Human non-small cell lung cancer cells A549 were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (GIBCO, Invitrogen, Grand Island, NY, USA) containing 10% fetal bovine serum (Solarbio, Beijing, China) and 1% penicillin-streptomycin (Solarbio, Beijing, China) at 37˚C, 5% CO2.
Cell transfection and grouping
Cells were passaged and inoculated on a 6-well plate. When the cells reached 70% confluence, transfection was performed according to the lentiviral transfection instruction (Shanghai Jikai Biotechnology Co., Ltd., Shanghai, China). The cells were divided into 6 groups: i) blank control group (BC), no treatment; ii) radiotherapy group (Rad), cells were exposed to a single dose of X-rays using a linear accelerator (RadSource, Suwanee, GA, USA) at a dose rate of 1.15 Gy/min and 160 kv X-ray energy,18 no transfection; iii) miR-381 overexpression negative control group (NC-1), cells were radiotherapy treatment and transfected with miR-381 scramble; iv) miR-381 overexpression group (miR-381), cells were radiotherapy treatment and transfected with miR-381 mimics; v) miR-381 silencing negative control group (NC-2), cells were radiotherapy treatment and transfected with miR-381 inhibitor negative control; and vi) miR-381 silencing group (si-miR), cells were radiotherapy treatment and transfected with miR-381 inhibitor.
Cell counting kit (CCK)-8 assay
The proliferation ability of cells was performed by the CCK-8 assay kit (Dojindo, Japan) according to the manufacturer’s protocol. Logarithmic growth phase cells were cultured in 96-well plates at a density of 2×104 cells/ml, 100 μl per well. After culture for 24 h, 48 h, 72 h, and 96 h, 10μl of CCK-8 solution was added into plates and then followed with incubation for 4 h at 37˚C, 5% CO2. The optical density (OD) was measured at 450 nm.
Colony formation assay
Logarithmic growth phase cells were digested with 0.25% trypsin and adjusted to 250 cells/ml. 2 ml/well cells were cultured in a 6-well plate at 37°C, 5% CO2 for 2-3 weeks and the fresh medium was changed every 3 days. The cells were fixed in methanol and each well was added with 1mL Ji Giemsa working fluid and stained for 30 min. After washed twice with ultrapure water, the record was imaged by a camera.
24 h after transfection, the cells were collected and resuspended with pre-chilled 1× PBS, and centrifuged at 1000 rpm for 5-10 min. After washing, 300 μl 1× binding buffer was added to the cells for suspension, and 5 μl Annexin V-FITC was added. The cells were mixed and incubated in dark for 15min. Then, 5 μl propidium iodide (PI) was added and incubated in the dark for 5 min. 200 μl 1× binding buffer was added prior to analysis using flow cytometry (Beckman Coulter, Brea, CA, USA). CellQuest software (BD Biosicences, San Diego, USA) was used to analyze the results.
TdT-mediated dUTP nick end labeling (TUNEL) staining
TUNEL apoptosis detection kit (C1098, Beyotime, Nanjing, China) was used to analyze the cells apoptosis. Cells were crawled and fixed with 4% paraformaldehyde for 30 min. After cultured in PBS containing 0.3% Triton X-100 at room temperature for 5 min, 0.3% H2O2 methanol solution was added to the cells and deactivated the samples for 20 min. TUNEL reaction mixed droplets were prepared and added to the specimen, reacted in a dark humid chamber at 37˚C for 1 h. After washed once with PBS, 0.3 ml labeled reaction stop solution dropwise were added to the cells and incubated at room temperature for 10 min. Then, the cells were incubated with 100 μl Streptavidin-HRP working solution at room temperature for 30 min. The cells were developed with DAB, then followed by hematoxylin counterstained, ethanol gradient dehydrated, xylene transparent, and neutral resin sealed. The apoptotic cells were observed under a ×400 optical microscope (BX50, Olympus, Japan) and counted by Aperio Imagescope 11.1 software. The cells were tan or brownish yellow and had morphological characteristics of apoptotic cells were judged as apoptotic positive cells. Apoptotic index (AI) = (the number of apoptotic positive cells ÷ the number of total cells) × 100%.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The cells were collected and centrifuged at 4°C, 12,000 rpm for 15 min. Total RNA was extracted with TRIzol kit (Takara, Dalian, China) (OD260/OD280 between 1.8-2.0, indicating that the RNA purity is acceptable). The cDNA was synthesized by the reverse transcription kit (Applied Biosystems, Waltham, MA, USA). Mastercycler® nexus X2 (Eppendorf, Hamburg, Germany) was used for RT-PCR. The data was processed by the 2-ΔΔCt method, and the relative expression level was calculated using U6 mRNA as an internal reference. The sequence of the primers (Shanghai Sheng gong Bioengineering Technology Service Co., Ltd., Shanghai, China) are as follows: miR-381, Forward: 5'-GTCTATACAAGGGCAAGCTCTC-3' and Reverse: 5'-ATCCATGACAGATCCCTACCG-3'. U6, Forward: 5'-GACCTCTATGCCAACACAGT-3', Reverse: 5'-AGTACTTGCGCTCAGGAGGA-3'.
