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. 2024 May 15;55(1):61.
doi: 10.1186/s13567-024-01309-7.

MiR-320 inhibits PRRSV replication by targeting PRRSV ORF6 and porcine CEBPB

Xiaoxiao Gao #  1 , Xiangbin You #  1   2 , Guowei Wang  1 , Mengtian Liu  1 , Longlong Ye  1 , Yufeng Meng  1   3   4 , Gan Luo  1   3   4 , Dequan Xu  1   3   4 , Min Liu  5
Affiliations

MiR-320 inhibits PRRSV replication by targeting PRRSV ORF6 and porcine CEBPB

Xiaoxiao Gao et al. Vet Res. .

Abstract

Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), results in huge economic losses to the world pig industry. MiRNAs have been reported to be involved in regulation of viral infection. In our study, miR-320 was one of 21 common differentially expressed miRNAs of Meishan, Pietrain, and Landrace pig breeds at 9-h post-infection (hpi). Bioinformatics and experiments found that PRRSV replication was inhibited by miR-320 through directly targeting PRRSV ORF6. In addition, the expression of CCAAT enhancer binding protein beta (CEBPB) was also inhibited by miR-320 by targeting the 3' UTR of CEBPB, which significantly promotes PRRSV replication. Intramuscular injection of pEGFP-N1-miR-320 verified that miR-320 significantly inhibited the replication of PRRSV and alleviated the symptoms caused by PRRSV in piglets. Taken together, miR-320 have significant roles in the infection and may be promising therapeutic target for PRRS.

Keywords: CCAAT enhancer binding protein beta; ORF6; PRRSV; miR-320; pig.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
MiR-320 was differentially expressed in mock vs PRSSV -infected PAMs from 4 pig breeds. MiR-320 expression levels were analyzed in miRNA-sequencing data of PRRSV-infected/mock-infected PAMs from Pietrain (P), Landrace (L), Qingping (QP), and Meishan (MS) pig breeds at 9 (A), 36 (B), and 60 (C) hpi. Mock-infected PAMs represent PAMs that were infected with culture medium and collected at 9, 36, and 60 h respectively, PRRSV-infected PAMs represent PAMs that were infected with PRRSV and collected at 9, 36, and 60 h respectively. the y-axes on the left present miR-320 RPKM (Reads Per Kilobase per Million mapped reads) expression levels. *p < 0.05, **p < 0.01.
Figure 2
Figure 2
MiR-320 inhibits the replication of PRRSV. Bioinformatical predication showed that ORF6 was a putative target mRNA of miR-320 (A). The dual luciferase reporter assay was used to detect the luciferase activity in cells co transfected with pmirGLO-ORF6-WT/ pmirGLO-ORF6-MUT and miR-320 mimics (B) or miR-320 inhibitor (C). Marc-145 cells were transfected with miR-320 mimics or miR-320 inhibitor to detect miR-320 expression (D). Marc-145 cells were transfected separately with miR-320 mimics and miR-320 inhibitor, and then infected with PRRSV (MOI = 0.1). The cells were harvested at 36 h post PRRSV infection, and qRT-PCR (E) and Western blot (F) was carried out to detect PRRSV replication. All values represent the mean ± SD of three independent experiments. *p < 0.05.
Figure 3
Figure 3
miR-320 regulate target mRNA CEBPB. Marc-145 cells were transfected with miR-320 to detect the expression of the potential target mRNAs (A). CEBPB protein expression was detected by Western blot after the transfection of Marc-145 cells with miR-320 mimics or miR-320 inhibitor (B). The potential target mRNAs predicted by bioinformatics for miR-320 (C). The dual luciferase reporter assay was used to detect the luciferase activity in cells co-transfected with miR-320 mimics/inhibitor and pmirGLO-CEBPB-WT (D) or pmirGLO-CEBPB-MUT (E). Marc-145 cells were transfected with si-CEBPB, and then infected with PRRSV (MOI = 0.1). The cells were harvested at 36 h post PRRSV infection, and qRT-PCR (F) and Western blot (G) was carried out to detect PRRSV replication. All values represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
miR-320 inhibits PRRSV replication in vivo. Marc-145 cells were transfected with pEGFP-N1-miR-320 or pEGFP-N1 (4 µg) to detect miR-320 expression (A) and the virus copy number (B). Daily average body temperatures (C) and weight gain (D) of piglets infected PRRSV after injection with plasmid pEGFP-N1 for the positive control group and pEGFP-N1-miR-320 for the experimental group. Virus copy number (E) and CEBPB mRNA expression (F) were detected in the lungs of piglets from the experimental group and positive control group. All values represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
Histopathological analyse of lungs of piglets from the control and experimental groups in vivo. Histological sections of lungs of piglets from the experimental group injected with pEGFP-N1-miR-320 and PRRSV infection (B1, B2, B3), the control group injected with pEGFP-N1 and PRRSV infection (A1, A2, A3) were stained with hematoxylin–eosin.
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