Cell lines and irradiation condition
Human non-small cell lung cancer A549 cell line, human lung epithelial HBE cell line and BEAS2B cell, human renal epithelial 293T cells were stored in our laboratory and authenticated by ATCC (American Type Culture Collection, USA). A549, BEAS2B and 293T cells were maintained in DMEM (HyClone) with 10% fetal bovine serum (Gibco, Scoresby, Victoria, Australia). In addition, 1% penicillin streptomycin-glutamine was added to the medium, and the cells were maintained at 37 °C in a humidified incubator containing 5% CO2.
γ-Radiation was performed using a 60Co source at the Institute of Radiation Medicine, Academy of Military Medical Sciences (Beijing, China). A549 and H1299 cells were sham-radiated or exposed to radiation at 6 Gy (dose rate: 127.15 cGy/min, source distance: 70 cm, voltage: 180 kV, current: 12.5mA). The cells were irradiated with 60Co γ-rays at room temperature as our team previous reports [4, 39]. To explain whether there exerted dose- and time-dependent manner, A549 cells were subjected to 60Co γ-rays irradiation at 4 Gy, 6 and 8 Gy, respectively (dose rate: 127.15 cGy/min, source distance: 70 cm, voltage: 180 kV, current: 12.5 mA) as our team previous reported [40,41,42].
Cell transfection
The cells were passaged the day before transfection. After the cells were grown to 60% density, siRNA (the sequence are listed in Additional file 1: Table S1) was conducted through the transient transfection using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Scrambled siRNA was used as the negative control. Forty-eight hours after transfection, the cells were collected for further experiments.
Cell proliferation analysis
For the cell proliferation analysis, cells were collected at passage 3–4, and inoculated in 96-well plates at a density of 3 × 103 cells/well. The effects of LGG on cell viability were detected using a standard cell counting kit-8 (CCK-8) according to the manufacturer’s instructions (CK04, DOJINDO, Japan). The optical density (OD) of the cells in each group was tested by measuring absorbance at 570 nm using a microplate reader.
Establishment of co-culture of cells and LGG
Prepared a concentration of 3.0 × 106 cells/ml cell suspension in the six well cell culture plate, discard the cell culture medium, add PBS solution for three times, and then put the 0.4 µM Transwell in the well of the culture plate. Then each well was divided into the upper chamber and the lower chamber. The upper chamber is the upper chamber, and the lower chamber is the inner chamber of the culture plate. Add new cell culture medium in the lower chamber. It is appropriate to just contact the basement membrane of the Transwell cell, which is about 2 ml. Add LGG bacteria diluted with the cell culture medium in the upper chamber, with a final concentration of 1012 CFU/ml at 37 ℃, 5% CO2, and co culture for 4 h, 12 and 24 h, respectively. Then removed Transwell cell, collected the bacterial suspension, centrifuge at 12,000 R/min for 10 min, discard the supernatant, separate the bacteria, add PBS solution for three times, calculated the final concentration of the bacterial solution.
Mice experiments design
Male C57BL/6 mice were purchased at 8 weeks of age from the Hunan SJA Laboratory Animal Co., LTD, China. All of the mice were housed at the Animal Laboratory Division, Xiangya School of Public Health, Central South University, China. All animal procedures and testing were conducted according to the National Legislation and local guidelines of the Laboratory Animals Center at the Central South University. The study and research protocols were approved by the Institutional Animal Review Board of Central South University (2020sydw0110). In addition, all animals in the study were treated humanely with regard to the alleviation of suffering. All the mice were maintained in a specific-pathogen-free (SPF) environment with controlled conditions of a 12 h light/dark cycle at 20–22 °C and 45 ± 5% humidity. After 1 week of acclimation, the mice were used for the study with the group design detailed below.