The cells were lysed, and centrifugated at 2000rpm for 20min. The protein concentration was measured by BCA kit (Solarbio, Beijing, China). 40 μg samples of each group were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Mini-protean-3, Bio-Rad, Hercules, CA, USA), and transferred to polyvinylidene difluoride (PVDF) membrane (Merck, Darmstadt, Germany). After blocking with 5% skimmed milk, the membranes were incubated overnight at 4˚C with primary rabbit anti-human antibodies against ROCK2 (1:1000, ab71589, Abcam), P65 (1:500, orb229138, Biorbyt, Cambridge, UK), p-P65 (1:500, orb304662, Biorbyt, Cambridge, UK), IκBα (1:500, orb223182, Biorbyt, Cambridge, UK), p-IκBα (1:500, orb223035, Biorbyt, Cambridge, UK), and β-actin (1:2000, orb178392, Biorbyt, Cambridge, UK). Subsequently, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:1000, #7074, Cell Signaling Technology, USA) was used. The membranes were observed and recorded by enhanced chemiluminescence (ECL) system (ImageQuant LAS 4000, General Electric Company, Fairfield, CT, USA). Protein expression levels were normalized with β-actin, scanned and quantified by Image J (NIH) software.
To verify the relationship between miR-381 and NF-κB signaling pathway, the cells were divided into 5 groups: i) blank control group (BC), no treatment; ii) radiotherapy group (Rad), cells were radiotherapy treatment and no transfection; iii) miR-381 mimic group (miR-381), cells were radiotherapy treatment and transfected with miR-381 mimics; iv) NF-κB inhibitor (PDTC, American Sigma) group (PDTC), cells were radiotherapy treatment and cultured with 100 μmol/L PDTC reagent; and v) miR-381 simulant + NF-κB activator (PMA, Sigma, USA) group (miR + PMA), cells were radiotherapy treatment and transfected with miR-381 mimics cultured with 100 μg/L PMA reagent. The above experiments were repeated.
Animals and ethics statement
36 female Balb/c nude mice (4 weeks old, 16-18 g weight) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., license number SCXK (Lu) 2014-0007. The feeding environment were maintained in the standard conditions of 26-28℃ temperature, 50-60% relative humidity, 12 h light/dark cycle with the freely accessed to drinking water and food. Animal experiments were conducted following the National Institute of Health (NIH) guidelines (NIH Pub. No. 85-23, revised 1996). The experiments have been reviewed and approved by the Animal Protection and Use Committee of Tianjin Medical University Cancer Institute and Hospital.
Construction and grouping of lung cancer xenograft model
The lung cancer cells A549 in logarithmic growth phase were digested with 0.25% trypsin, collected and counted. The cell concentration was adjusted to 2.5×106 cells/ ml, and 0.2 ml was inoculated into the soft skin of the right forelimb back of the nude mouse. 36 mice were randomly divided into 6 group (n=6): i) blank control group (BC); ii) radiotherapy group (Rad); iii) miR-381 mimic group (miR-381); iv) Radiotherapy + miR-381 overexpression negative control group (NC-1); v) Radiotherapy + miR-381 silent group (si-miR), and vi) Radiotherapy + miR-381 silent negative control group (NC-2). One week later, except for the control group, the other nude mice were irradiated with 6MV X-rays at a dose rate of 2 Gy/min. A lead plate with a distance of 100 cm and the latter 3 mm covered other parts of the nude mice, and the dose was 10 Gy.
Tumor volume calculation
The tumor's long diameter (L) and short diameter (W) were measured every week with vernier caliper. The tumor volume (V) = (L x W2)/2. Tumor was measured on a weekly basis for 4 weeks and the tumor growth curve was drawn. 4 weeks later, nude mice were anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital (40 mg/kg, New Asiatic Pharmaceutical, China) and sacrificed by cervical dislocation.
After conventional sectioning of the tumor tissue, the slices were baked, dewaxed with xylene, and sequentially hydrated with a gradient ethanol solution. 3% H2O2 methanol solution was used to inactivate processing for 20 min. The slices were heated with citrate buffer (pH 6.0) for 10 min and sealed with 5% BSA for 20 min. After blocking with 5% goat serum (Gibco, USA) for 20 min, the slices were incubated with rabbit anti-human PCNA (1: 500, orb251877, Biorbyt, Cambridge, UK) polyclonal antibody overnight at 4 °C. Then, the goat anti-rabbit IgG (1: 1000, ABIN101988, antibodies-online, Germany) labeled with horseradish peroxidase was used for secondary antibody incubation. 3,3’-Diaminobenzidine staining, hematoxylin counterstaining, dehydration and sealed. The expression of PCNA in each group was observed under a × 400 light microscope (Olympus, Japan) and count using AperioImagescope 11.1 software.
Tumor tissue was ground, homogenized, and centrifuged at 10,000 rpm for 10 min. The primary antibodies were rabbit anti-human Bax (1:500, orb224426, Biorbyt, Cambridge, UK), Bcl-2 (1:500, orb228150, Biorbyt, Cambridge, UK), and Caspase-3 (1:500, orb10231, Biorbyt, Cambridge, UK). Other experimental steps were the same as above western blot.
Double luciferase reporter assay
The wild type and mutant 3'UTRs of ROCK2 were amplified in pGL3/luciferase vector (Promega, Madison, WI, USA) and cloned to the downstream of the luciferase gene. The cells were tested for luciferase activity using a dual luciferase reporter system (Promega) 48 h after transfection according to the instructions.
SPSS19.0 statistical software was used to analyze the data. The results were expressed as mean ± standard deviation (SD). Data analysis between the two groups using t test. Means of analysis between multiple groups were analyzed by single factor analysis of variance (ANOVA), and subsequent analysis was performed by LSD test. P < 0.05 indicates that the difference is statistically significant.