Mice, cell lines and reagents
Animal experimental protocols were performed in 8–12-week-old female C57BL/6 mice, which obtained from the Institute of Laboratory Animal Science (Chinese academy sciences, Shanghai) and were bred and maintained under specific pathogen-free conditions. All experiment protocols were approved by Medical Experimental Animal Center of Southeast University.
Lewis lung carcinoma cell line was purchased from ATCC (USA) and cultured in RPMI 1640 medium containing 10 % fetal bovine serum. After grew against the wall of flask, formed a monolayer, the cells were digested by 0.25 % trypsin to prepare cell suspensions at 1 × 106/ml.
RPMI 1640 medium and fetal bovine serum were purchased from Gibco (Invitrogen Company, USA). Lipofectamine 2000 transfection reagent was obtained from Invitrogen Life Technologies (Grand Island, NY, USA). Anti-Ku70 antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
Tumor xenograft and irradiation therapy
For tumor transplantation, lewis lung carcinoma cells were transfected with or without miR-545 inhibitor and then suspended in 100 μL PBS and mixed with 20 % matrigel. Tumors were generated by prepared 2 × 106 cells/ml cells subcutaneously injected into the abdomen of C57BL/6 mice. Seven days after the cell inoculated, the mice were divided into two group including control group and radiotherapy group. For radiotherapy, tumors inoculation sites were received a single fraction of 12 Gy/1f/1d. In brief, mice in control group and radiation therapy group were anesthetized by intraperitoneal injection with 10 % chloral hydrate (350 mg/kg). The four limbs were fixed with medical proof fabric. The body surface of tumor location was marked by marker pen and compensated with 1 cm petrolatum gauze. X-ray form 6MV-X (Elekta Limited, West Sussex, UK) vertically irradiated target area under condition of 100 cm Source-skin distance and 12Gy/1f/1d. The irradiated vision is 2.0 cm × 2.0 cm. Notably, mice were kept waking state during radiotherapy. During therapy, the tumor volume was measured using calipers as length × width × depth per two days. Sixteen days after inoculation, animals were euthanized and tumors were removed for Quantitative Real-Time PCR or western blot assay.
Cell treatment with miRNA inhibitor or mimics
Lewis lung carcinoma cells were treated with miR-545 mimic or miR-545 inhibitor (Ambion Pre-miR miRNA Precursors, Life Technologies) using Oligofectamine (Life Technologies) according to the manufacturer’s instructions. Both miRNA mimics negative control (mimic-NC) and miRNA inhibitor negative control (inhibitor-NC) were severed as negative controls in the experiments respectively. Further analysis of the samples (infection or RNA isolation) was performed at 48 h post-transfection. Lewis lung cancer cells were irradiated after 6 h when pretreated with miR-545-mimics, miR-545-inhibitors, or their negative controls. The sequence for this experiment as follows:
Sense strand 5′-UCAGCAAACAUUUAUUGUGUGC-3′
Anti-sense strand 5′-GCACACAATAAATGTTTGCTGA-3′
MTT proliferation assay
Lewis lung carcinoma cells were seeded at a density of 1400 cells/well. On the next day, cells were irradiated with dose 5 Gy for 24 h. After that, 50 mL of 5 mg/mL 3-(4,5-Dimethylthiazol-2-yl) 22,5-diphenyltetrazolium bromide (MTT) was added to wells for 4 h, and then the media were replaced with DMSO for analysis of optical density using a mQuant Microplate Spectrophotometer (BioTek, UK) at a wavelength of 540 nm.
Quantitative Real-time PCR
Total RNA was extracted from the Lewis lung carcinoma cells with the Trizol Reagent (Invitrogen, Carlsbad, CA, USA). The mature miR-545 and Ku70 mRNA were quantified by using Quantitative Real-Time PCR (qRT-PCR) assays with fluorescent nucleic acid dye. Each sample (1 μg) was reverse-transcribed into cDNA by using the RealMasterMix First Strand cDNA Synthesis Kit (Tiangen). Real-time PCR was conducted by using SYBR Premix ExTagTM (Takara) according to the manufacturer’s protocols and performed in the Applied Biosystems 7500 Real-time PCR system. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. The miRNA expression levels were normalized to U6 RNA and the Ku70 mRNA levels were normalized to actin mRNA. All reactions were run in triplicate.
