miR-214-5p/C1QTNF1 axis enhances PCV2 replication through promoting autophagy by targeting AKT/mTOR signaling pathway

Abstract

Porcine circovirus type 2 (PCV2) is the causative agent of PCV2-associated disease, which causes a relevant economic impact on the global swine industry. Accumulating data have indicated host microRNAs play essential roles in numerous virus replication of pigs, while their roles in PCV2 replication remain unclear. Herein, we demonstrated that PCV2 infection downregulated the expression of miR-214-5p in PK15 cells, and miR-214-5p promoted PCV2 replication. C1q/tumor necrosis factor-related protein 1 (C1QTNF1) was then identified as a target gene of miR-214-5p, and C1QTNF1 suppressed PCV2 replication. Interestingly, miR-214-5p/C1QTNF1 axis negatively regulated AKT/mTOR signaling, and then enhanced PCV2 replication through promoting autophagy in PK15 cells. Collectively, our findings provide insight into the mechanism of PCV2 replication and highlight miR-214-5p and C1QTNF1 as potential novel targets for the treatment of PCV2 infection.

Keywords: AKT/mTOR signaling pathway; Autophagy; C1QTNF1; PCV2; Pigs; miR-214–5p.

Fig. 1

miR-214–5p promotes PCV2 replication in PK-15 cells. (A) PK15 cells were infected with PCV2 for indicated times, and the relative miR-214–5p expressions were quantified by qPCR. (B) Transfection efficiency of miR-214–5p mimics and miR-214–5p inhibitor was determined using qPCR. After miR-214–5p mimics or miR-214–5p inhibitor transfection, PK15 cells were infected with PCV2 (MOI = 1) for 48 h, PCV2 viral DNA copies were detected by qPCR (C), levels of CAP protein expression were detected by western blotting for three replications (D), and the number of PCV2 virus particles was observed by immunofluorescence (E). Mimics: cells transfected with the miR-214–5p mimics, Inhibitor: cells transfected with the miR-214–5p inhibitor, Mimics-NC and Inhibitor-NC: cells transfected with the NC of which targeting none of the genomic sequences. Data are presented as the mean ± SD of three independent experiments (n = 3). ** P <0.01.

Fig. 2

C1QTNF1 gene is a target of miR-214–5p. (A) PK15 cells were infected with PCV2 for indicated times, and the relative C1QTNF1 expression was quantified by qPCR. (B) A correlation analysis was performed between the relative expression level of miR-214–5p and C1QTNF1 in PK15 cells at different times after PCV2 infection. After miR-214–5p mimics transfection, the mRNA expression level of C1QTNF1 was detected by qPCR (C), and levels of C1QTNF1 protein expression were detected by western blotting (D). (E) Schematic diagram of the binding sites for miR-214–5p and C1QTNF1. (F) The luciferase reporter plasmids including the wild-type or mutant nucleotide type sequences of the C1QTNF1 3′ UTR region were constructed. (G) A dual luciferase assay was performed to reveal that the C1QTNF1 gene is a target of miR-214–5p. Mimics: cells transfected with the miR-214–5p mimics, NC: cells transfected with the mimics NC of which targeting none of the genomic sequences. Data are presented as the mean ± SD of three independent experiments (n = 3). ** P <0.01.

Fig. 3

C1QTNF1 suppresses PCV2 replication in PK-15 cells. Overexpression efficiency of C1QTNF1 was determined using qPCR (A) and western blotting (B). Interference efficiency of three siRNAs was detected by qPCR (C) and the specific inhibition of C1QTNF1 protein expression by the first siRNA was confirmed by western blotting (D). After C1QTNF1 overexpression cell line or cells transfected with the siRNA infected with PCV2 (MOI = 1) at 48 hpi, PCV2 viral DNA copies were detected by qPCR (E), levels of CAP protein expression were detected by western blotting for three replications (F), and the number of PCV2 virus particles was observed by immunofluorescence (G). OE-NC: vector-transfected cells, OE: C1QTNF1 overexpression cell line, Si-NC: cells transfected with the Si-NC, C1QTNF1-Si: cells transfected with the siRNA. Data are presented as the mean ± SD of three independent experiments (n = 3). * P <0.05, ** P <0.01.

Fig. 4

miR-214–5p/C1QTNF1 axis negatively regulates AKT/mTOR pathway. (A) The activity of the AKT/mTOR pathway was detected by western blotting after miR-214–5p mimics transfection and PCV2 infection. (B) The activity of the AKT/mTOR pathway was detected by western blotting after miR-214–5p mimics transfection and C1QTNF1 overexpression. Mimics: cells transfected with the miR-214–5p mimics, OE: C1QTNF1 overexpression cell line. Capital letters correspond to significant differences (P <0.01).

Fig. 5

miR-214–5p/C1QTNF1 Axis enhances PCV2 replication through promoting autophagy by targeting AKT/mTOR pathway. (A) The LC3 levels were detected by western blotting after miR-214–5p mimics transfection and PCV2 infection. (B) The confocal fluorescent images of PK15 cells expressing mRFP-GFP-LC3. (C) The LC3 levels were detected by western blotting after miR-214–5p mimics transfection and C1QTNF1 overexpression. (D) The inhibition efficiency of LC3 following 3-MA treatment was detected by western blotting. After miR-214–5p mimics transfection and PCV2 infection, PCV2 viral DNA copies were detected by qPCR (E), and levels of CAP protein expression were detected by western blotting (F). Mimics: cells transfected with the miR-214–5p mimics, NC: cells transfected with the mimics NC of which targeting none of the genomic sequences. Data are presented as the mean ± SD of three independent experiments (n = 3). Capital letters correspond to significant differences (P <0.01), ** P <0.01.

Fig. 6

A working model summarizing the role and mechanism of the miR-214–5p/C1QTNF1 axis regulating PCV2 replication. Pig miR-214–5p was identified as a host regulator associated with PCV2 susceptibility in PK15 cells. miR-214–5p enhances PCV2 replication via promoting autophagy by inhibiting the C1QTNF1-based AKT/mTOR signaling.