Mir-331-3p Inhibits PRRSV-2 Replication and Lung Injury by Targeting PRRSV-2 ORF1b and Porcine TNF- α

Abstract

Porcine reproductive and respiratory syndrome (PRRS) caused by a single-stranded RNA virus (PRRSV) is a highly infectious respiratory disease and leads to huge economic losses to the swine industry worldwide. To investigate the role of miRNAs in the infection and lung injury induced by PRRSV, the differentially expressed miRNAs (DE-miRs) were isolated from PRRSV-2 infected/mock-infected PAMs of Meishan, Landrace, Pietrain, and Qingping pigs at 9, 36, and 60 hpi. Mir-331-3p was the only common DE-miR in each set of miRNA expression profile at 36 hpi. Mir-210 was one of 7 common DE-miRs between PRRSV infected and mock-infected PAMs of Meishan, Pietrain, and Qingping pigs at 60 hpi. Mir-331-3p/mir-210 could target PRRSV-2 ORF1b, bind and downregulate porcine TNF-α/STAT1 expression, and inhibit PRRSV-2 replication, respectively. Furthermore, STAT1 and TNF-α could mediate the transcriptional activation of MCP-1, VCAM-1, and ICAM-1. STAT1 could also upregulate the expression of TNF-α by binding to its promoter region. In vivo, pEGFP-N1-mir-331-3p could significantly reduce viral replication and pathological changes in PRRSV-2 infected piglets. Taken together, Mir-331-3p/mir-210 have significant roles in the infection and lung injury caused by PRRSV-2, and they may be promising therapeutic targets for PRRS and lung injury/inflammation.

Keywords: ORF1b; PRRSV; STAT1; TNF-α; lung injury; miR-210; miR-331-3p.

Figure 1

DE-miRs in PRRSV-infected/mock-infected PAMs from 4 pig breeds. Venn diagrams of DE-miRs between PRRSV-infected and mock-infected PAMs of Pietrain (P), Landrace (L), Qingping (QP), and Meishan (MS) pigs at 9, 36, and 60 hpi (A–C). Mir-331-3p expression levels were analyzed in miRNA-sequencing data of PRRSV-infected/mock-infected PAMs from 4 breeds of pigs at 9, 36, and 60 hpi (D–F). Mir-210 expression levels were analyzed in miRNA-sequencing data of PRRSV-infected/mock-infected PAMs from 4 breeds of pigs at 9, 36, and 60 hpi (G–I). All values represent the mean ± s.d. of three independent experiments. **p < 0.01.

Figure 2

Mir-331-3p and mir-210 inhibit PRRSV-2 replication. Bioinformatical predication showed that ORF1b was a putative target gene of mir-331-3p and mir-210 (A). The dual luciferase reporter assay was verify the bind of mir-331-3p and ORF1b in cells co-transfected with ORF1b-331-WT/ORF1b-331-MUT and mir-331-3p mimics (B) or mir-331-3p inhibitor (C). The dual luciferase reporter assay was used to verify the bind of mir-210 and ORF1b in cells co-transfected with ORF1b-210-WT/ORF1b-210-MUT and mir-210 mimics (D) or mir-210 inhibitor (E). ORF1b expression was quantitatively analyzed in Marc-145 cells infected with PRRSV-2 after transfection with mimics/inhibitor of mir-331-3p (F) or mir-210 (G). Marc-145 cells were transfected separately with mir-331-3p mimics and mir-331-3p inhibitor, and then infected with PRRSV-2 (MOI = 0.1). The cells were harvested at 36 h post PRRSV-2 infection, and qRT-PCR (H,J), and western blot (I,K) was carried out to detect PRRSV-2 replication. Meanwhile, Marc-145 cells were transfected separately with mir-210 mimics and mir-210 inhibitor, and then infected with PRRSV-2 (MOI = 0.1). The cells were harvested at 36 h post PRRSV-2 infection, and qRT-PCR (L,N) and western blot (M,O) was carried out to detect PRRSV-2 replication. All values represent the mean ± s.d. of three independent experiments.*p < 0.05, **p < 0.01.