Bioinformatics analysis of miR-545 target in Ku70
Based on bioinformatics analysis, we predicted that hsa-miR-545 can bind with the 3′UTR region of Ku70 by using four common websites (Target Scan: http://www.targetscan.org/, miRanda: http://www.microrna.org/, Microcosm: http://www.ebi.ac.uk/enright-srv/microcosm/cgi-bin/targets/v5/search.pl, and PITA: http://genie.weizmann.ac.il/) (Fig. 3a).
Plasmid construction and luciferase reporter assays
For Ku70 3′UTR reporter assay, Lewis lung carcinoma cells were placed in 24-well plates (1 × 105 cells per well) and then cotransfected with the pGL3 Luciferase Reporter Vectors (Promega, WI, USA) according to manufactures’ protocol. The mimics and inhibitors of hsa-miR-545 and their negative controls (RIBO Bio, Guangzhou, P.R. China) were cotransfected with the reporter plasmids at a final concentration of 100 nmol/μl. 48 h after transfection in Lewis lung cancer cells, luciferase activity in lysates was measured with a Dual-Luciferase Reporter Assay System (Promega, WI, USA) followed by the manufacture’s suggestions and normalized against the activity of the pRL-SV40. Independent triplicate experiments were performed for each plasmid construct.
The TUNEL reaction which based on labeling of DNA strand breaks was conducted to detect cells apoptosis and performed with an in situ cell detection kit (Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer’s instruction. In brief, sections in 2 changes of xylene for 5 min each, and hydrate with 2 changes of 100 % ethanol for 3 min each, and 95 % ethanol for 1 min followed by rinsing in distilled water. After pretreated with proteinase K, cells were cultured in TdT Reaction Buffer for 10 min and then conducted TdT Reaction in TdT Reaction Mixture for 1–2 h at 37–40 °C in humidified chamber. The reaction was stopped with washing buffer and the sample was resuspended in PBS containing FITC-Avidin D for 30 min at room temperature. The reaction mixture was counterstained with PI or DAPI for 20 min. TUNEL-positive nuclei (stained brown) were counted by Motic Images Advanced 3.2 in 10 random fields (×200), and then averaged.
Proteins in Cells were extracted by inM-PER mammalian protein extraction reagent (Pierce Biotechnology) followed by centrifugation at 15 000 g for 10 min. Protein concentration of cell lysates was measured by using DC protein assay kit (Bio-Rad). Proteins (10–20 mg) were separated by 10 % SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane from Bio-Rad (Hercules, CA, USA). Two hours after blocking in 5 % nonfat milk, the protein blots were then incubated with primary antibodies in 3 % bovine serum albumin at 4 °C overnight, followed by incubation with secondary antibodies at room temperature for 2 h. The protein signals were detected by ECL method.
Lewis lung carcinoma cells were treated with Ku70 siRNA or scrambled siRNA as negative control according to the manufacturer’s instructions. Briefly, siRNA and OligofectAmine were mixed separately with OptiMem and incubated for 15 min at room temperature. And then, these reagents were combined and incubated for 15 min before adding to the cells in RPMI 1640 without penicillin and streptomycin. Notably, Ku70 siRNA was cotransfected with miR-545 inhibitor. Twenty-four hours after transfection, the cells were irradiated with a single dose of 5 Gy/1f/1d for 24 h. And then the cell viability and apoptosis were examined accordingly.
All statistical analyses were performed using SPSS 16.0 software (Chicago, IL). Student’s t test was used to analyze the significance between two groups. Error bars represent SD. P-Values < 0.05 were considered statistically significant.