Pol-miR-7133 and pol-miR-3p-9227 of Japanese flounder Paralichthys olivaceus modulate Streptococcus iniae infection through regulation of the common target gene LAMP2

Abstract

MicroRNAs (miRNAs) are small, non-coding endogenous RNAs that play critical roles in diverse cellular processes through regulating target gene expression. In the present study, we identified two Japanese flounder (Paralichthys olivaceus) miRNAs, i.e., pol-miR-7133 and pol-miR-3p-9227, and examined the functions of the miRNAs and their target gene in association with the infection of Streptococcus iniae. pol-miR-7133 and pol-miR-3p-9227 were identified as miRNAs downregulated during S. iniae infection. Both miRNAs targeted the gene of lysosome-associated membrane protein 2 (LAMP2) isoform 3 of flounder (named PoLAMP23) by interaction with the 3′-untranslated region (3’-UTR) of PoLAMP2–3. Both in vivo and in vitro studies showed that PoLAMP2–3 expression negatively correlated with pol-miR-7133 and pol-miR-3p-9227 expression. Since to date no study on fish LAMP2 had been documented, we investigated the biological property of PoLAMP2–3. We found that PoLAMP2–3 expression occurred in multiple tissues and was most abundant in liver. PoLAMP2–3 is a highly acidic transmembrane protein and was observed to localize in both lysosome and cellular membrane. In vitro studies showed that overexpression of pol-miR-7133 and pol-miR-3p-9227 or interference with PoLAMP2-3 expression significantly enhanced S. iniae infection in flounder cells. Consistently, in vivo studies showed that knockdown of pol-miR-7133 and pol-miR-3p-9227, or overexpression of PoLAMP2-3, inhibited S. iniae infection in flounder, whereas knockdown of PoLAMP2-3 in flounder facilitated S. iniae infection. Together these results revealed for the first time an important immune function of fish LAMP2 and indicated that pol-miR-7133 and pol-miR-3p-9227, via LAMP2, play a vital role in anti-bacterial immunity in fish.