A MicroRNA-Mediated Positive Feedback Regulatory Loop of the NF-κB Pathway in Litopenaeus vannamei

Abstract

In the evolutionarily conserved canonical NF-κB pathway, degradation of the NF-κB inhibitor IκB in the cytoplasmic NF-κB/IκB complex allows the liberated NF-κB to translocate into the nucleus to activate various target genes. The regulatory mechanism governing this process needs further investigation. In this study, a novel microRNA, temporarily named miR-1959, was first identified from an invertebrate Litopenaeus vannamei. miR-1959 targets the 3′-untranslated region of the IκB homolog Cactus gene and reduces the protein level of Cactus in vivo, whereas the NF-κB homolog Dorsal directly binds the miR-1959 promoter to activate its transcription. Therefore, miR-1959 mediates a positive feedback regulatory loop, in that Dorsal activates miR-1959 expression, and in turn, miR-1959 inhibits the expression of Cactus, further leading to enhanced activation of Dorsal. Moreover, miR-1959 regulates the expression of many antimicrobial peptides in vivo and is involved in antibacterial immunity. To our knowledge, it is the first discovery of a microRNA-mediated feedback loop that directly regulates the NF-κB/IκB complex. This positive feedback loop could collaborate with the known NF-κB/IκB negative loop to generate a dynamic balance to regulate the activity of NF-κB, thus constituting an effective regulatory mechanism at the critical node of the NF-κB pathway.

RNAi screening identifies a new Toll from shrimp Litopenaeus vannamei that restricts WSSV infection through activating Dorsal to induce antimicrobial peptides

“…To further confirm the above results, we probed Dorsal translocation from the cytoplasm to the nucleus upon WSSV infection by using an L . vannamei Dorsal specific antibody prepared in our previous study [ 49 ]. In good agreement with the results of immunofluorescence staining, we were able to detect more nuclear import of Dorsal in shrimp hemocytes along with the times of WSSV infection from 0 h to 6 h, while at 6 hpi only a slight signal of Dorsal can be detected in the cytoplasm ( Fig 5C ).…”

Identification and profiling of growth-related microRNAs in Chinese perch (Siniperca chuatsi)

“…It has been well known that miRNAs are involved in various biological processes in eukaryotes, including cellular proliferation, development, differentiation, metabolism, oncogenesis, and apoptosis [3–6]. They negatively regulate gene expression through binding to the 3′ untranslated regions (UTRs), 5’UTRs, or the coding regions of target mRNA [7–9]. Since the first discovery of the miRNAs lin-4 and let-7 in Caenorhabditis elegans in 1993 [10], numerous miRNAs have been identified in mammals, plants, insects, worms, and viruses [11–14].…”

RNAi screening identifies a new Toll from shrimp that restricts WSSV infection through activating Dorsal to induce antimicrobial peptides

“…To gain more information on nuclear translocation of Dorsal upon WSSV infection, we firstly detected the dynamics of Dorsal translocation at several times, and we observed that shrimp hemocytes exhibited gradually and significantly increased nuclear translocation levels of Dorsal (53%, 88% and 94%) compared to the control hemocytes treated with PBS (Normal 0 h, 20%) at 1 h, 3 h and 6 h after WSSV challenge, respectively (Figures 5B and b). To further confirm the above results, we probed Dorsal translocation from the cytoplasm to the nucleus upon WSSV infection by using an L. vannamei Dorsal specific antibody prepared in our previous study (44). In good agreement with the results of immunofluorescence staining, we were able to detect more nuclear import of Dorsal in shrimp hemocytes along with the times of WSSV infection from 0 h to 6 h, and at 6 hpi almost no signal of Dorsal can be detected in the cytoplasm (Figure 5C).…”

“…The polyclonal antibodies for L. vannamei Dorsal and Cactus were produced in guinea pigs and rabbits, respectively, by GL Biochem antibody manufacturing company (China) from our previous study (44). Polyclonal rabbit anti-NF-κB p65 (phospho S276) antibody (ab194726, Abcam) was used to detect the phosphorylated shrimp Dorsal.…”