2. Materials and Methods
All experiments involving the use of laboratory animals were performed according to National Institutes of Health Laboratory Animal Care and Use Guidelines and were approved by theInstitutional Animal Care and Use Committee ofHuazhong University of Science and Technology.
We used C57BL/6J mice as donors and BALB/c mice as receptors for lung allograft transplantation. The mice selected in this experiment were all specific-pathogen-free (SPF) grade(male, 24±2g, and 6 to 8 weeks old). All mice were provided by the Beijing Vital LiHua Company.
2.1Constructionan of experimental model
Ten BALB/c mice were randomly divided into two groups (transplanted and control). Left LT was performed in mice in the transplanted group according to the procedure described by Zheng et al (15). Mice in the control group only underwent left thoracotomy without LT and were sutured after 70 min. All mice were sacrificed 24 h after the surgery. The left lung specimens were stored in the refrigerator at –80°C for further analysis.
2.2 Construction of small RNA libraries and sequence analysis
The total RNA was extracted according to the protocol provided by Wuhan Fakang Biological Co. Ltd. (Wuhan, China). The HiSeq platform was used for sequencing and calculating the sequence length distribution. Conserved miRNA were identified using miRBase 21, an miRNA database. Other small non-coding RNAs, such as rRNA, tRNA, snoRNA, and snRNA, were identified using the Genbank database (http://blast.ncbi.nlm.nih.gov) and the Rfam database (http://sanger.ac.uk/software/Rfam). The unannotated RNA sequences were used to predicted novel miRNAs using Mireap. The frequency of DE miRNAs was identified using DESeq2 and the formula log2 (fold changes)>1 and p <0.05. MiRanda was used to predict the target genes of DE miRNAs that were mapped to the gene ontology (GO) database (http://www.geneontology.org), the number of genes corresponding to significantly enriched GO terms was calculated. Kyoto Encyclopedia of Genes and Genomes (KEGG) is a utility database for understanding the advanced functions and biological systems and genome sequences and retrieving molecular data. The target genes were mapped to the KEGG database (http://www.genome.jp/kegg/) to predict the enriched pathways.
2.3 Experimental groupings.
BALB/c mice were randomly divided into four groups.Antagomir-122(Ruibo Biotechnology Co., Ltd. Guangzhou, China ) 20nmol/L was injected into the caudal vein of mice immediately after the chest operation in one transplantation group and one control group. The other two groups (one transplantation group and one control group) were simultaneously injected with 1.5 mL of 0.9% normal saline. All mice were sacrificed 24 h later.
2.4 Pulmonary tissue hematoxylin-eosin staining, assessment of lung injury score, and detection of lung W/D weight ratio
The left lung tissue sample were embedded in paraffin, sectioned, stained with hematoxylin-eosin (H&E), and observed under the microscope (H&E ×400) to assess the lung tissue injury. Next, the lung W/D weight ratio was calculated.
2.5 Detection of miRNA-122 expression
The expression of miRNA122 was detected by the TaqMan® MicroRNA assay.
2.6 ELISA and western blotting
The TNF-α and MPO enzyme activity were detected using an enzyme-linked immuneosorbent assay (ELISA) kit after isolating and extracting the proteins from mouse lung tissues. The protein expression of TLR2, TLR4 and interleukin (IL)-10 by western blotting (Wuhan Fakang biology Co. Ltd, China).
2. 7 Statistical analysis
All data were analyzed using the SPSS 22.0 statistical software. The measured data are expressed as mean ± standard deviation. Ap < 0.05 was considered significant.