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.