Clinical specimen collection
Totally 30 NSCLC tissues and matched adjacent normal tissues were harvested from patients underwent surgery in the Second Xiangya Hospital of Central South University from August 2013 to April 2014 and immediately frozen at -80 °C after operation. The patients included 17 males and 13 females (with 18 of them aged greater than 60 years, and 12 aged less than 60 years). All tumor samples were confirmed by three pathologists and classified in accordance with the 7th edition of the American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) system. Patients diagnosed histopathologically and with complete follow-up and imaging records were included. Patients with secondary tumors or the history of other tumors and those received any radiation therapy or chemotherapy before surgery were excluded. According to the 7th edition of AJCC-TNM system, 16 patients were in class Ⅰ, while 14 in class II or III. Meanwhile, there were 4, 12, 9 and 5 patients in N0, N1, N2 and N3, respectively on the basis of the TNM system. The collection of clinical tissues was permitted by Research Ethics Committee of the Second Xiangya Hospital of Central South University and written informed consent was gathered from all enrolled patients.
Cell culture and treatment
NSCLC cell lines A549, H1650 and SK-MES-1 as well as normal lung cell line WI-38 used for the experiment were from American Type Culture Collection, Manassas, VA, USA. The cells were all cultivated in Roswell Park Memorial Institute (RPMI)-1640 culture medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. The culture condition was controlled at 37 °C with 5% CO2.
miR-130a-5p mimic/inhibitor, pcDNA RUNX2, short hairpin RNAs (shRNAs) targeting RUNX2 (sh-RUNX21#, 2#, 3#), pcDNA STK32A, sh-STK32A (1#, 2#, 3#) were from GenePharma Corporation (Shanghai, China). After 48 h of co-culture transfection, a medium containing 3 µg/mL perimycin was used to screen stable cell lines for 10 to 14 days. The transfection efficiency was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
RNA isolation and RT-qPCR analysis
Total RNA was isolated from cells and tissues with Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized with PrimeScript™ RT Master Mix (Takara Holdings Inc., Kyoto, Japan). RT-qPCR was performed on the StepOnePlus real-time PCR system (Life Technologies, Foster, CA, USA) using SYBR Premix ExTaq™ II (TaKaRa). The mRNA and miRNA expression were respectively normalized to β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The differential expression of miRNA and mRNA was calculated by the 2−ΔΔCt formula. The primers applied were as follows: miR-130a-5p, forward primer, 5’-ACACTCCAGCTGGGGCTCTTTTCACATTGT-3’, reverse primer, 5’-CTCAACTGGTGTCGTGGAGTC GGCAATTCAGTTGAG AGTAGCAC-3’; RUNX2, forward primer, 5’-TCCACACCATTAGGGACCATC-3’, reverse primer, 5’- TGCTAATGCTTCGTGTTTCCA-3’; STK32A, forward primer, 5’-GCATGGTCTGTAGGGCCTTG-3’, reverse primer, 5’- TGGGGGTAACTTCAACTGCC-3’; GAPDH, forward primer, 5’-GCACCGTCAAGGCTGAGAAC’, reverse primer, 5’- TGGTGAAGACGCCAGTGGA-3’; U6, forward primer, 5’- GCTTCGGCAGCACATATACTAAAA-3’, reverse primer, 5’-GCTTCGGCAGCACATATACTAAAAT − 3’.
Cell counting kit-8 (CCK-8) assay
Cell proliferation was determined by a CCK-8 assay. The cells in each well were treated with 10 µL CCK-8 solution (Beyotime Biotechnology Co., Ltd., Shanghai, China) at 37 °C for 2 h, and then the optical density (OD) value at 450 nm was measured with a microplate reader (BioTek Instruments, Winooski, VT, USA).
Colony formation assay
The cells were detached with trypsin and seeded into a 6-well plate at 200 cells each well. The cells were grown at 37 °C in a Dulbecco’s modified Eagle’s medium with 10% FBS with 5% CO2, with the medium renewed once in 3 days. After 2 weeks, the colonies formed were fixed with methanol for 15 minutes at room temperature and stained with 1% crystal violet for 30 minutes. Under a light microscope (× 100, Olympus Optical Co., Ltd., Tokyo, Japan), the number of colonies (more than 50 cells) was calculated.
A 24-well Transwell plate (8 µm, Costar Technologies, Inc., Coppell, TX, USA) was used for Transwell assays. The cells and serum-free medium were added to the apical chamber and the RPMI-1640 medium was supplemented to the basolateral chamber with 10% FBS. When the plates were incubated for 16 h at 37 °C with 5% CO2, the cells on the lower side of the chamber were stained with crystal violet. Five random fields on the lower side of the chamber were selected for quantification. Diluted Matrigel (50 µL, BD Biosciences, San Jose, CA, USA) was pre-coated on the apical chamber for the invasion assay.