To investigate the effects of LGG or SNHG17 on radiation-induced EMT progress, mice were divided into five groups: control group (Con), radiation-induced lung fibrosis group with 10 Gy radiation treated once time (10 Gy), mice treated with LGG prior to received 10 Gy radiation on lung (10 Gy + LGG), mice treated with siSNHG17 through tail vein injection prior to received 10 Gy radiation on lung (10 Gy + siSNHG17), mice treated with LGG and siSNHG17 prior to received 10 Gy radiation on lung (10 Gy + LGG + siSNHG17). The LGG was administrated for 7 days prior to radiation via water, and the concentration of bacterial suspension was 4.5 × 109 CFU/ml per mouse. Each group was enrolled 10 mice. Mice were subjected to 60Co γ-rays irradiation at 10 Gy on the lung part and the other parts of mice were shielded with 10 cm thick lead bricks (dose rate: 200 cGy/min, source-skin-distance: 100 cm, voltage: 180 kV, current: 12.5 mA, the field size: 3 cm × 40 cm) at room temperature at the Institute of Radiation Medicine, Academy of Military Medical Sciences (Beijing, China) [40, 43]. Body weight, food intake and water consumption were recorded. The mice were killed at the 7th weeks to collect tissues for further detection of various parameters.
RNA isolation and real-time PCR
Total RNA was extracted from cells by using a Total RNA extraction kit (Vazyme, China) according to the manufacturer’s instructions. After the quality and quantity of the extracted RNA were confirmed by a nucleic acid quantitative detector (NanoDrop 2000c, USA), complementary DNA (cDNA) was synthesized using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, China) according to the manufacturer’s instructions. The Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China) was used for real-time PCR analysis on a PCR platform (Bio-Rad CFX96 Touch, USA) to determine the expression level of lncRNA SNHG17. Then, the relative expression of lncRNA SNHG17 was calculated by the 2−ΔΔCT value method, and GAPDH was used as a housekeeping gene. The specific primers for lncRNA SNHG17, PTBP1 and GAPDH used for RT-PCR are listed in Additional file 1: Table S3. All the primers were all synthesized by Sangon (Shanghai, China). Each PCR amplification was performed in triplicate to verify the results. Primers for PCR is listed in Additional file 1: Table S3.
Western blotting
Cells in the logarithmic growth phase were placed in a 35 mm dish at the appropriate density and cultured in an incubator. Proteins were extracted from irradiated cells by using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Taiwan, China) according to the manufacturer’s instructions. Equal amounts of proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes (Millipore, USA). 5% skimmed milk was used to block the membranes for 1 h, and then, the membranes were probed overnight at 4 °C with the primary antibodies listed in Additional file 1: Table S3. Next, the membranes were incubated with specific secondary antibodies (ZSGB-BIO, Beijing, China) for 1 h at room temperature. Peroxidase labeling was visualized via enhanced chemiluminescence labeling using an ECL Western blotting detection system (Thermo Fisher Scientific, Waltham).The details of antibodies used in this study are listed in Additional file 1: Table S4.
Fluorescence in situ hybridization (FISH)
Cy3-labeled lncRNA SNHG17 probes were designed and synthesized by RiboBio (Guangzhou, China). The probe signals were determined with a Fluorescent In situ Hybridization Kit (RiboBio, Guangzhou, China) following the manufacturer’s guidelines. Images were taken under an immunofluorescence confocal microscope (Crest Optics X-Ligt V3, Italia).
Immunofluorescence staining
Cells plated on 22 × 22 mm2 cover slips in 6-well plates were irradiated and fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized in 0.25% Triton X-100 buffer for 30 min and then blocked in 3% BSA for 30 min at room temperature. Then, the cells were incubated with a PTBP1 monoclonal antibody (Sino Biological, Beijing, China) and Notch1 antibody (Santa Cruz) antibody at 4 °C overnight and washed twice with PBS. Subsequently, the cells were incubated with a FITC-labeled anti-mouse antibody (Invitroge, USA) and a Texas Red-labeled anti-rabbit antibody (Invitroge, USA) at room temperature for 2 h. The slides were finally fixed with a fluorescent sealer containing DAPI (ZSGB-BIO, Beijing, China). Images were obtained under a confocal microscope (Crest Optics X-Ligt V3, Italia) with the NIS-Elements Viewer 4.20 capture system. We observed 3 high-power visual fields (100× oil lens) were randomly selected from each slice to observe the confocal.
