Animals
The animal model of RILI was established according to previous studies,which have been verified by H&E staining and Masson-staining(9, 10). Female Balb/c mice, 8 weeks, were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China), Shanghai, China. Mice were housed five per cage and kept under standard laboratory conditions (22±2℃, 55±10% humidity, 12-12 hours/light-dark cycle). All animal experimental procedures and protocols were conducted according to the guidelines of our institutional animal care and use committee and were approved with the reference number of 20150404A114 by Department of Laboratory Animal Science.
Irradiation protocol
Six mice were treated in two groups: Control (non‐irradiated) group and IR group(irradiated). The mice of the control group only receive sham irradiation, while the mice of the IR group received thoracic irradiation. Radiation of mice was provided by Small Animal Radiation Research Platform (SARRP, FUSCC, Shanghai, China). All the six Mice were induced and maintained anesthesia with isopentane (increasing from 0.5% to 2.0% in 8min for inducing and 1.5% for maintaining) and then were positioned with a self-designed box to fix their neck and stretch the body. For the IR group, the chest of each mouse was irradiated with a single dose of 12 Gy exposure using a 6 MV linear accelerator (Siemens Primus-Hi) while for the control group each received 0 Gy exposure. The dose rate was 2 Gy/min. The source-surface distance was 1 m, and the size of the radiation field was 2.5 × 15 cm. The head and the abdomen were shielded by 2.5 cm thick lead blocks to protect other body parts from irradiation. All mice were killed at day 3 after irradiation. Once a mouse was killed, the lung was taken and frozen in −80°C fridge immediately.
H&E staining and evaluation
A part of lung samples from 3 irradiated and 3 control mice were processed to make paraffin blocks, then the tissue sections were deparaffinized by xylene and rehydration in a series of alcohol. After wash, tissue sections were processed as followed: 10min haematine, 1-3s wash, 1-3s 1% Ethanol hydrochloride, 10-30s wash, 1-3min 0.5% eosin, 1-2s wash, 1-2s 80% alcohol, 2-3s 95% alcohol, 3-5s 95% alcohol, 5-10min 100% alcohol, 5-10min xylene carbonate, 2min xylene (I), 2min xylene (II), 2min xylene (III). After drying for some time, neutral balsam was used for mounting. The evaluation of the H&E results were performed according to the previous reported scoring system(11). The pneumonia alterations were defined as thickened alveolar walls, widening of the interlobular septa, alveolar septal edema and massive inflammatory cells infiltration in the alveoli. Three randomly selected fields of each mouse were scored by a pathologists in our hospital and scored as follow: 0, no pneumonia; 1+, pneumonia alterations in 1-20% of the field; 2+, pneumonia alterations in 21-50% of the field; 3+, pneumonia alterations in 51-100% of the field.
RNA library construction and sequencing
Lung samples were proceeded by Trizol (Invitrogen, Carlsbad, CA, USA) to isolate and purify total RNA. NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) was used to quantify RNA amount and purity. RNA integrity was assessed using Agilent 2100 with RIN number >7.0. To deplete ribosomal RNA, Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, USA) was used. The remaining RNAs were fragmented into small pieces by divalent cations at high temperature. Then, cDNAs were created by reverse-transcription of these cleaved RNA fragments. The cDNAs was then used for synthesizing U-labeled second-stranded DNAs with RNase H, E. coli DNA polymerase I, and dUTP. Then, an A-base is added to the blunt ends of each strand, preparing them for ligating to the indexed adapters, which each contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single-or dual-index adapters are ligated to the fragments, and AMPureXP beads were used to perform size selection. After the using heat-labile UDG enzyme on the U-labeled second-stranded DNAs, PCR was proceeded with following settings: 3 mins, 95 ℃ for initial denaturation; 15 secs, 98℃ for 8 cycles of denaturation; 15secs, 60℃ for annealing; 30 secs, 72℃ for extension; 5 mins, 72℃ for final extension. The insert size of the final cDNA library was proximately 300 bp in average. Finally, Illumina Hiseq 4000 (LC Bio, China) was used to perform paired-end sequencing.
The quantity and purity of each RNA sample was verified using Bioanalyzer 2100 with RIN number > 7.0 and RNA 6000 Nano LabChip Kit (Aglient, Santa Clara, CA, USA) with 28S/18S ratio between 1.8 and 2.2. Approximately 1 μg of total RNA was used to prepare miRNA library according to the protocol of TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA).
Differential expression analysis of mRNA, circRNAs and miRNAs
Cutadapt (12) was used to remove the reads with low quality or undetermined bases and adaptor contamination. After verifying sequence quality by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), Bowtie2 (13) and topaht2 (14) were used to map reads to the genome of mice. Remaining reads (unmapped reads) were still mapped to genome using tophat-fusion. CIRCExplorer was used to denovo assemble the mapped reads to circRNAs at first; Then, back splicing reads were identified in unmapped reads by tophat-fusion and CIRCExplorer. All samples were generated unique circRNAs. CircRNA expressions from different samples or groups were caculated by scripts in house. The differentially expression was defined as an srpbm log2 (fold change) >1 or log2 (fold change) <-1 along with p value<0.05 by R package – EdgeR.
As for miRNAs, raw reads were subjected to ACGT101-miR (LC Sciences, Houston, Texas, USA) to remove low complexity, junk, adapter dimers, common RNA families (snRNA, snoRNA, rRNA, tRNA,) and repeats. Subsequently, known miRNAs and novel miRNAs were identified in miRbase 22.0 using BLAST search. Finally, we defined differential expressed miRNAs according to normalized deep-sequencing counts by Chi-squared nXn test, Fisher exact test, Student t test, Chi-squared 2X2 test, or ANOVA with a p value less than 0.05.
circRNA‐miRNA‐mRNA network construction and functional analysis
To explore the function of circRNAs as ceRNAs, we predicted the target miRNAs of circRNAs using MiRanda (3.3a) and Targetscan (5.0) according to the protocol of softwares with a maximum binding free energy less than -20 along with a Targetscan score more than 50. After the prediction, miRNAs which could bind with differentially expressed circRNAs were selected to further predict the target mRNAs. Then, the significantly differentially expressed gene symbols were mapped into the terms in GO database and pathways in KEGG database. Based on this, 4 parameters were calculated including S (significantly differentially expressed gene symbols of certain GO term or pathway), TS (total gene symbols that are significantly differentially expressed), B (total gene symbols of certain GO term or pathway), TB (total gene symbols), then hypergeometric test was performed to find the most significantly enriched terms or pathways compared with the genome background. Then, Cytoscape3.5.1 was used to display the circRNA-miRNA-mRNA networks.
Statistics
FPKM (15) were calculated by StringTie to quantify the expression level of mRNAs and circRNAs. The differentially expression was defined as an FPKM log2 (fold change) >1 or log2 (fold change) <-1 along with p value<0.05 by R package – Ballgown (16). Differentially expressed miRNAs were defined according to normalized deep-sequencing counts by Chi-squared nXn test, Fisher exact test, Student t test, Chi-squared 2X2 test, or ANOVA with a p value less than 0.05.