IL-6 Regulates MMP-10 Expression via JAK2/STAT3 Signaling Pathway in a Human Lung Adenocarcinoma Cell Line

Materials and Methods

Cell line and reagents. The human lung adenocarcinoma cell line, A549, was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science (SIBS, CAS, Shanghai, China). Bovine serum albumin (BSA), fetal calf serum (FCS) and RPMI-1640 culture medium were purchased from the Gibco Corporation (Carlsbad, CA, USA). IL-6 was obtained from PeproTech Inc (Rocky Hill, New jersey, USA) and AG490, a specific JAK2 inhibitor from the Alexis Corporation (Plymouth Meeting, PA, USA). The total RNA purification kit was purchased from the Shenergy Biocolor BioScience and Technology Company, Shanghai, China. The first strand cDNA synthesis kit was obtained from Fermantas, Vilnius, Lithuania. The primers and TaqMan probes of glyceraldehyde-3 3-phosphate dehydrogenase (GAPDH), JAK2, STAT3 and MMP-10 were synthesized by the Sangon Biological Engineer Technology and Services Limited Corporation, Shanghai, China. The whole-cell extraction kit and mouse anti human MMP-10 monoclonal antibody was obtained from Chemicon International Inc, (Billerica, MA, USA). The ECL chemiluminescence kit, mouse anti-human GAPDH and peroxidase conjugated goat anti-mouse IgG were purchased from the MultiSciences Biotech Co, Ltd, Hangzhou, China. The LightCycler real-time RT-PCR System was from Roche Applied Science, Mannheim, Germany. Electroblotting apparatus, gel imaging system and Quantity One software v4.6.2 were obtained from Bio-Rad, Hercules, CA, USA. Polyvinylidene difluoride (PVDF) membranes were obtained from the Millipore Company, Billerica, MA, USA.

Cell cultures. The A549 cells were cultured in RPMI-1640 with 10% FCS in the presence of benzylpenicillin (100 U/ml) and streptomycin (100 μg/ml) under standard culture conditions (5% CO₂, 37°C). The cells were seeded into 6-well cell culture clusters and allowed to grow to 50-70% confluence. Prior to the experiments, the cells were washed twice with phosphate-buffered saline (PBS) and once with serum-free RPMI-1640. The experimental medium RPMI-1640 with 10% FCS and different concentrations of IL-6 (0, 12.5, 25 or 50 ng/ml) with or without AG490 (50 μM) was added. The cells were cultured at 37°C for 24 h.

Total RNA extraction and RT-PCR. The total RNA was extracted using the total RNA purification kit according to the manufacturer's instructions. The purity and concentration of the total RNA were determined by spectrophotometer. Only samples with optical density (OD) 260/280 ratios from 1.80 to 2.00 were chosen. Six μl total RNA were reverse transcribed into cDNA by using the First strand cDNA synthetic kit according to the manufacturer's instructions. The synthetic cDNA were preserved at −30°C. The primers and TaqMan probes of GAPDH, JAK2, STAT3 and MMP-10 were designed according to the National Center for Biotechnology Information (NCBI) database (NM_002046, NM_004972, NM_003150 and NM_002425, respectively) by using Primer Primier 5.0 software (Palo Alto, CA, USA) (Table I). All the PCRs were performed on a LightCycler in a final volume of 25 μl. Optimum reaction conditions were obtained with 2.5 μl of 10×PCR buffer, 1.5 μl of 25 mM MgCl₂, 0.5 μl of 10 mM 4×dNTPs, 0.25 μl of 5 U/μl common Taq DNA polymerase, 0.1 μl of 100 μM specific sense primer(s), 0.1 μl of 100 μM specific antisense primer(s), 0.1 μl of 100 μM specific probe(s) and 2 μl template cDNA. Finally, 17.95 μl ddH₂O was added to the reaction mixture. The cycling conditions for GAPDH, JAK2, STAT3 and MMP-10 were as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 15 s, and 60°C for 45 s, collecting the fluorescence signal at 60°C. From each amplification plot, a threshold cycle (Ct) value was calculated, then according to the 2(-Delta Delta Ct) method, the relative mRNA levels of JAK2, STAT3 and MMP-10 were calculated (19).

Western blotting analysis. After being plated at the appropriate seeding density, the cells were incubated with IL-6 (25 ng/ml) in the presence or absence of AG490 (50 μM) for 24 h, the total intracellular proteins were extracted and the total protein concentrations were determined according to the manufacturer's instruction for the whole cell extraction. For immunoblotting, cell lysates containing 30 μg of total protein were applied and separated on the SDS-PAGE. The proteins were transferred to PVDF membranes using an electroblotting apparatus. The membranes were blocked in blocking solution BSA/tris buffered saline (TBS)-T (TBS containing 4% Tween 20 and 3% BSA) overnight at 4°C. The blots were incubated with primary antibody (mouse anti-human MMP-10) for 2 h at room temperature and washed three times with TBS-T. The blots were then incubated with secondary antibody (peroxidase-conjugated goat anti-mouse IgG) for 2 h at room temperature, washed three times with TBS-T, and then developed using a ECL chemiluminescence kit and exposed to the BioMax Film (Kodak, Rochester, NY, USA). The same blot was stripped and reprobed for GAPDH.

Statistical analyses. Statistical analysis was performed with Graphpad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA). The results are expressed as means±SE. Two comparisons were analyzed by Student's t-test. Multiple comparisons were analyzed by the one-way ANOVA/Tukey. Significance was established at a p-value less than 0.05.