The human fibroblasts WI-38 and IMR-90 cell lines were purchased from American type culture collection (ATCC), and cultured at 37 °C in a humidified atmosphere containing 5% carbon dioxide with F-12K (Hyclone, Beijing, China) supplemented with 20% FBS (Gibco, Invitrogen, Carlsbad, CA, USA).
The human fibroblasts WI-38 and IMR-90 cells were irradiated with 137Cs at the doses of 0 Gy (control), 4, 6, 8, 10 and 12 Gy respectively. Dose rate was 77 cGy/min and SSD was 10 cm.
According to standard procedures of western blotting, the protein samples extracted from the fibroblasts WI-38 and IMR-90 cells after receiving irradiation which were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA). After blocking with 5% non-fat milk in TBST for 1 h at room temperature, these membranes were incubated with the following primary antibodies (1:1000) at 4 °C overnight: anti-VTN monoclonal antibody (mAb) (Abcam, Cambridge, MA, US), anti-collagen I mAb (Abcam, Cambridge, MA, US), anti-collagen III mAb (Abcam, Cambridge, MA, US), anti-α-SMA mAb (CST, Beverly, MA, USA), anti-TGF-β mAb (Biolegend, San Diego, CA, USA), anti-p-ERK mAb (Santa, Santa Cruz, CA, USA), anti-ERK mAb (Pierce, Cruz, CA, USA), anti-p-AKT mAb (CST, Beverly, MA, USA), anti-AKT mAb (CST, Beverly, MA, USA), anti-p-JNK mAb (CST, Beverly, MA, USA), anti-JNK mAb (CST, Beverly, MA, USA), anti-GAPDH mAb (Sigma-Aldrich, St. Louis, MO, USA) and anti-β-actin mAb (Bioworld Technology, Inc. St. Louis, MO, USA). After washing three times with TBST, the membranes were incubated with secondary antibodies conjugated with HRP (1:10,000) for 1 h at room temperature. At last, these membranes were visualized using enhanced chemiluminescence reagents (Pierce, Cruz, CA, USA) according to the manufacturer’s instructions.
The protein levels of VTN, collagen I, collagen III, Hydroxyproline and α-SMA in WI-38 and IMR-90 cells after receiving irradiation were detected with TMB enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, GIBCO, Carlsbad, CA, USA) according to the manufacturer’s instructions. The absorbance was measured by a microplate reader (Bio-Rad, Hercules, CA, USA).
RNA extraction and quantitative real-time PCR
Total RNA from cultured WI-38 and IMR-90 cells or tissue samples after received irradiation was extracted by using RNA-Trizol reagent (Invitrogen, Gibco, Carlsbad, CA, USA), and reversely transcribed into cDNA using extraction kit (Amresco, Solon, HO, USA). Primers for quantitative RT-PCR (qRT-PCR) were listed in Additional file 1: Table S1. PCR process and data collection were performed on the 7900HT Fast Real-Time PCR system (Applied Biosystem, Carlsbad, CA, USA) according to the manufacturer’s protocol and GAPDH was used as the reference gene.
Vector construction, lentivirus production and transduction
The cDNA sequence of VTN was amplified from pGEM-VTN and constructed into the vector pCDH to generate pCDH-VTN. The vector pCDH-VTN went through initial bacterial colony, PCR filtering, doubled digestion and gene sequencing assessment, and then was used to prepare lentivirus by co-transfecting into 293T cells with liposome. Three siRNA sequences targeted at VTN were designed by RNAi designer, and synthesized as follows in Additional file 1: Table S2. The siRNA sequences were inserted into PLVX vector to generate PLVX-si-RNA-VTN. A mixture of pGEM-VTN or PLVX-si-RNA-VTN, psPAX2 and pMDG2 were co-transfected into 293T cells using lipofectamine 2000 reagent to produce lentivirus. WI-38 cells were infected with the recombinant lentivirus-transducing units and 8 μg/mL polybrene (Sigma, St. Louis, MO, USA).
In vivo study
This study was approved by the Ethic Committee of Shanghai Chest Hospital, Shanghai Jiao Tong University (050432-4-1008A) and all the experiment procedures involved in animals were conducted according to the Guideline of Animal Care and Use Committee of Shanghai Chest Hospital, Shanghai Jiao Tong University. Sixty male C57 black 6 (C57 BL/6) mice with 3 months old which were purchased from the Shanghai Model Organisms Center, Inc., (Shanghai, China) were randomly divided into 5 groups, including normal group (for the mice without irradiation), model group (for the mice with irradiation only), sc-RNA group (for the mice with empty virus with irradiation), OE-VTN group (for the mice infected with VTN-overexpressing lentivirus with irradiation) and the siRNA group (for the mice infected with VTN-si-RNA lentivirus with irradiation). Saline (for model group), empty lentivirus (the vacant group as negative control). VTN-overexpressing lentivirus (for the OE-VTN group) and VTN-si-RNA lentivirus (for the siRNA group) were given to C57 BL/6 mice (12-week-old C57 BL/6, male, 12 mice/group, 1 × 107 TU/mL) via tail vein injection, respectively. After transfected for 48 h, the mice in the 4 irradiation groups received a single dose of 12 Gy irradiation at the whole semi-thorax by using 6 MeV linear accelerator (ClinaciX, Varian), once only a mouse could receive irradiation. SSD was 100 cm, dose rate was 600 cGy/min, and irradiation length was 1 cm. After 8 or 12 weeks of irradiation, mice were sacrificed. Their lungs were obtained surgically and then subjected to hematoxylin and eosin (H&E) staining. The levels of hydroxyproline were evaluated by alkaline hydrolysed sample assay according to protocol.
Immunohistochemical staining and fibrosis score
Immunohistochemical staining was operated according to standard procedures. In brief, the lung tissues from all sacrificed rats were firstly fixed with 4% polyformaldehyde (PFA). Then they were produced into 3.5 μm sections, respectively. Tissue slides were incubated at 4 °C overnight with anti-VTN mAb (1:50) (Abcam, Cambridge, MA, US), anti-collagen I mAb (1:50) (Abcam, Cambridge, MA, US). Lastly, these sections were observed under a microscope and five random fields were chosen to evaluate the fibrosis score. Scores were measured by the cell cytoplasm staining patterns of the lung tissues as described: score 0, absent staining; score 1, light yellow staining; score 2, light brown staining; score 3, dark brown staining. Fibrosis scores were analyzed by two senior and experienced pathologists in single-blind review and evaluated the pathological changes according to the method proposed by Phillips et al. .
All experiments were repeated at least three times in this study. The statistical analysis was performed by using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). All data were shown as mean ± standard deviation (SD) unless otherwise noted. The main statistical methods were t-test and one-way ANOVA. p < 0.05 was considered as statistically significant.