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).