RNA immunoprecipitation (RIP)
Cells were used to perform RNA immunoprecipitation (RIP) experiments using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. Cells were spread in a 60 mm dish at the appropriate density and were irradiated after they had grown to the appropriate amount. Then, the cells were rinsed with PBS and centrifuged at 1500 rpm for 5 min at 4 °C, and the supernatant was discarded. Next, the cells were re-suspended in 100 µl of RIP lysis buffer and pipetted until homogeneous on ice for 5 min. Magnetic beads were washed with RIP wash buffer and incubated with 5 µg of the anti-IgG antibody (Millipore, Bedford, MA), anti-PTBP1 antibody (Thermo Fisher, USA) for 2 h at room temperature in 100 µl of RIP wash buffer. After a brief centrifugation, the supernatant was discarded, and the unbound protein antibodies on the magnetic beads were washed away with RIP wash buffer. Then, we centrifuged the cell lysate at 4 °C and 12,000 rpm for 10 min and collected 100 µl of the supernatant. The supernatant was incubated with 900 µl of IP buffer containing magnetic beads conjugated with different antibodies at 4 °C overnight. Before incubation, 10 µl of the sample buffer was removed and marked as the input for later Western blot experiments. The sample buffer was then washed with RIP wash buffer and was used in subsequent Western blot experiments for verification after heat denaturation. At the same time, 150 µl of Proteinase K buffer was added to the sample buffer to dissolve protein. Then, immunoprecipitated RNA was isolated, and co-precipitated RNAs were detected by qRT-PCR.
RNA pulldown assay
The cDNA sequence of SNHG17 and different fragments were cloned into pCDNA3.1 (+). Biotin-labeled RNAs were transcribed in vitro using a biotin-labeling mix and T7 polymerase in the linearized pCDNA3.1 (+) plasmid following the manufacturer’s instructions (Large Scale RNA ProductionSystem-T7, Promega). For the RNA pulldown assay, cells were treated with the RNA 3′-End Desthiobiotinylation Kit and Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, USA). Cells were rinsed with PBS and then re-suspended in 1 ml ice-cold PBS. Then, we centrifuged the suspension at 4 °C and 1000 rpm for 3 min. Next, the cell pellet was suspended in 400 µl of dilution buffer (with a protease inhibitor cocktail) and centrifuged at 4 °C and 12,000 rpm for 10 min. The cell supernatant was collected for use in the next experiment. Pierce nucleic acid compatible streptavidin magnetic beads were washed twice with wash buffer to remove the stock solution and were re-suspended in RNA capture buffer. We added labeled biotin-lncRNA SNHG17 to the beads and incubated them for 15–30 min. Then, the beads were washed twice with wash buffer and re-suspended in Protein-RNA Binding Buffer. Next, we constructed the RNA pulldown reaction system according to the manufacturer’s instructions and incubated the beads at 4 °C for 2 h in a rotary shaker. Finally, we eluted the proteins with 50 µl biotin elution buffer after washing the beads with wash buffer and detected the proteins by SDS-PAGE and mass spectrometry analysis.
Cell-cycle analysis
PI/RNase Staining Solution kit (CY2001-P, Sungene Biotech, China) was used. The cells were seeded into 35 mm culture dishes at a density of 70–80% per dish. The cells were transfected or not transfected with siRNA, subjected to irradiation 12 h after the pretreatment, and harvested at the indicated timepoints (0, 2, 4, 6, 8, or 12 h) after irradiation. After the medium was removed, the cells were treated with RNase A (62 µg/ml) and incubated at 37 °C for 30 min. The cells were stained with propidium iodide (PI) solution, and the cell-cycle distribution was analyzed by flow cytometry (Agilent NovoCyte, USA). G2/M assay was based on the cell cycle detection.
