General information comparison
The patient’s basic information was recorded before the patients were admitted into the hospital. The 49 patients with dry eye disease in the hospital were included in the model group, including 32 males and 17 females, and the average age of 37.4 ± 10.8 years. The body’s BMI value was 22.75 ± 3.14, while the control group was 53 healthy subjects who passed the physical examination in the hospital, including 20 males and 33 females, average age was 38.2±10.7, body BMI is also within the normal range. There was no statistical difference There was no statistical difference between the general information of the model combination control groups: they were comparable. See Table 1 for details.
MiRNA-146a-5p was low-expression in the patients with dry eye disease
To explore the expression of miRNA-146-5p in the patients with dry eye disease, the blood of the model and the control group one was selected, the expression of miRNA-146-A-5p was detected by RT-PCR. The result showed that miRNA-146a-5p had low-expression in model group compared with the control group. The difference between the two groups was statistically significant (Figure 1). It indicated that miRNA-146-5p may be the marker of dry eye disease.
IRAK1 Is the Target Gene Regulated by miRNA-146a-5p
To predict the target genes of miRNA-146a-5p, the databases were used. We can know that there are 7 genes regulated by miRNA-146a-5p determined by miRDB, miRWalk, PicTar, TargetScan databases (Figure 2A), including IRAK1, TRAF-6, NOVA1 and so on. Among them, IRAK1 is related to inflammatory response, while inflammatory response would lead to the dry eye disease. Therefore, we will focus on the specific mechanism of dry eye disease with miRNA-146a-5p and IRAK1 and explore the potential mechanism.
We explore the targeting relationship between miRNA-146a-5p and IRAK1 by establishing the network using cytoscape3.6.1. The network showed that there are numerous genes regulated by miRNA-146a-5p, and IRAK1 was one of them (Figure 2B).
IRAK1 Is the Target Gene Regulated by miRNA-146a-5p
To determine whether the inhibition of IRAK1 by miR-146a-5p occurred via these predicted miR-146a-5p binding sites, the combining site was mutated. Luciferase reporter assays indicated that the IRAK1 mutant 3ʹ-UTR interrupted miR-146a-5p-mediated repression, it indicated that miRNA-146a-5p could regulate the expression of IRAK1 directly (Figure 3).
MiRNA-146a-5p Inhibits Inflammatory Release by Targeting IRAK1 mRNA
Then we determined the expression of IRAK1 when miRNA-146a-5p with inhibition treatment (Figure 4A), we found that compared with the control group, the expression of miRNA-146a-5p was lower and IRAK1 higher at the same time in the model group. While the inhibitor group showed that the inhibition of miRNA-146a-5p would lead to the increase of IRAK1 mRNA, and when the expression of miRNA-146a-5p is rising, the IRAK1 mRNA would decrease at the same time. The relationship between miRNA-146a-5p and IRAK1 were negatively correlated. These results suggested that the highly conserved sequence of the IRAK1 3ʹ-UTR is the major miR-146a-5p binding site, and further confirmed that miR-146a-5p directly inhibits IRAK1(Figure 4B).
Then we detected the expression of IL-6, calprotectin and TNF-α (Figure 4A), the result showed that the expressions of IL-6, calprotectin and TNF-α were higher in the model group compared with the control group, and the expressions of IL-6, calprotectin and TNF-α in inhibitor groups were lower obviously (Figure 4B). It indicated that the up-regulating miRNA-146a-5p and targeting IRAK1 mRNA may inhibit inflammatory release.
At last, we detected the relationship of miRNA-146a-5p, IRAK1, calprotectin, IL-6 and TNF-α mRNA, the result showed that miRNA-146a-5p was negatively related with IRAK1, while the expression of IL-6, calprotectin, TNF-α had positive relation with IRAK1 and negatively related with miRNA-146a-5p (Figure 4C).
MiRNA-146a-5p attenuates inflammatory response by regulating TRAF-6 protein
In order to prove the effects of miRNA-146a-5p on the expression of IRAK1 protein, Western Blot was used. The IRAK1 protein was significantly increased in the model group compared with the control group while the expression of IRAK1 protein was the highest in the inhibitor group (Figure 5A), and the difference was statistically significant (P<0.01). MiRNA-146a-5p inhibitor attenuates the inhibitory effect of miR-146a-5p on IRAK1 protein expression.
At last, we detected the relationship of IRAK1, IL-6 and TNF-α proteins, the result showed that miRNA-146a-5p was negatively correlated to IRAK1, while the expression of calprotectin, IL-6 and TNF-α proteins had positive correlation with IRAK1 (Figure 5B).