MicroRNA-146a promotes red spotted grouper nervous necrosis virus (RGNNV) replication by targeting TRAF6 in orange spotted grouper, Epinephelus coioides.

MicroRNA-146a (miR-146a) has been demonstrated to function as a negative regulator of cellular immune responses against pathogens in mammals, however, little information focused on its functions in lower vertebrates. In this study, we investigated the regulatory roles of orange spotted grouper, Epinephelus coioides miR-146a during red spotted grouper nervous necrosis virus (RGNNV) infection. During RGNNV infection in grouper spleen (GS) cells, the endogenous expression level of miR-146a and tumor necrosis factor receptor-associated factor 6 (TRAF6) significantly increased along with the infection time. Overexpression of miR-146a significantly facilitated viral infection, evidenced by the increased transcription of viral CP and RdRp genes, while miR-146a knockdown by specific inhibitors decreased RGNNV replication. Using pMIR-REPORT Luciferase system, we found that the 3' untranslated region (UTR) of grouper TRAF6 could be specifically targeted by miR-146a. Further studies showed that its downstream target gene pro-inflammatory cytokines, including TNF-α, IL-8 and IL-1β, were all significantly decreased in miR-146a mimic transfected cells, but increased in miR-146a inhibitors transfected cells during RGNNV infection. Thus, our results suggested and verified that holding the level of miR-146a exerted crucial roles in RGNNV infection through TRAF6-mediated inflammatory response.