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.

Keywords: Paralichthys olivaceus; apoptosis; autophagy; immune defense; microRNA; pathogen infection; phosphatase and tensin homolog.

Figure 1

Expression of pol–miR-novel_547 (A) and phosphatase and tensin (PTEN) homolog of flounder (PoPTEN) (B) in response to bacterial and viral infection. Flounder were infected with or without (control) Edwardsiella tarda or megalocytivirus, and the expressions of pol–miR-novel_547 (A) and PoPTEN (B) in kidney were determined by quantitative real time RT-PCR at different times. hpi, hour post-infection; dpi, day post-infection. Values are the means of triplicate experiments and shown as means ± SD. * p < 0.05; ** p < 0.01.

Figure 2

Regulation of PoPTEN expression by pol-miR-novel_547. (A) HEK293T cells were transfected with pPTEN-Report in the absence (control) or presence of pol-miR-novel_547 mimic, pol-miR-NC (negative control of pol-miR-novel_547), or pol-miR-novel_547 mimic-M (mutant of pol-miR-novel_547). Relative luciferase activity was determined at 24 h after transfection. (B,C) FG-9307 cells were transfected with or without (control) pol-miR-novel_547 mimic or pol-miR-NC, and PoPTEN expression was determined by qRT-PCR (B) or Western blot (C) with β-actin as a loading control. The values of (A,B) are the means of triplicate experiments and shown as means ± SEM. * p < 0.05; ** p < 0.01.

Figure 3

In vitro effects of pol-miR-novel_547 and PoPTEN on bacterial infection in flounder cells. (A) FG-9307 cells were transfected with or without (control) pol-miR-novel_547 mimic or pol-miR-NC and then infected with Edwardsiella tarda. Intracellular bacterial number was determined at different hours post infection (hpi) and shown as CFU (colony forming unit). (B) FG-9307 cells were transfected with or without (control) siRNA-PoPTEN or siRNA-NC and then infected with E. tarda. Intracellular bacterial number was determined as above. In both panels, values are the means of triplicate experiments and shown as means ± SEM. ** p < 0.01.

Figure 4

In vivo effects of PoPTEN on bacterial infection in flounder. For PoPTEN overexpression, flounder were treated with or without (control) pPoPTEN or the control plasmid pCN3 (A), for PoPTEN knockdown, flounder were treated with or without (control) pPoPTENsi or the control plasmid pPoPTENsiC (B). The fish were infected with Edwardsiella tarda, and bacterial numbers in kidney, spleen, and liver were determined at different times. Values are the means of triplicate experiments and shown as means ± SD. * p < 0.05; ** p < 0.01.

Figure 5

The expression of autophagy-associated genes in flounder cells with PoPTEN knockdown (A) and pol-miR-novel_547 overexpression (B). (A) FG-9307 cells were transfected with or without (control) siRNA-PoPTEN or siRNA-NC for 24 h. (B) FG-9307 cells were transfected with or without (control) pol–miR-novel_547 mimic or pol-miR-NC for 24 h. For both panels, the expressions of ATG5, AKT, mTOR, and beclin 1 were determined by qRT-PCR. Values are the means of triplicate experiments and shown as means ± SEM. * p < 0.05; ** p < 0.01.

Figure 6

In vivo expression of autophagy-associated genes in flounder with PoPTEN knockdown (A) and overexpression (B). (A) Flounder were administered with or without (control) pPoPTENsi or pPoPTENsiC for 7 days. (B) Flounder were administered with or without (control) pPoPTEN or pCN3 for 7 days. For both panels, the expressions of ATG5, AKT, mTOR, and beclin1 in kidney were determined by qRT-PCR. Values are the means of triplicate experiments and shown as means ± SD. * p < 0.05; ** p < 0.01.

Figure 7

Effects of PoPTEN and pol-miR-novel_547 on the expression of autophagy-related genes in mammalian and fish cells. (A) HEK293T cells were transfected with or without (control) pPoPTEN or pCN3 for 24 h. (B) FG-9307 cells were transfected with or without (control) pol-miR-novel_547 inhibitor or pol-miR-NC inhibitor for 24 h. For both panels, PTEN, AKT, p-AKT (phosphorylated AKT), beclin 1, and LC3 proteins were detected by Western blot. β-actin was used as a loading control.

Figure 8

The effect of PoPTEN on apoptosis. FG-9307 cells were transfected with or without (control) pol-miR-novel_547 inhibitor or pol-miR inhibitor NC for 24 h. (A) PoPTEN expression in the cells was determined by qRT-PCR. Values are the means of triplicate experiments and shown as means ± SEM. **, p < 0.01. (B) The transfected cells were labeled with annexin V-FITC and propidium iodide, and apoptosis was determined by flow cytometry.