3.4 RNA sequencing of sample of impingement knee cartilage
injury in rabbits and TLR8 target prediction.
Related studies have reported that the TLRs receptor-mediated innate
immune responses are mostly mediated by miRNAs. It has been reported
that
TLR8
and its endogenous ligand miR-21 contribute to neuropathic pain in
murine DRG26.There is
no literature report that the congenital immune response of
TLR8-mediated cartilage injury is mediated by which miRNA species.
Therefor we performed transcriptome sequencing of specimens from the
rabbit cartilage damage model. The expression level of miRNA was
detected in samples of control cartilage and in cartilage 1 week after
injury. DESeq (Version 1.18.0, Anders S and Huber W, 2010) was used to
analyze the differential expression of miRNA (Fig.4A). According to the
expression quantity ratio difference (| log2FoldChange
| & gt; 1) and significant difference in expression (p-value;
0.05) Differential conserved mi-RNAs were screened. The results showed
that, 21 miRNA down-regulated and 26 mRNA up—regulated, among which
mir-12093-3p, mir-99a-5p, mir-873-5p, mir-452-5p, and mir-1-3p were the
top 5 miRNAs with the most significantly down-regulated expression,
mir-12093-3p and mir-133-3p had no relevant information in the database.
Therefore, mir-99a-5p with the highest degree of difference was selected
for verification. Then through the NCBI database search and blast
comparison found that mir-99a-5p may bind to TLR8. We can see that there
is a conserved binding site in both rabbits and humans (Fig. 4B). To
further understand the changes of in the mir-99a-5p human body, the
blood samples were collected from patients who diagnosed with cartilage
injury and normal patients respectively, and then analyze the level of
mir-99a-5p. Thirty-two patients were included in each group, and their
clinical data can be seen in Table1(Fig. S5). The blood level of
mir-99a-5p was 1.03 ± 0.04 and 0.31 ± 0.02 in group of normal and injury
respectively, p<0.05(Fig. 4C).
In order to verify the interaction between mir-99a-5p and TLR8, we first
using RNA pull-down techniques find when mir-99a-5p as the probe, it
could be seen that TLR8 in mir-99a-5p
probe group was significantly enriched and the average CT values was
17.93, significantly decreased compared with the cell lysis
buffer(Fig.S6). After taken agarose gel electrophoresis, and gray values
of electrophoresis bands were calculated. The results showed that the
amplification bands of Mir-99a-5p probe group were significantly
stronger than cell lysates (Fig.4.D,E). Then the TLR8 probe was labeled
with the same method, and mir-99a-5P in the TLR8 probe group was
significantly enriched, and the gray value of the amplified band in the
TLR8 probe group was significantly stronger than that in the cell lysate
(Fig.4.F,G).Therefor through the positive and negative RNA pull down
experiments of mir-99a-5p and TLR8, it is likely that mir-99a-5p binds
and interacts with TLR8.
To further clarify the relationship between them, we performed a double
luciferase assay. The results show that the average fluorescence value
ratio of Psichec2-TLR8-WT+miR-99a group is 0.33, much lower than the
other groups(P<0.05) (Fig.4H),which suggested that the
activity of luciferase decreased after TLR8 was combined with
mir-99a-5p, indicating the interaction of the 3-terminal non-coding
region of mir-99a-5p and TLR8. At the same time, we also carried out
double fluorescence on cells,
found that the expression of TLR8 increased and mir-99a-5p decreased at
the same time, and the position of the cells was similar(Fig.4I).To
further illustrate the changes of related immune-involved factors in
cells after cartilage injury, we selected NF-κB factor for detection and
found that it is activated after injury and transferred to the nucleus
(Fig.4J).This suggests that TLR8 activation in cartilage injury can
mediate inflammatory molecular pathways.
3.5 miR-99a-5p negatively regulates TLR8 in the
occurrence of impingement cartilage injury of rabbit knee joint.
To gain further understanding of the downstream molecular mechanisms of
cartilage damage underlying TLR8 activation, we injected mir-99a-5p as
well as the overexpression and inhibitory vector of TLR8 in the
auricular vein of model rabbits, related inflammatory properties and
apoptosis-related indicators were detected one week later(Fig.5A).Animal
models were divided into six groups, depending on the rabbit treatment
pattern,from no treatment in the control group to overexpression of
mir-99a-5p mimic and TLR8 after cartilage injury(Fig.5B).All groups of
animals underwent further testing one week after receiving the
corresponding treatment, PCR analysis of cartilage tissue in each group
showed when the TLR8 overexpression or mir-99a-5p decline, the
MyD88(Fig.5C),IRF7(Fig.5D),NF-κB(Fig.5E) also increased expression, but
Iκ-Bα is decrease when TLR8
overexpression(Fig.5F). They are accompanied by TLR8 or mir-99a-5p
changes, suggesting them involved in the activation of the signaling
pathway during cartilage injury. We further detected the apoptosis
related factors of IL-6, TNF, caspase-9 and BCL2, and found that with
the TLR8 overexpression or mir-99a-5p decline, IL-6, TNF and caspase-9
is become higher expression (Fig.5G-I), however the BCL2 expression is
opposite (Fig.5J). As above to the changes of TLR8 and mir-99a-5p,
indicating that they may involve in various regulation and effects in
cartilage injury. At the same time, WB test was used to further confirm
the protein expression levels of MyD88,IRF7,NF-κB , Iκ-Bα and the
results were basically consistent with the PCR results.(Fig.5K). Base on
this study, we can infer that in the cartilage repair model, mir-99a-5p
negatively regulates the expression of TLR8 and affect the occurrence of
chondrocyte innate immune response(Fig.5L).