Regulatory role of novel miR-56 on the expression of immunerelated genes in Macrobrachium rosenbergii

【Objective】 To analyze the regulatory role of novel miR-56 on immune-related genes in Macrobrachium rosenbergii, which could provide theoretical basis for further exploring the involvement of miRNAs in the immune regulation mechanism in M. rosenbergii. 【Method】 The expression changes of novel miR-56 in the gill tissues of Macrobrachium rosenbergii after Vibrio parahaemolyticus infection were detected by real-time fluorescence quantitative PCR. The potential target gene TLR1 of novel miR-56 was predicted and screened by RNAhybrid, and dual-luciferase reporter gene assay verified the targeting relationship between novel miR-56 and target gene TLR1. The expression of novel miR-56 was interfered by in vitro injection of agomiR-56 and antagomiR-56 at different concentrations（0.6, 1.0 and 1.5 μg/g）, the optimal injection concentration of agomiR-56 and antagomiR-56 was screened, and changes in the expression of immunerelated genes of Macrobrachium rosenbergii were detected by rel-time fluorescence quantitative PCR. 【Result】 After infection with Vibrio parahaemolyticus, the relative expression level of novel miR-56 in the gill tissues of Macrobrachium rosenbergii showed a gradual upward trend with the progression of infection. At 72 h post-infection, the relative expression level of novel miR-56 was approximately as 6.18 times as that before infection（0 h）. TLR1 was the target gene of novel miR-56, and the binding site between them was located in the 3’ untranslated region（3’-UTR） of TLR1 gene. Moreover, the overexpression of novel miR-56 could inhibit the expression of TLR1 gene. Intracardiac injection of agomiR-56 at doses of 0.6, 1.0 and 1.5 μg/g into Macrobrachium rosenbergii significantly inhibited the expression of TLR1 gene（P＜0.05, the same below）, with the optimal inhibitory effect observed at 1.5 μg/g agomiR-56. The relative expression levels of other immune-related genes in Macrobrachium rosenbergii（including Spz, TLR2, TLR3, MyD88, IMD, Crustin1, Wntless, ALF1 and proPO） also significantly decreased after agomiR-56 injection. In contrast, the results of the antagomiR-56 injection experiment showed the opposite trend. Injection of different doses of antagomiR-56 significantly promoted the expression of TLR1 gene in Macrobrachium rosenbergii. With the increase of injection dose, the promoting effect on TLR1 gene in Macrobrachium rosenbergii expression first increased and then decreased, and 1.0 μg/g was the optimal in vitro injection dose of antagomiR-56. The relative expression levels of other immune-related genes in in Macrobrachium rosenbergii significantly increased after antagomiR-56 injection. 【Conclusion】 TLR1 is a target gene of novel miR-56. The binding site is located in the 3’-UTR of TLR1 gene. Novel miR-56 regulates Toll signaling pathway by targeting and inhibiting TLR1 gene, and has the function of inhibiting the expression of immune-related genes in Macrobrachium rosenbergii.