MicroRNA-induced negative regulation of TLR-5 in grass carp, Ctenopharyngodon idella

Abstract

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in antibacterial defence in fish has not been fully determined. Here, we identified that nine miRNAs are differentially expressed in kidney between susceptible and resistant grass carp strains. Analysis of spatial and temporal miRNA expression patterns suggests that cid-miRn-115 and miR-142a-3p are potential regulators of anti-bacterial activity. Overexpressing of cid-miRn-115 and miR-142a-3p results in a visible change in Ctenopharyngodon idella kidney (CIK) cells immune effector activity. Bioinformatics analysis and overexpressing assay shows that cid-miRn-115 and miR-142a-3p directly regulate tlr5 expression. cid-miRn-115 and miR-142a-3p overexpressing leads to a significant decrease in tlr5 expression in CIK, thereby repressing its downstream genes, such as il-1β, il-8 and tnf-α. These findings provide a novel insight into the determination of anti-bacterial compounds in grass carp.

Figure 1. Length distribution and reads of small RNAs from A. hydrophila- susceptible (SGC) and-resistant grass carp (RGC) libraries.

Figure 2. Predicted hairpin structures of grass carp coding candidate miRNAs.

Dominant forms of the mature miRNAs are indicated in red. A: cid-miRn-100, B: cid-miRn-101, C: cid-miRn-102.

Figure 3. qPCR of conserved miRNAs in the A. hydrophila-susceptible (SGC) and -resistant grass carp (RGC) libraries.

The expression of selected miRNAs in SGC or RGC was validated by qPCR amplification, using a specific primer for each miRNA. The fold change in expression was calculated based on the level of miR-192 expression (used as a control for the normalization). The data were obtained from three independent experiments (mean ± SD). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. TLR5 is a potential target of miRNA regulation.

(A) The alignment between miR142a-3p, miR-21, miR-223, cid-miRn-115, cid-miRn-131 and the 3′-UTR segment of tlr5. (B) CIK was exposed to FLG22 for 0 h, 24 h, 36 h and 48 h. The expression of Citlr5 in CIK was detected using real-time PCR. 18S rRNA expression was detected as internal control for mRNA. MiR-192 expression was detected as the internal control for miRNA. (C) MiRNA samples were extracted from different tissues, including kidney, spleen, intestine, liver, gill and blood. MiRNA expression was detected by qRT-PCR. MiR-192 was used as loading control. The data were obtained from three independent experiments (mean ± SD). *P < 0.05.

Figure 5. cid-miRn-115 and miR-142a-3p overexpression changes the expression of Citlr5 and its downstream genes.

CIK received cid-miRn-115 and miR-142a-3p agomir at a dose of 50 nM for the indicated times. The relative mRNA expression of Citlr5 (A) and of TLR5 downstream genes, including Ciil-1β, Ciil-8 and Citnf-α (B and C) was detected using real-time PCR. 18S rRNA expression was used as internal control for mRNA. MiR-192 was detected as the loading control. The data were obtained from three independent experiments (mean ± SD). *P < 0.05.

Figure 6. Effect of cid-miRn-115, miR-142a-3p and pEGFP-CiTLR5 on A. hydrophila invasion.

CIK cells were transfected with plasmid pEGFP- pEGFP-CiTLR5 or cid-miRn-115 and miR-142a-3p agomir. After 24 h, the cells were infected with A. hydrophila. The invasion number was counted as the number of entered bacteria. Data are presented as mean ± SE (n = 3). *P < 0.05.