Phosphatase and Tensin Homolog (PTEN) of Japanese Flounder—Its Regulation by miRNA and Role in Autophagy, Apoptosis and Pathogen Infection

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs with important roles in diverse biological processes including immunity. Japanese flounder (Paralichthys olivaceus) is an aquaculture fish species susceptible to the infection of bacterial and viral pathogens including Edwardsiella tarda. In a previous study, pol-miR-novel_547, a novel miRNA of flounder with unknown function, was found to be induced by E. tarda. In the present study, we investigated the regulation and function of pol-miR-novel_547 and its target gene. We found that pol-miR-novel_547 was regulated differently by E. tarda and the viral pathogen megalocytivirus, and pol-miR-novel_547 repressed the expression of PTEN (phosphatase and tensin homolog) of flounder (PoPTEN). PoPTEN is ubiquitously expressed in multiple tissues of flounder and responded to bacterial and viral infections. Interference with PoPTEN expression in flounder cells directly or via pol-miR-novel_547 promoted E. tarda invasion. Consistently, in vivo knockdown of PoPTEN enhanced E. tarda dissemination in flounder tissues, whereas in vivo overexpression of PoPTEN attenuated E. tarda dissemination but facilitated megalocytivirus replication. Further in vitro and in vivo studies showed that PoPTEN affected autophagy activation via the AKT/mTOR pathway and also modulated the process of apoptosis. Together these results reveal for the first time a critical role of fish PTEN and its regulatory miRNA in pathogen infection, autophagy, and apoptosis.

A Novel microRNA of Japanese Flounder Regulates Antimicrobial Immunity Involving a Bacteria-Binding CSF3

“…To construct the plasmid pPoCSF3-1-Report, the coding sequence of PoCSF3-1 was amplified by PCR with primers PoCSF3-1-CDS-F1 and PoCSF3-1-CDS-R1 ( Table S1 ). The PCR product was inserted into pmirGLO (Promega, USA) at between the Nhe I and Sal I sites as reported previously ( 41 ). To construct the plasmid pPoCSF3-1si, which expresses a PoCSF3-1 specific siRNA, a siRNA targeting PoCSF3-1 was chemically synthesized (TsingKe, China) and inserted into pRNAT-CMV3.1 (GenScript, USA) at between the BamH I and Alf II sites as reported previously ( 41 ).…”

“…The PCR product was inserted into pmirGLO (Promega, USA) at between the Nhe I and Sal I sites as reported previously ( 41 ). To construct the plasmid pPoCSF3-1si, which expresses a PoCSF3-1 specific siRNA, a siRNA targeting PoCSF3-1 was chemically synthesized (TsingKe, China) and inserted into pRNAT-CMV3.1 (GenScript, USA) at between the BamH I and Alf II sites as reported previously ( 41 ). pPoCSF3-1siC, which expresses a nonspecific siRNA, was constructed similarly.…”

“…Luciferase reporter assay was performed as reported previously ( 41 ). Briefly, HEK293T cells were distributed in 24-well plates (about 80% confluence) and transfected with pPoCSF3-1-Report (control), pPoCSF3-1-Report plus pol-miR-novel_642 mimic, pPoCSF3-1-Report plus miR-C, or pPoCSF3-1-Report plus pol-miR-novel_642M.…”

“…To examine the regulation of PoCSF3-1 by pol-miR-novel-642 in flounder cells, FG-9307 cells were distributed in 6-well plates (about 80% confluence) and transfected with or without pol-miR-novel_642 mimic or miR-C for 24 h. qRT-PCR was performed to determine PoCSF3-1 mRNA expression with primers as described above. Western blot was performed to determine PoCSF3-1 protein expression as reported previously ( 41 ). Briefly, the transfected cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, China) on ice for 30 min.…”

“…The fish were infected with E. tarda or megalocytivirus as above. At 12 h, 24 h, and 48 h post E. tarda infection, the numbers of E. tarda in kidney, spleen and liver were determined by plate count as previously reported ( 41 ). At 6 d and 8 d post-megalocytivirus infection, the viral loads in kidney were determined by absolute quantitative real time RT-PCR as reported previously ( 16 ).…”