Thirty-five pairs of resected specimens were selected randomly, all of them were non-small cell lung cancer patients with complete clinical data and confirmed by pathological diagnosis. The average age of the patients was (46.2 ± 8.1) years old. All patients had not received any form of anti-cancer treatment three months before the operation. Each specimen included lung cancer tissues and lung tissues more than 5 cm away from the cancer focus. This study protocol was approved by the Ethics Committee of Huaihe Hospital of Henan University (Kaifeng, China), and written informed consent was obtained from each participant.
Cell lines, Transfection and interference
The human NSCLC cell lines A549, SPC-A1, PC9, H2170 and SK-MES-1 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Gibco, Rockville, MD) containing 10 % fetal bovine serum and 100 U/ml penicillin and 100 μg/mL streptomycin (Sigma, St. Louis, MO, USA) with 5% CO2 at 37 °C. NC-mimic, miR-185-5p mimic, pcDNA3.1 and pcDNA-RAB35 were purchased from RiboBio Co., Ltd (Guangzhou, China) and transfected using RiboBio Transfection Kit (RiboBio Co., Ltd). Small interfering RNA of RAB35 (siRAB35) and scramble siRNA of RAB35 (Scramble) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All transfection reagent transfected into cells using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
RNA extraction and Quantitative Real-Time PCR Analysis
Total RNA was isolated from osteoarthritis chondrocytes by using the TRIzol (Invitrogen, Carlsbad, CA, USA)). Single-stranded cDNA was synthesized with the PrimeScript Reagent Kit (Promega, USA). Real-time PCR was conducted by using SYBR Premix Ex TaqTM Kit (Applied Biosystems, Foster City, CA, USA). The reaction was run in ABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA). GAPDH was used as an endogenous control. The RT PCR cycling conditions consisted of: 95 ˚C for 10 min; then 35 cycle amplification for 20 s at 95 ˚C, 30 s at 55 ˚C, 15 s at 72 ˚C; followed by 1min at 72 ˚C. The primers used in this study were synthesized from Sangon Biotech (Shanghai, China). The level of mRNA was normalized to β-actin expression using the 2-ΔΔCt method.
Cell proliferation assay
The A549 cells or H2170 cells cultured for the indicated periods of time (0～4days), and cell viability was detected using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions (Beyotime Institute of Biotechnology, Shanghai, China) and read using a microplate reader (Synergy HT, BioTek), at 450 nm.
The A549 cells and H2170 cells were maintained in RPMI supplemented with 10% FBS, and 1×105 cells were plated on BD BioCoat MATRIGEL Invasion Chambers with Matrigel and control inserts with polyethylene terephthalate membrane (BD Biosciences, San Jose, CA, USA) to assess the cell migratory and invasive abilities as previously described.
Luciferase Reporter Gene Analysis
The putative relationship between miR-185-5p and RAB35 were predicted using TargetScan (http://www.targetscan.org/). The wild type and mutant 3'-UTR sequences of RAB35 were cloned into the pcDNA3.1 (+) vector (Cosmogenetech, Seoul, Korea) containing the luciferase reporter gene, respectively. HEK293 cells were seeded in 24-well plates, and when grown to approximately 70% confluence, co-transfected with luciferase plasmid and miR-185-5p mimics or NC mimics using Lipofectamine 2000. After 24 hours of transfection, the luciferase reporter activity was measured by Dual-Luciferase Reporter Assay System (Promega, Madison, WI) under the manufacturer's instructions.
Protein homogenates from A549 cells and H2170 cells were extracted as previously described. Briefly, the cells were lysed for 20 min on ice in ice-cold lysis buffer (Roche). The lysates were centrifuged at 12,000 × g for 20 min at 4◦C to obtain a clear lysate. The protein content of each sample was determined using the BCA Protein Assay Kit (Thermo Scientific). Then, equal amounts of proteins(12μg/lane) were separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA).The membranes were blocked in 5% (w/v) nonfat dry milk in TBST (Tris-buffered saline–0.1% Tween) at 25 °C for 3 h and then incubated with the following primary antibodies: β-actin (1:800, Abcam, EPR16769), RAB35 (1:700, Abcam, ab152138), TSG101 (1:1500, Abcam, ab125011), CD63 (1:1000, Abcam, EPR21151), HSP70 (1:1000, Abcam, EPR16892). The bands were visualized using horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (1:2,000, Boster) prior to the ECL protocol (Amersham Biosciences, Piscataway, NJ, USA).
Exosome isolation and purification
Exosomes were isolated from the supernatant either of the A549 or H2170 cell culture media. In brief, 2×106 cells were plated in a Petri dish with RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. The following day, the medium was changed to exosome-free RPMI supplemented with 10% exosome-depleted FBS. The culture medium was collected until the cells reached 70～80% confluence and exosomes were isolated by ultracentrifugation with a rotor (L8-80M; Beckman, Brea, CA, USA) at the centrifugal force of 110,000 × g for 70 min. Exosomes were re-washed in PBS at 110,000 × g for 70 min to eliminate contaminating proteins, then re-suspended in 100 µl PBS and immediately tested or stored at -80˚C for further analysis.
Exosome enzyme treatment
The exosomes extracted from A549 cells and H2170 cells were treated with 2 μg/ml RNase, which was hydrolyzed at 37 ℃ for 10 min, and then the reaction was terminated with RNase inhibitor (10U/ml). Then, protease K was added and hydrolyzed at 37 ℃ for 60 min. Then the temperature was adjusted to 60 ℃, and the protease was inactivated for 10 minutes. After centrifugation at 110,000 × g for 70 min in 4 ℃, the supernatant was discarded, and the PBS solution was resuspended and precipitated as the exosomes.
All statistical analyses were performed using the SPSS software (ver. 13.0; SPSS, Chicago, IL). The quantitative data derived from three independent experiments are expressed as mean ± SEM. Significance was determined by a one-way ANOVA with the Student paired t-test. Values of P < 0.05 were considered statistically significant.