Cells in the logarithmic growth phase were seeded on the 6-well plate and subjected to propidium iodide (PI)/Annexin-V staining. After a 15-minute incubation at room temperature in darkness, the cells were resuspended into cell suspension with 300 µL 1 × binding buffer in the dark and then transferred into a 5 mL flow tube. The number of apoptotic cells was detected by a flow cytometer (ABI Company, Oyster Bay, N.Y., USA) within one hour.
Western blot analysis
The protein was extracted using immunoprecipitation lysis buffer (P0013, Beyotime Biotechnology, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride (ST506, Beyotime). After full lysis, the cells were centrifuged for 3–5 min at 10000–14000 × g to harvest the supernatant. A bicinchoninic acid kit (P0009, Beyotime) was applied to determine the protein concentrations. After isolation with sodium dedecyl sulfate polyacrylamide gel electrophoresis and membrane transferring, the membranes were sealed for 1 h with Tris buffered saline with Tween containing 5% bovine serum albumin at room temperature on a shaker. The membranes were then probed with the primary antibodies at 4 °C overnight and with the secondary antibody for 4 h at 4 °C. The immunoreactive bands were visualized using chemiluminescence reagents (WBKLS0100, Millipore Corp, Billerica, MA, USA). The primary antibodies included E-cadherin (1:30000, ab40772), N-cadherin (1:100, ab18203), vimentin (1:3000, ab92547), RUNX 2 (1:100, ab23981), GAPDH (1:2500, ab9485) and the corresponding horseradish peroxidase-labelled secondary antibody (1:50000, ab205718). All antibodies above were from Abcam (Cambridge, UK).
Luciferase reporter assay
The binding relationship between miR-130a-5p and RUNX2 was detected using dual luciferase reporter assays. The wild-type (WT) RUNX2 sequence, 3’untraslated region (3’UTR) sequences of RUNX2 containing the predicted binding sites for miR-130a-5p, or mutated RUNX2 sequence, 3’UTR sequences of RUNX2 without miR-130a-5p binding sites were inserted into pMIR-REPORT™ promoter vector (Thermo Fisher Scientific Inc., Waltham, MA, USA). Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA).
Chromatin immunoprecipitation (ChIP)
The cells were treated with 37% formaldehyde (1% final concentration). The ultrasonic breaker was then set to 4.5 s per ultrasonic cycle with 9-s intervals with 14 cycles to break the chromatin. Protein-DNA complexes were immunoprecipitated using RUNX2 antibody (ab23981, Abcam) or IgG (ab125900, Abcam) as a control. The immunoprecipitated DNA was purified and tested by qPCR. STK32A promoter fragment containing RUNX2 elements were amplified using STK32A-1 forward primer 5’-CAGTCAGTGGAGCTC-3’, reverse primer 5’-AGCTTGTCCAGG-3’; STK32A-2 forward primer 5’-ACTTGGCAGCCCAGACCTGAGCAT-3’, reverse primer 5’-AGTACCAGACGACTCACGTAGCC-3’; STK32A-3 forward primer 5’-ACAGTTCCGGAAGCC-3’, reverse primer 5’-GACCTACGTTCCGACT-3’; STK32A-4 forward primer 5’-ACTGACGTG TACCCC-3’, forward primer 5’-ACCCTC GCTA GCAC-3’..
In vivo study
Five-week-old female athymic (nu/nu) BALB/c mice were ordered from the animal center of the Second Xiangya Hospital of Central South University. Approximately 5 × 106 A549 cells in 200 µL phosphate buffered saline transfected with negative control (NC) mimic, miR-130a-5p alone or in the presence of pcDNA or pcDNA RUNX2 were injected subcutaneously into the axilla of the BALB/c nude mice (n = 3). Tumor growth was determined as the formula 0.5 × length × (width)2. After 4 weeks, the mice were subjected to an intravenous injection of Barbiturate at 100 mg/kg for euthanasia. Euthanasia was considered to be successful if there was no cardiac arrest, no spontaneous breath for 2 to 3 min and no blinking reflex in mice. The in vivo studies were carried out as per the institutional ethics guidelines involving animal experiments, which were accepted by the Animal Management Committee of the Second Xiangya Hospital of Central South University.
Values are displayed in the form of mean ± standard deviation (SD). SPSS 22.0 (IBM Corp., Armonk, NY, USA) was applied for statistical analyses. Differences were compared using unpaired t test, one-way or two-way analysis of variance (ANOVA) along with a post-hoc Tukey’s test. For the five-year follow-up survival, log-rank test was used for analysis. p < 0.05 was indicative of a statistically significant difference.