fixed in 4% polyformaldehyde (PFA). IHC staining was performed according to the manufacturer’s protocol. The deparaffinized and rehydrated histologic sections were aimmersed in 10 mM citrate buffer for heat-induced antigen retrieval. The slides were stained using antibodies against CD163 (ab182422, Abcam) and Ki67 (ab15580, Abcam).
Animals
Male BALB/c nude mice (6∼8 weeks) were raised strictly in the pathogen-free animal house in accordance with feeding standards, which was approved by the Institutional Animal Care and Research Advisory Committee of Affiliated Tumor Hospital of Harbin Medical University.
Xenograft models
To assess the effects of exosomal ANLN-210–activiated M2 polarized macrophages on HNSCC tumor growth in vivo, 1.5 × 107 SCC-9 cells were mixed in a 4:1 ratio with the macrophages transfected with exosomes with different treatment as experiment designed. Then the cell mixture was co-injected into the flanks of the nude mice. After 3 weeks, the mice were euthanized and the tumors were obtained and fixed in formaldehyde for and IHC staining.
Statistical analysis
All the experiments were repeated at least three times. The data were analyzed using SPSS 20.0 and GraphPad Prism 7. The P value <0.05 was considered to be significant.