3. Results
3.1 Sequencing and data analysis
Deep sequencing using the Illumina platform, revealed 26,078,258 and 27,278,394 raw reads in the experimental and control groups, respectively. The data were sorted and the adaptors were removed to filter the low-quality reads including duplicate sequences. The selected sequence nucleotides were between 18 and 36 in length. We obtained 20,613,567 and 22,970,296 clean reads in each group, accounting for 79.4% and 84.2% of the total reads, respectively. Duplicate data in two groups were named as common read. We obtained 91,269 unique small RNAs between the two groups (9.6% of the total). In addition, the experimental and control groups had 449,114 and 408,513 unique small RNAs, which accounted for about 47.3% and 43.1% of the total small RNAs, respectively(Fig.1a ).
We analyzed small RNAs with 18 to 36 nucleotides in length. We found that RNAs with in length from 20 to 23 nucleotides in the RNA libraries of experimental and control groups constituted the majority. In addition, the majority of the RNAs had a length of 22 nucleotides, followed by RNAs with a length of 21 nucleotides (Fig.1b ).We compared the clean reads with the genome and obtained the distribution of small RNAs. The genome comparison results were combined with the sequencing data of annotated genes to identify the corresponding mature miRNAs. Figure 1c summarizes the comparison and annotation of all small RNAs and RNAs. Small RNA sequences were matched to different fragments (Fig.1c ).
Twelve DE miRNAs were selected (Table.1 ) and 2,476 corresponding target genes were predicted using Miranda. The predicted target genes were divided into 6,063 terms by GO analysis, of which 4,633 terms were annotated to biological processes, 576 terms were annotated to cell components, and 854 terms were classified as molecular functions (Fig.2 ).
The KEGG pathway analysis annotated 1,554 predicted target genes to biological processes including signal transduction, cell-cell interactions and metabolic pathways. Thirty-seven genes were annotated to the PI3K–Akt signaling pathway (ko04151), the most widely used annotation pathway. accounting for approximately 2.38% of the total annotated genes; This was followed by 27 genes annotated to the Ras signaling pathway (ko04014) accounting for about 1.74% of the total; 27 genes annotated to cell phagocytosis (ko04144), accounting for about 1.74% of the total; and 26 genes annotated to cell adhesion (ko04510), accounting for about 1.67% of the total(Fig.3 ).
3.2 Relative expression of miRNA122
We compared the expression of miRNA-122 in each group following inhibition. The expression of miRNA122 was significantly increased in the transplanted group compared with that in the non-transplanted group (p <0.05). The expression of miRNA-122 in the group treated with mir-122 antagomir was significantly reduced compared with that in the control group (p <0.05) (Fig.4 ).
3.3 Study of lung tissue damage
Compared with the control group, the degree of damage to the lung tissues was significantly reduced in the LT + antagomir group (p <0.05) (Fig.5 ). The lung injury score was calculated using the method proposed by Smith, et al (16). We observed significantly reduced lung injury score and W/D weight ratio (p <0.05) (Fig.4 ) in the LT + antagomir group. Indicating that the inhibition of miRNA-122 reduced the early-stage lung injury after transplantation
3.4 Expression of TNF-α and IL-10,MPO activity
The expression of TNF-α and the activity of MPO were significantly decreased (p <0.05), whereas the expression of IL-10 was significantly increased (p <0.05) in the antagomir-122 treated group compared with the control group(Fig.4 and Fig.6 .). These data suggest that miRNA122promotes early IR injury after LT.
3.5 Relative expression of TLR2/4 proteins in lung tissue
Compared with the control group, the antagomir-122 treated group showed a significant decrease in the level of TLR2/4 proteins (Fig.6 ). These data demonstrate that the inhibition of miRNA-122 repressed activation of the TLR signaling pathway.