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.