Apoptosis assay
Fluorescein Isothiocyanate (FITC)-Annexin V Apoptosis Detection Kit was used to detect cell apoptosis according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA, USA). Briefly, we collected the cell culture supernatant 24 h after irradiation. After digesting the cells with trypsin (without EDTA), the cells were centrifuged. The supernatant was collected and washed three times with PBS. Then, we re-suspended the cells in binding buffer at a concentration of approximately 5 × 105 cells/ml and added FITC-conjugated Annexin V and propidium iodide (PI) solution according to the manufacturer’s instructions. The cells were incubated at room temperature for 15 min in the dark. Then, the cells were subjected to flow cytometry analysis (Agilent NovoCyte, USA).
Scratch wound healing migration
Cells were seeded into 6-well plates and grown to approximately 90% confluence. Cell monolayers were scratched with a 20-µl sterile pipette tip. Cells were rinsed with phosphate-buffered saline and cultured in DMEM supplemented with 1% fetal bovine serum. Cell migration was photographed 0 and 48 h after scratching using an inverted microscope (Olympus, Tokyo, Japan).
Fluorescence in situ hybridization (FISH)
Cy3-labeled lncRNA SNHG17 probes were designed and synthesized by RiboBio (Guangzhou, China). The probe signals were determined with a Fluorescent In situ Hybridization Kit (RiboBio, Guangzhou, China) following the manufacturer’s guidelines. Images were taken under an immunofluorescence confocal microscope (Crest Optics X-Ligt V3, Italia).
Microarray analysis of lncRNAs
The lncRNA profile between irradiated A549 MLE-12 cells with or without LGG treatment post 6 Gy radiation was performed at oeBiotech Biotechnology Corporation (Shanghai, China). Agilent lncRNA microarray (Agilent Technologies, USA) was used in the analysis. According to the manufacturer’s protocol, lncRNAs were labeled and hybridized with lncRNA complete Labeling and Hybridization kit. Data normalization and processing were performed using Quantile algorithm, Gene Spring Software 12.6 (Agilent Technologies, USA). The differential expression of lncRNAs was performed via the Pearson’s correlation analysis with Cluster 3.0 and TreeView software, and the differentially expressed genes (DEGs) were identified to have at least |logFC| > 2, p value < 5% in expression.
Electron microscopy for structural analysis of the lung
TEM analysis was performed after collection of the lung tissues by Shiyanjia Lab (www.shiyanjia.com). The tissues were split and treated in a cold fixative solution composed of 2.5% glutaraldehyde at 4 °C for 4 h. After washing with PBS, the specimens were post-fixed in 1% OsO4 at 4 °C for 1 h and washed again with PBS. A graded series of ethanol solutions was used for further dehydration, and the specimens were transferred to be incubated. TEM was performed with a JEM-2100 F at 80 kV, and images were acquired using a side-inserted BioScan camera.
Online available databases
SNHG expression in various cancers was evaluated using the TCGA database (https://cistrome.shinyapps.io/timer/). The GEPIA database (http://gepia.cancer-pku.cn/index. html) was used to analyze RNA sequencing data from normal and tumor tissue samples from the TCGA and GTEx projects. We also used the GEPIA website to generate overall free survival rates. UCSC (http://genome.ucsc.edu/) and ALGGEN (http://alggen.lsi.upc.es/) databases were used to obtain the potential transcriptional binding sites in the promoter of genes. GeneMainia (http://genemania.org/) and Hitpredict (http://www.hitpredict.org/) databases was used to predict the potential interaction proteins with SNHG17.
Statistical analysis
All experiments were performed with at least three independent experiments. In general, Student’s two-tailed unpaired t test was used to compare differences between two groups. One-way analysis of variance followed by the Newman–Keuls multiple comparison test were used to compare more than two groups. All data are expressed as the means ± standard deviation (SD) for each experiment. A p value of < 0.05 was considered to indicate a statistically significant result. GraphPad Prism 6 Software (GraphPad Software Inc., La Jolla, CA) was utilized for all statistical analyses and construction of graphs.