Figure 3

Mir-331-3p and mir-210 regulate target genes TNF-α and STAT1, respectively. Marc-145 cells were transfected with mir-331-3p to detect the expression of the potential target genes (A). TNF-α expression was detected by western blot in Marc-145 cells transfected with mir-331-3p mimics (B) or mir-331-3p inhibitor (C). The dual luciferase reporter assay was used to verify the bind of mir-331-3p and TNF-α in cells co-transfected with mir-331-3p mimics/inhibitor and TNF-α-WT (D) or TNF-α-MUT (E). Bioinformatical predication showed that TNF-α and STAT1 were the target gene of mir-331-3p and mir-210, respectively (F). Marc-145 cells were transfected with mir-210 mimics to detect the expression of the potential target genes (G). STAT1 protein expression was detected by western blot in Marc-145 cells transfected with mir-210 mimics (H) or mir-210 inhibitor (I). The dual luciferase reporter assay was used to verify the bind of mir-210 and STAT1 in cells co-transfected with mir-210 mimics/inhibitor and STAT1-WT (J) or STAT1-MUT (K). All values represent the mean ± s.d. of three independent experiments. *p < 0.05.

Figure 4

Mir-331-3p and mir-210 are involved in the regulation of genes that cause lung injury. Marc-145 cells were transfected with mir-210 mimics (A) or mir-331-3p mimics (B), infected with the PRRSV-2 for 36 h, and the expressions of MCP-1, ICAM-1, and VCAM-1 were detected by qRT-PCR. Knockdown of STAT1 inhibited TNF-α mRNA expression (C) and protein expression (D). Knockdown of STAT1 also inhibited MCP-1, ICAM-1, and VCAM-1 mRNA expression (E). The siSTAT1 was co-transfected with PGL3-TNF-α into Marc-145 cells and reduced the luciferase activity (F). All values represent the mean ± s.d. of three independent experiments. *p < 0.05, **p < 0.01.

Figure 5

Mir-331-3p inhibits PRRSV-2 replication and lung injury in vivo. Marc-145 cells were transfected with pEGFP-N1-mir-331-3p or pEGFP-N1 (4 μg) to detect mir-331-3p expression (A) and the effect on PRRSV-2 replication (B). The expression of mir-331-3p (C), daily average rectal temperatures, (D) and average daily weight gain (E) of piglets infected PRRSV-2 after injection with plasmid pEGFP-N1 for the negative control group and pEGFP-N1-mir-331-3p for the experimental group. The Ct value of PRRSV ORF7 (F), TNF-α mRNA expression (G), TNF-α protein expression (H), MCP-1, VCAM-1, and ICAM-1 (I) mRNA expression was detected in the lungs of piglets from the experimental group and negative control group. All values represent the mean ± s.d. of three independent experiments. *p < 0.05, **p < 0.01.

Figure 6

The pathological changes of lung surface of piglets from the experimental group and negative control group. After lung removal, the lung surface was rinsed with PBS, and then the lungs were placed on a white background plate and photographed. All six lungs are photographed at once.

Figure 7

Histopathological and immunohistochemical analyses of lungs of piglet from the experimental group and negative control group. Histological sections of lungs of piglets from the experimental group injected with pEGFP-N1-mir-331-3p (A1–A3) and negative control group injected with pEGFP-N1 (B1–B3) were stained with hematoxylin-eosin. TNF-α (C1,C2) were immunohistochemically localized in porcine lungs from negative control group and the experimental group, respectively. TNF-positively stained cells are in reddish brown color. PAMs were labeled with short arrows, while alveolar epithelial cells were labeled with long arrows.

Figure 8

The regulation diagram of mir-210 and mir-331-3p inhibits PRRSV-2 replication and lung injury.